Dr Corinne Dorel

Dr Sze Chun

Claire Perrin

Yann Ferrandez

Ang Kian

Feng Shu

Han Sujun

Jia Jingyu

Lin Min

Xia

Nan Yang

Shalini Ratnasingam

Byan Ley

Corinne Chew

Keywords: E. coli, multispecies biofilm, mucin, curli, GFP.. E. coli, biofilm multi-espèce, mucine, curli, GFP

PhD life has been a different one. Although difficult at times, it has been an enriching and memorable journey,

Résumé

Les bactéries dans la nature existent principalement en biofilm, qui est une communauté structurée et adhérente de microbes enveloppés dans des matrices polymériques.

Dans le corps humain, la plupart de biofilms sont composés de microorganismes commensaux et le tractus gastro-intestinal est le site le plus fortement colonisé. L'attachement bactérien à la couche de gel de mucus couvrant l'épithélium intestinal est fondamental à l'établissement d'une microflore commensale stable. Cependant, les interactions entre les bactéries et le gel de mucus restent mal décrites. En plus, la complexité et la diversité du microbiote intestinal luimême est un obstacle pour les analyses de son fonctionnement biologique. Les fonctions du microbiote sont le produit de communautés bactériennes complexes, et des interactions entres les différentes espèces qui les composent. De nouvelles approches sont nécessaires pour étudier la génétique de l'espèce la plus étudiée du microbiote de l'intestin humain, Escherichia coli. Cette thèse est consacrée à l'exploration de la réponse transcriptionnelle d'E. coli à différents facteurs présents dans l'intestin humain à travers la réalisation de 3 objectifs principaux. La première partie de mon travail concerne la conception et l'optimisation d'outils génétiques permettant de détecter E. coli au sein de biofilms multi-espèces tout en mesurant simultanément l'activité d'un gène d'intérêt. L'utilisation du gène codant la protéine fluorescente verte (GFP) et de ses dérivés a permis d'importantes avancées sur le marquage des cellules entières ainsi que le suivi d'activité transcriptionnelle. Par contre, l'utilisation de marqueurs fluorescents rouges s'est révélée décevante. Dans un deuxième temps, grâce aux outils mis au point dans la première partie de mon travail, l'influence de la mucine sur la capacité d'E. coli à former des biofilm a pu être étudiée. J'ai montré que la mucine augmente la formation du biofilm d'E. coli par modulation transcriptionnelle de structures d'adhérences telles que les curli et les pili de type 1. Enfin, l'influence de la culture en biofilms multiespèces constitués d'E. coli et de bactéries commensales (K. pneumoniae and E. faecalis) sur la croissance de chacun des partenaires a été analysée, en focalisant notre attention sur l'influence possible de structures d'adhérence telles que les curli. Les résultats indiquent que la production de curli en biofilm augmente le développement d'E. coli en co-culture avec K. pneumoniae alors qu'elle favorise l'interaction synergique entre E. coli Chapter 1 Overview

List of Figures

Biofilm in the environment

Bacteria, the most abundant life on earth, have undergone millions of years of evolution, adapting to their changing environment. It is now well accepted that bacteria in nature mostly exist not in the free-swimming planktonic form, but rather the biofilm form, which describes matrix-enclosed bacterial population adherent to each other and/or surfaces or interfaces [START_REF] Stoodley | Biofilms as complex differentiated communities[END_REF]. They are widespread in nature, and their existence can be traced in soils, water, sediments, and various parts of the human body including the skin, mouth and gut. Biofilm bacteria can also be found on surfaces of man-made structures which are in contact with fluids, such as pipe-lines, ship hulls, air-conditioning ducts and water-holding tanks. Since the observation of biofilms by [START_REF] Zobell | OBSERVATIONS ON THE MULTIPLICATION OF BACTERIA IN DIFFERENT VOLUMES OF STORED SEA WATER AND THE INFLUENCE OF OXYGEN TENSION AND SOLID SURFACES[END_REF][START_REF] Zobell | OBSERVATIONS ON THE MULTIPLICATION OF BACTERIA IN DIFFERENT VOLUMES OF STORED SEA WATER AND THE INFLUENCE OF OXYGEN TENSION AND SOLID SURFACES[END_REF][START_REF] Zobell | OBSERVATIONS ON THE MULTIPLICATION OF BACTERIA IN DIFFERENT VOLUMES OF STORED SEA WATER AND THE INFLUENCE OF OXYGEN TENSION AND SOLID SURFACES[END_REF], this form of bacterial existence has been studied with steadily increasing intensity, particularly in the last three decades, driven mainly by the need to deal with problems resulting from their colonization. For example, biofilm growth causes clogging of medical devices, exhibits more resistance to antibiotic treatments than the planktonic forms, and is responsible for much of the structural biofouling in maritime industries [START_REF] Costerton | Bacterial biofilms: a common cause of persistent infections[END_REF][START_REF] Stewart | Antibiotic resistance of bacteria in biofilms[END_REF][START_REF] Bak | UVC fluencies for preventative treatment of Pseudomonas aeruginosa contaminated polymer tubes[END_REF][START_REF] Salta | Designing biomimetic antifouling surfaces[END_REF][START_REF] Bushnak | Adhesion of microorganisms to bovine submaxillary mucin coatings: effect of coating deposition conditions[END_REF]. Whatever damage they cause and via whatever means, the characteristics of biofilm bacteria that differentiate them from their planktonic counterparts will come into play. A biofilm population is vastly more heterogeneous than a planktonic one -in terms of morphology, structure, microenvironment as well as the physiological and metabolic states of individual bacterial cells. More importantly, bacteria within a biofilm exhibit coordinated multicellular behaviour, and this is expected to be even more complex if the biofilm contains more than one species, as often is the case in natural or environmental settings.

The cellular coordination, both within and between species, seems to be promoted by non-optimal growth conditions or even by cellular stresses, and confers better adaptation to and protection from harsh environment [START_REF] Stewart | Antibiotic resistance of bacteria in biofilms[END_REF][START_REF] Stewart | Multicellular resistance: biofilms[END_REF][START_REF] Engelberg-Kulka | mazEF: a chromosomal toxin-antitoxin module that triggers programmed cell death in bacteria[END_REF][START_REF] Macfarlane | Microbial biofilms in the human gastrointestinal tract[END_REF][START_REF] Landini | Cross-talk mechanisms in biofilm formation and responses to environmental and physiological stress in Escherichia coli[END_REF]. The main extracellular structures known to be involved are labelled below each step [START_REF] Stoodley | Biofilms as complex differentiated communities[END_REF][START_REF] Kaplan | Biofilm dispersal: mechanisms, clinical implications, and potential therapeutic uses[END_REF].

Biofilm development involves many extracellular structures

Indeed, bacteria in biofilms have to face stresses that arise from higher cell density, greater oxygen limitation, and higher-osmolarity conditions than in the planktonic condition (Prigent-Combaret et al., 1999). The consequential differences in the physiological state of cells are reflected by substantial changes in their gene expression pattern, observed in both functional genomic and gene fusion studies of several bacterial species (Prigent-Combaret & Lejeune, 1999;[START_REF] Whiteley | Gene expression in Pseudomonas aeruginosa biofilms[END_REF][START_REF] Sauer | Characterization of phenotypic changes in Pseudomonas putida in response to surface-associated growth[END_REF][START_REF] Sauer | Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm[END_REF]. In the instance of Escherichia coli, for example, up to 38% of its genome expression is affected by biofilm formation, and a significant part of E. coli strain K-12 biofilm-related genes are in fact stationary phase induced (Prigent-Combaret et al., 1999;Schembri et al., 2003b;[START_REF] Beloin | Global impact of mature biofilm lifestyle on Escherichia coli K-12 gene expression[END_REF]. This change of lifestyle from planktonic to sessile state is a well-coordinated process involving many genes with various functions, but a major part is mediated through the expression of extracellular structures, which contribute to the intrinsic property of biofilm, i.e. adherence. Their expression is tightly regulated along the course of biofilm development and -based mainly on research work on Pseudomonas spp -can be categorized into five stages (Figure 1.1) [START_REF] Van Houdt | Role of bacterial cell surface structures in Escherichia coli biofilm formation[END_REF]. Different species and different experimental conditions can lead to variations in these generalised developmental steps. As illustrated, the whole life cycle of biofilm involves the concerted action of many extracellular factors such as flagella, type 1 pili, conjugative pili, curli, Antigen 43 (Ag43), β-1,6-Nacetylglucosamine (PGA), cellulose, colanic acid, lipopolysaccharides, capsules and DNA. Here I present a brief description on the function and regulation of a few of these structures especially with reference to E. coli, which are relevant to the scope of my work.

Cellulose and colanic acid

In the development and maturation of biofilm structures, exopolysaccharides such as cellulose, colanic acid and PGA are very important, as they form the bulk of the biofilm structural matrix. Here we will focus on cellulose and colanic acid because of their relevance to the regulation and synthesis of curli. IgaA/YrfF appears to inhibit the activation of RcsC (Meberg et al., 2001;Tierrez & Garcia-del Portillo, 2004). The RcsB-dependent regulation of some genes requires an auxiliary protein, RcsA, an unstable cytoplasmic protein that is degraded by the Lon (and ClpYQ) protease. The Rcs phosphorelay activates colanic acid synthesis (cps), while represses the expression of flagella (flhDC) and curli (csgD). Figure modified from [START_REF] Huang | The role of the Rcs phosphorelay in Enterobacteriaceae[END_REF] Cellulose, better known for its role as the main component of plant cell wall, is essentially a homopolysaccharide consisting of D-glucopyranose units linked by β-14 glycosidic bonds. In bacteria, it is produced by many Enterobacteriaceae, including commensal and pathogenic E. coli [START_REF] Zogaj | The multicellular morphotypes of Salmonella typhimurium and Escherichia coli produce cellulose as the second component of the extracellular matrix[END_REF][START_REF] Zogaj | Production of cellulose and curli fimbriae by members of the family Enterobacteriaceae isolated from the human gastrointestinal tract[END_REF][START_REF] Da Re | A CsgD-Independent Pathway for Cellulose Production and Biofilm Formation in Escherichia coli[END_REF]. In these bacteria, cellulose production can lead to the formation of a rigid biofilm at the air-liquid interface, the characteristics of which varies depending on the strains and environmental conditions [START_REF] Beloin | Escherichia coli biofilms[END_REF]. Cellulose synthesis genes are located in two divergently organised operons, bcsABZC and bcsEFG, which are constitutively transcribed [START_REF] Zogaj | The multicellular morphotypes of Salmonella typhimurium and Escherichia coli produce cellulose as the second component of the extracellular matrix[END_REF][START_REF] Solano | Genetic analysis of Salmonella enteritidis biofilm formation: critical role of cellulose[END_REF]. However, cellulose synthesis per se is activated by YaiC (AdrA in Salmonella) at a posttranscriptional level, by controlling the synthesis of the ubiquitous bacterial second messenger bis-(3'-5')-cyclic-diguanosine monophosphate (c-di-GMP). Cellulose does not contribute to early biofilm formation, and its co-expression with curli, under the control of CsgD, may even impair biofilm formation in certain conditions [START_REF] Gualdi | Cellulose modulates biofilm formation by counteracting curli-mediated colonization of solid surfaces in Escherichia coli[END_REF], although the co-existence of cellulose and curli can result in the formation of hydrophobic extracellular matrix [START_REF] Zogaj | The multicellular morphotypes of Salmonella typhimurium and Escherichia coli produce cellulose as the second component of the extracellular matrix[END_REF].

Colanic acid or M antigen is a negatively charged extracellular polysaccharide that is produced by E. coli and other species of the Enterobacteriaceae. It forms a protective layer around the bacterial cell in response to certain environmental stress, and is not produced in rich medium at 37°C [START_REF] Beloin | Escherichia coli biofilms[END_REF]. Colanic acid is not required during initial attachment to abiotic surface but is important for the complex 3D structure and depth of biofilm, and its synthesis is consistently up-regulated in biofilms (Prigent-Combaret et al., 1999;Danese et al., 2000b;[START_REF] Hanna | Role of capsular colanic acid in adhesion of uropathogenic Escherichia coli[END_REF]. Colanic acid synthesis by the wca operon (formerly named cps) is induced by the Rcs phosphorelay involving RcsC/RcsD/RcsB and the auxiliary co-activator RcsA (Figure 1.2) [START_REF] Huang | The role of the Rcs phosphorelay in Enterobacteriaceae[END_REF].

The signals activating the Rcs system are not well characterized. Environmental conditions such as desiccation, osmotic shock and growth at low temperature (20°C) with glucose as a carbon source have been reported to activate the Rcs phosphorelay [START_REF] Ophir | A role for exopolysaccharides in the protection of microorganisms from desiccation[END_REF][START_REF] Sledjeski | Osmotic shock induction of capsule synthesis in Escherichia coli K-12[END_REF][START_REF] Hagiwara | Genome-wide analyses revealing a signaling network of the RcsC-YojN-RcsB phosphorelay system in Escherichia coli[END_REF]. The Rcs system not only activates colanic acid that is important for biofilm maturation, but also represses the expression of cell surface structures involved in motility and attachment such as flagella, Ag43 and curli (Figure 1.2), thereby controlling the transition from attached cells to mature biofilm (Francez-Charlot et al., 2003;[START_REF] Ferrieres | The RcsC sensor kinase is required for normal biofilm formation in Escherichia coli K-12 and controls the expression of a regulon in response to growth on a solid surface[END_REF].

Flagella

During the first step of biofilm formation, the planktonic bacterium approaches solid surfaces and initial reversible attachment occurs. This has to take place in the presence of not only the passive movement of Brownian or gravitational forces, but also repulsive electrostatic and hydrodynamic forces around the surfaces. One means by which the planktonic bacteria can overcome these obstacles to arrive at the surface [START_REF] Donlan | Biofilms: microbial life on surfaces[END_REF] is via the use of their motility appendage, flagella. In Gram-negative bacteria such as E.

coli and Salmonella, flagella enable them to swim in liquid or semi-liquid medium. Pratt and Kolter showed that motility, but not chemotaxis, was required for initial cell-surface contact, possibly by overcoming the repulsive forces [START_REF] Pratt | Genetic analysis of Escherichia coli biofilm formation: roles of flagella, motility, chemotaxis and type I pili[END_REF]. Wood and co-workers confirmed using different motility mutants that biofilm formation ability of E.

coli K-12 directly correlated with their ability to swim [START_REF] Wood | Motility influences biofilm architecture in Escherichia coli[END_REF]. Flagella synthesis in E. coli and Salmonella involves fliC, encoding the building block of the flagella filament, flagellin [START_REF] Fernandez | Secretion and assembly of regular surface structures in Gram-negative bacteria[END_REF] and flhDC, which encodes the master regulator required for the expression of all other genes of the flagellar regulon [START_REF] Soutourina | Regulation cascade of flagellar expression in Gramnegative bacteria[END_REF]. However, in E. coli strains that overproduce curli (see Section 1.1.1.5), flagella are dispensable for biofilm formation and development [START_REF] Prigent-Combaret | Developmental pathway for biofilm formation in curli-producing Escherichia coli strains: role of flagella, curli and colanic acid[END_REF]. It has been demonstrated that inverse regulatory coordination of flagella and surface adherence structures such as curli exist to facilitate bacteria's change of lifestyle [START_REF] Pesavento | Inverse regulatory coordination of motility and curli-mediated adhesion in Escherichia coli[END_REF].

Whereas some adherence factors are restricted to specific E. coli pathotypes, being located on plasmids or pathogenicity islands, the E. coli species core genome contains general colonization factors such as Ag43, type 1 pili or curli [START_REF] Jubelin | CpxR/OmpR interplay regulates curli gene expression in response to osmolarity in Escherichia coli[END_REF]. The following section focuses on these common adherence factors.

Antigen 43

Biofilm development undergoes a stage of microcolony formation (Figure 1.1, stage 3) which results in the formation of early biofilm architecture. This is probably the stage that involves the most surface factors [START_REF] Van Houdt | Role of bacterial cell surface structures in Escherichia coli biofilm formation[END_REF], one of which is the outer membrane protein Antigen 43 (Ag43) in E. coli, described as "the most abundant phase-varying outer membrane protein" [START_REF] Henderson | The major phase-variable outer membrane protein of Escherichia coli structurally resembles the immunoglobulin A1 protease class of exported protein and is regulated by a novel mechanism involving Dam and oxyR[END_REF]. It is encoded by a single gene originally designated "flu" for "fluffing", because a change in this gene prevented bacteria flocculation in planktonic culture and altered the morphology of colonies on agar plate [START_REF] Diderichsen | flu, a metastable gene controlling surface properties of Escherichia coli[END_REF]. In recent years, this gene has frequently been referred to as agn43, which will also be the name used in this work. Ag43 is a self recognising surface autotransporter protein, which possesses both receptor target and receptor recognition domains [START_REF] Klemm | Structure-function analysis of the self-recognizing Antigen 43 autotransporter protein from Escherichia coli[END_REF]. It promotes cell-cell adhesion by an intercellular handshake mechanism [START_REF] Hasman | Antigen-43-mediated autoaggregation of Escherichia coli is blocked by fimbriation[END_REF], thus giving the "clump" formation phenotype in liquid cultures. Although Ag43 is apparently not involved in nonspecific initial adhesion to abiotic surfaces (Kjaergaard et al., 2000a), it appears capable of promoting multispecies biofilm formation, for example, between E. coli and P.

aeruginosa (Danese et al., 2000a;Kjaergaard et al., 2000b). Clinical studies demonstrated that Ag43 proteins promoted long-term persistence of uropathogenic E. coli isolates in the urinary tract [START_REF] Ulett | Functional Analysis of Antigen 43 in Uropathogenic Escherichia coli Reveals a Role in Long-Term Persistence in the Urinary Tract[END_REF][START_REF] Luthje | Ag43 Promotes Persistence of Uropathogenic Escherichia coli Isolates in the Urinary Tract[END_REF].

The expression of Ag43 is phase variable, governed by the concerted action of both the DNA-methylating enzyme deoxyadenosine methylase (Dam) for activation and the transcriptional regulator OxyR for repression [START_REF] Haagmans | Phase variation of Ag43 in Escherichia coli: Dam-dependent methylation abrogates OxyR binding and OxyR-mediated repression of transcription[END_REF]Schembri et al., 2003a). The coordinated regulation of agn43 and the other phase variable regulated fim cluster gene has earlier been reported and hypothesised as due to steric hindrance of Ag43-Ag43 interaction by type 1 pili [START_REF] Hasman | Antigen-43-mediated autoaggregation of Escherichia coli is blocked by fimbriation[END_REF]. However, it was later found that type 1 pili expression is in fact dominant to Ag43 and that the expression of type 1 pili per se constitutes a signal transduction mechanism that affects the thiol-disulfide status of OxyR, the thiol form of which represses agn43 expression (Figure 1.3) [START_REF] Schembri | Coordinate gene regulation by fimbriae-induced signal transduction[END_REF].

Type 1 pili

During the irreversible attachment of bacteria to surface (Figure 1.1, stage 2), surface factors such as type 1 pili and curli come into play. Type 1 pili are the most common adhesins expressed by both commensal and pathogenic E. coli isolates [START_REF] Sauer | Bacterial pili: molecular mechanisms of pathogenesis[END_REF]. They are filamentous adhesins with a tubular structure about 7 nm in diameter and approximately 1 µm long, composed mainly of the structural subunit protein FimA and the mannose-specific adhesin located at the tip of the pilus, FimH (Figure 1.4). The adaptor subunits FimG and FimF connect FimA and FimH, while the transmembrane usher FimD and chaperone FimC mediate pilus type I pili fibre formation [START_REF] Hahn | Exploring the 3D molecular architecture of Escherichia coli type 1 pili[END_REF][START_REF] Remaut | Fiber formation across the bacterial outer membrane by the chaperone/usher pathway[END_REF][START_REF] Waksman | Structural biology of the chaperone-usher pathway of pilus biogenesis[END_REF].

FimH adhesin binds to eukaryotic mannose oligosaccharides [START_REF] Duncan | The distinct binding specificities exhibited by enterobacterial type 1 fimbriae are determined by their fimbrial shafts[END_REF], and plays a role in the formation of secreted IgA mediated biofilm within the gut [START_REF] Bollinger | Human secretory immunoglobulin A may contribute to biofilm formation in the gut[END_REF][START_REF] Orndorff | Immunoglobulin-mediated agglutination of and biofilm formation by Escherichia coli K-12 require the type 1 pilus fiber[END_REF][START_REF] Bollinger | Secretory IgA and mucin-mediated biofilm formation by environmental strains of Escherichia coli: role of type 1 pili[END_REF]. Type 1 pili also mediates bacterial invasion by facilitating adherence of uropathogenic E. coli to superficial bladder epithelial cells. Interactions between FimH and bladder epithelial cells induce host signalling cascades that may trigger cell apoptosis [START_REF] Schilling | Structure and function of Escherichia coli type 1 pili: new insight into the pathogenesis of urinary tract infections[END_REF]. In addition to eukaryotic cell components, FimH adhesin also binds to abiotic surfaces through nonspecific binding [START_REF] Pratt | Genetic analysis of Escherichia coli biofilm formation: roles of flagella, motility, chemotaxis and type I pili[END_REF][START_REF] Beloin | Global impact of mature biofilm lifestyle on Escherichia coli K-12 gene expression[END_REF].

Type 1 pili expression is induced during adhesion and biofilm formation at early and late stages (Schembri et al., 2003b;[START_REF] Ren | Gene expression in Escherichia coli biofilms[END_REF]. It was thought that the highest probability of type 1 expression occurs at 37°C, in rich amino-acid-replete medium (Gally et al., 1993). However, it was later shown that fim cluster expression increased in biofilms grown in minimal medium in continuous flow chamber cultures (Schembri et al., 2003b). The expression of fimA and fimH are regulated by the fim regulatory cluster in a phase variable fashion [START_REF] Van Der Woude | Re-examining the role and random nature of phase variation[END_REF]. The promoter for fimA is located in a short segment of invertible DNA called fim switch (fimS), the orientation of which determines whether fimA is transcribed (ON phase) or not (OFF phase) [START_REF] Abraham | An invertible element of DNA controls phase variation of type 1 fimbriae of Escherichia coli[END_REF]. The inversion of switch is catalysed by two tyrosine-class recombinases FimE and FimB. While FimE promotes primarily the ON-to-OFF inversion, FimB inverts the switch in both directions. When they are co-expressed, FimE is dominant over FimB and turns the switch to OFF phase [START_REF] Klemm | Two regulatory fim genes, fimB and fimE, control the phase variation of type 1 fimbriae in Escherichia coli[END_REF]Gally et al., 1996;[START_REF] Blomfield | Integration host factor stimulates both FimB-and FimE-mediated site-specific DNA inversion that controls phase variation of type 1 fimbriae expression in Escherichia coli[END_REF].

The fim switch is also regulated by global regulators including integration host factor (IHF), leucine-responsive regulatory protein (Lrp) and histone-like nucleoid structuring protein (H-NS) (van der [START_REF] Van Der Woude | Phase and antigenic variation in bacteria[END_REF]. Recently fimEand fimB-independent fimS phase variation by tyrosine site-specific recombinase HbiF has been reported in E.

coli K1, which causes meningitis [START_REF] Xie | HbiF regulates type 1 fimbriation independently of FimB and FimE[END_REF], and by two recombinases encoded by ipuA and ibpA in UPEC CFT073 [START_REF] Bryan | Regulation of type 1 fimbriae by unlinked FimB-and FimE-like recombinases in uropathogenic Escherichia coli strain CFT073[END_REF].

Expression of fimbriae and adhesins in E. coli appears coordinated. Coordination between type 1 pili and Ag43 production is the best characterized (see above), but coordination with other fimbriae found in uropathogenic E. coli, such as P and F1C

fimbriae was also shown [START_REF] Holden | Demonstration of regulatory cross-talk between P fimbriae and type 1 fimbriae in uropathogenic Escherichia coli[END_REF][START_REF] Lindberg | Regulatory Interactions among adhesin gene systems of uropathogenic Escherichia coli[END_REF].

Curli

Another extracellular structure important for stable bacteria attachment to surfaces is curli.

Curli are thin proteinaceous fimbriae initially identified in E. coli [START_REF] Olsen | Fibronectin binding mediated by a novel class of surface organelles on Escherichia coli[END_REF], and subsequently also found to be produced by other Enterobacteriaceae such as Salmonella [START_REF] Collinson | Thin, aggregative fimbriae mediate binding of Salmonella enteritidis to fibronectin[END_REF], Shigella, Citrobacter and Enterobacter [START_REF] Smyth | Fimbrial adhesins: similarities and variations in structure and biogenesis[END_REF]. Six proteins encoded by two divergent operons direct curli formation (Figure 1.5).

Curli fibers are composed of a major subunit CsgA and a minor subunit CsgB. CsgA remains unpolymerized until it encounters the surface nucleator CsgB, which initiates CsgA polymerization. CsgD is a transcriptional activator for the csgBA operon. CsgG, CsgE, and CsgF are structural accessory proteins involved in secretion and stabilization of the fibre subunits and modulation of fiber assembly. CsgG is proposed to be the curli secretion apparatus that directs the secretion of CsgA, CsgB, and CsgF across the outer membrane [START_REF] Barnhart | Curli biogenesis and function[END_REF][START_REF] Epstein | Polymerizing the fibre between bacteria and host cells: the biogenesis of functional amyloid fibres[END_REF] (Figure 1.6).

These structures are known for three main functions. First, curli fibres mediate cellsurface and cell-cell interactions, thus promoting biofilm formation on abiotic surfaces such as sand, glass or biomaterials [START_REF] Vidal | Isolation of an Escherichia coli K-12 mutant strain able to form biofilms on inert surfaces: involvement of a new ompR allele that increases curli expression[END_REF][START_REF] Cookson | The role of type 1 and curli fimbriae of Shiga toxin-producing Escherichia coli in adherence to abiotic surfaces[END_REF][START_REF] Uhlich | Analyses of the red-dry-rough phenotype of an Escherichia coli O157:H7 strain and its role in biofilm formation and resistance to antibacterial agents[END_REF].

Second, curli production is often associated with virulence. In food contamination, adhesion of the O157:H7 strain (Shiga toxin producing strain of E. coli) to tomato skin, spinach leaves and roots of alfalfa sprouts has been shown to be mediated by curli [START_REF] Jeter | Characterization of the binding of diarrheagenic strains of E. coli to plant surfaces and the role of curli in the interaction of the bacteria with alfalfa sprouts[END_REF]. These adhesion properties enhance both bacterial survival in harsh environmental conditions as well as bacterial dissemination. Moreover, several clues also indicate that curli actively participate in mammalian host colonization. Curli bind to human host proteins including fibronectin and plasminogen [START_REF] Olsen | Fibronectin binding mediated by a novel class of surface organelles on Escherichia coli[END_REF][START_REF] Ben Nasr | Assembly of human contact phase proteins and release of bradykinin at the surface of curli-expressing Escherichia coli[END_REF] and mediate internalisation of E. coli by eukaryotic cells (Gophna et al., 2001;[START_REF] Gophna | Role of fibronectin in curlimediated internalization[END_REF][START_REF] Wang | Impact of biofilm matrix components on interaction of commensal Escherichia coli with the gastrointestinal cell line HT-29[END_REF]. Antibodies to CsgA were present in the sera from sepsis patients. E. coli isolates from blood of these patients expressed curli at 37°C in vitro, and curli-expressing E. coli K-12 strains induced higher levels of proinflammatory cytokines in human macrophages [START_REF] Bian | Expression of and cytokine activation by Escherichia coli curli fibers in human sepsis[END_REF], increased the release in human plasma of bradykinin -a potent inducer of fever, pain and hypotension [START_REF] Herwald | Activation of the contact-phase system on bacterial surfaces--a clue to serious complications in infectious diseases[END_REF] -and resulted in a decreased blood pressure in systemically infected mice compared to isogenic curli deficient mutants [START_REF] Bian | Activation of inducible nitric oxide synthase/nitric oxide by curli fibers leads to a fall in blood pressure during systemic Escherichia coli infection in mice[END_REF].

Third, curli fibres provide protection to the bacteria in harsh environment such as from antibacterial agents like chlorine or quaternary ammonium sanitizer [START_REF] Ryu | Biofilm formation by Escherichia coli O157:H7 on stainless steel: effect of exopolysaccharide and Curli production on its resistance to chlorine[END_REF][START_REF] Uhlich | Analyses of the red-dry-rough phenotype of an Escherichia coli O157:H7 strain and its role in biofilm formation and resistance to antibacterial agents[END_REF]. Formation of biofilm by a strain expressing curli may confer resistance to heavy metals by retarding metal diffusion [START_REF] Hu | Determination of spatial distributions of zinc and active biomass in microbial biofilms by two-photon laser scanning microscopy[END_REF][START_REF] Hu | Spatial distributions of copper in microbial biofilms by scanning electrochemical microscopy[END_REF][START_REF] Perrin | Nickel promotes biofilm formation by Escherichia coli K-12 strains that produce curli[END_REF][START_REF] Hidalgo | Curli Produced by Escherichia coli PHL628 Provide Protection from Hg(II)[END_REF]. Curli's protection function is associated with its distinct biochemical and biophysical properties, the most characteristic of which is its remarkable resistance to chemical and thermal denaturation (Gebbink et al., 2005), as swell as metal sorbing [START_REF] Hidalgo | Curli Produced by Escherichia coli PHL628 Provide Protection from Hg(II)[END_REF]. The benefits enjoyed by curli producing bacteria have been conferred by the amyloid properties of these astonishing structures.

Amyloids [START_REF] Chapman | Role of Escherichia coli curli operons in directing amyloid fiber formation[END_REF] in human are traditionally associated with neurodegenerative diseases including Alzheimers and Parkinson's diseases [START_REF] Cohen | Therapeutic approaches to protein-misfolding diseases[END_REF], and are the product of protein misfolding [START_REF] Chiti | Protein misfolding, functional amyloid, and human disease[END_REF]. In contrast, bacterial amyloid fibres such as curli are actively synthesized and assembled under tight regulation [START_REF] Wang | Gatekeeper residues in the major curlin subunit modulate bacterial amyloid fiber biogenesis[END_REF]. To appreciate the sophistication in the regulation of curli expression, one needs to look at the genetic components involved (Figure 1.5). First and foremost, genes for curli production are organised in two divergently transcribed operons csgBA and csgDEFG. Expression of curli is cryptic in most E. coli laboratory strains due to csgD promoter silencing [START_REF] Hammar | Expression of two csg operons is required for production of fibronectin-and congo red-binding curli polymers in Escherichia coli K-12[END_REF].

Control of curli production by environmental or clinical isolates occurs mainly at this regulatory step rather than by loss or acquisition of the curli genes.

1.1.1.6 The complex regulation of curli synthesis

Environmental conditions

Temperature, osmolarity and slow growth are key factors for curli-dependent host colonization. It is generally considered that curli expression occurs at temperature below 30°C [START_REF] Olsen | Fibronectin binding mediated by a novel class of surface organelles on Escherichia coli[END_REF], and the most common conditions recognised in laboratory E.

coli strains for maximal curli expression are growth below 30°C, in low osmolarity, and nutrient-limiting media [START_REF] Vidal | Isolation of an Escherichia coli K-12 mutant strain able to form biofilms on inert surfaces: involvement of a new ompR allele that increases curli expression[END_REF][START_REF] Jubelin | CpxR/OmpR interplay regulates curli gene expression in response to osmolarity in Escherichia coli[END_REF]. Moreover, curli expression takes place during entry into stationary phase [START_REF] Olsen | The RpoS sigma factor relieves H-NS-mediated transcriptional repression of csgA, the subunit gene of fibronectin-binding curli in Escherichia coli[END_REF]. However, Kikuchi and colleagues have shown, surprisingly, that E. coli K-12 strain in biofilm can produce curli at 37°C [START_REF] Kikuchi | Curli fibers are required for development of biofilm architecture in Escherichia coli K-12 and enhance bacterial adherence to human uroepithelial cells[END_REF]. Apparently, over expression or mutations in the csgD promoter region can also lead to curli expression at 37°C [START_REF] Uhlich | Mutations in the csgD promoter associated with variations in curli expression in certain strains of Escherichia coli O157:H7[END_REF][START_REF] Gualdi | Cellulose modulates biofilm formation by counteracting curli-mediated colonization of solid surfaces in Escherichia coli[END_REF]. Furthermore, clinical and environmental isolates of E. coli have been shown to synthesize curli at 37°C [START_REF] Bian | Expression of and cytokine activation by Escherichia coli curli fibers in human sepsis[END_REF][START_REF] Beloin | Escherichia coli biofilms[END_REF][START_REF] Saldana | Synergistic role of curli and cellulose in cell adherence and biofilm formation of attaching and effacing Escherichia coli and identification of Fis as a negative regulator of curli[END_REF]. From the published data, the temperature regulation of curli appears to be strain specific and may be quickly modified by regulatory mutations. Oxygen tension is also involved in CsgD-mediated curli regulation, with the highest csgD expression found in micro-aerobic conditions in rich medium (Gerstel et al., 2006), and favourable csgD expression under aerobic atmosphere in minimum medium [START_REF] Smith | The Molecular Genetics of Bioadhesion and Biofilm Formation[END_REF].

Genetic regulation

Genetic regulation of curli production is subject to some variations depending on species and strains, but is globally well conserved. More than ten transcriptional factors regulate the transcriptional activator CsgD of the structural curli operon in a complex interplay.

These regulators can be categorized into 4 groups.

The first group constitutes global regulators. In this group, Nucleoid-Associated Proteins (NAPSs) including H-NS [START_REF] Dorman | H-NS: a universal regulator for a dynamic genome[END_REF], IHF (Gerstel & Romling, 2001;Gerstel et al., 2006) and FIS [START_REF] Saldana | Synergistic role of curli and cellulose in cell adherence and biofilm formation of attaching and effacing Escherichia coli and identification of Fis as a negative regulator of curli[END_REF] play an important role in the curli genes transcription. Moreover, RpoS (σ S ), a general stress sigma factor controls many stationary phase-inducible genes including the curli genes [START_REF] Arnqvist | Sigma S-dependent growth-phase induction of the csgBA promoter in Escherichia coli can be achieved in vivo by sigma 70 in the absence of the nucleoid-associated protein H-NS[END_REF] with the cooperation of Crl [START_REF] Bougdour | Crl, a low temperature-induced protein in Escherichia coli that binds directly to the stationary phase sigma subunit of RNA polymerase[END_REF].

The second group consists of regulators which are members of signal transduction pathways, such as OmpR [START_REF] Vidal | Isolation of an Escherichia coli K-12 mutant strain able to form biofilms on inert surfaces: involvement of a new ompR allele that increases curli expression[END_REF], CpxR (Dorel 1999), RcsB [START_REF] Vianney | Escherichia coli tol and rcs genes participate in the complex network affecting curli synthesis[END_REF], and RstA (Ogasawara et al., 2007b;Ogasawara et al., 2010a). A model integrating interplay between several of these signal transduction pathway was proposed in Jubelin et al., 2005[START_REF] Jubelin | CpxR/OmpR interplay regulates curli gene expression in response to osmolarity in Escherichia coli[END_REF] and in Ogasawara et al., 2010(Ogasawara et al., 2010a).

When osmolarity is low to moderate, OmpR is activated, which activates csgD promoter, and CsgD in turn activates the transcription of csgBA [START_REF] Prigent-Combaret | Complex regulatory network controls initial adhesion and biofilm formation in Escherichia coli via regulation of the csgD gene[END_REF]. On the other hand, the Cpx system, that responds to envelope stresses such as high osmolarity, overproduction and misfolding of membrane proteins and elevated pH [START_REF] Raivio | Periplasmic stress and ECF sigma factors[END_REF], as well as the Rcs system that also responds to envelope stress regulates csgBA expression negatively and will turn off the curli production in fixed cells [START_REF] Dorel | Involvement of the Cpx signal transduction pathway of E. coli in biofilm formation[END_REF]. Vidal and co-worker have discovered that a point mutation at the 234 position of the ompR gene, leading to a leucine to arginine residue change at position 43, resulted in a constitutively active OmpR that mediates high expression of csgBA and elevated curli production [START_REF] Vidal | Isolation of an Escherichia coli K-12 mutant strain able to form biofilms on inert surfaces: involvement of a new ompR allele that increases curli expression[END_REF]. This mutation is termed "ompR234". E. coli strains that normally do not make biofilms would form biofilm with the incorporation of the ompR234 mutation that causes curli overproduction. module that specifically and exclusively regulates the transcription of csgD [START_REF] Weber | Cyclic-di-GMP-mediated signalling within the σS network of Escherichia coli[END_REF]. YegE and YhjH antagonistically modulate csgD expression [START_REF] Pesavento | Inverse regulatory coordination of motility and curli-mediated adhesion in Escherichia coli[END_REF]. YddV and Dos proteins play an important role in fine tuning the expression of curli-encoding genes in response to oxygen availability [START_REF] Tagliabue | The yddV-dos operon controls biofilm formation through the regulation of genes encoding curli fibers' subunits in aerobically growing Escherichia coli[END_REF]. YeaP may regulate csgBA expression transcriptionally or post-transcriptionally [START_REF] Sommerfeldt | Gene expression patterns and differential input into curli fimbriae regulation of all GGDEF/EAL domain proteins in Escherichia coli[END_REF].

Regulators affecting only a small number of genes, such as MlrA [START_REF] Brown | MlrA, a novel regulator of curli (AgF) and extracellular matrix synthesis by Escherichia coli and Salmonella enterica serovar Typhimurium[END_REF]Ogasawara et al., 2010b), constitute a third group.

Lastly, beside these conventional transcriptional regulators, proteins involved in c-di-GMP metabolism indirectly but undoubtedly affect the production of curli. The c-di-GMP is a secondary messenger widely used by bacteria and invoved in the transition between planktonic and sessile life style [START_REF] Simm | GGDEF and EAL domains inversely regulate cyclic di-GMP levels and transition from sessility to motility[END_REF]. The production of curli fibers appears strongly stimulated by c-di-GMP [START_REF] Weber | Cyclic-di-GMP-mediated signalling within the σS network of Escherichia coli[END_REF]. Six different genes encoding c-di-GMP-related proteins are involved in curli gene regulation [START_REF] Sommerfeldt | Gene expression patterns and differential input into curli fimbriae regulation of all GGDEF/EAL domain proteins in Escherichia coli[END_REF]. A model summarizing gene expression regulation of curli genes by diguanylate cyclases (DGC) and phosphodiesterase (PDE) proteins was proposed [START_REF] Tagliabue | The yddV-dos operon controls biofilm formation through the regulation of genes encoding curli fibers' subunits in aerobically growing Escherichia coli[END_REF] and shown in (Figure 1.7).

In addition to control by transcriptional regulators, post-transcriptional control is involved in curli expression, and occurs via two antisense RNAs that target the transcriptional regulator CsgD to inhibit curli synthesis [START_REF] Holmqvist | Two antisense RNAs target the transcriptional regulator CsgD to inhibit curli synthesis[END_REF].

Talking about curli regulation, one has to recall that evidence exists for regulatory relationships between flagella and fimbriae, between flagella and capsule, and between different types of fimbriae (reviewed in [START_REF] Pruss | A Complex Transcription Network Controls the Early Stages of Biofilm Development by Escherichia coli[END_REF]. Therefore, such a complexity of the curli transcriptional regulatory network is not surprising. Having learnt the advantages of bacteria survival in biofilm and its life cycle involving extracellular structures, we tend to correlate chronic or recurrent infections in the human body with bacterial biofilm persistence due to their ability to express surface adhesion structures and form biofilms. However, it should not be neglected that the biofilm formation ability of E. coli isolates is not solely associated with their pathogenecity or surface adhesins, but rather dependent on their growth condition, to which different isolates respond differently [START_REF] Reisner | In vitro biofilm formation of commensal and pathogenic Escherichia coli strains: impact of environmental and genetic factors[END_REF]. Dong and Schellhorn have shown that the nature of the RpoS-controlled regulon in minimal media was substantially different from that expressed in rich media [START_REF] Dong | Control of RpoS in global gene expression of Escherichia coli in minimal media[END_REF]. For example, in minimal media, genes coding for flagella synthesis and motility are down regulated compared to that in rich media, while agn43 is up regulated. Therefore, caution should be exercised when it comes to analysis of biofilm data. Differential regulatory responses between different strains and/or different conditions are not surprising due to the complexity of genetic and environmental factors in biofilm formation of E. coli.

Biofilm development is a dynamic and adaptive process

Biofilm in the human body

Besides the harsh environment in nature, bacteria also inhabit the warm and nutrient-rich human body. Due to bacteria's requirement of moisture for survival, they mainly live on cutaneous and mucosal surfaces, of which the gastrointestinal tracts are the most heavily colonised. Sterile at birth, the human colon is colonised with bacteria during parturition (Macfarlane & McBain, 1999), and the first colonisers are generally facultative anaerobes, such as enterococci and enterobacteria, followed by obligate anaerobes. Upon weaning, a complex adult type microbiota is established [START_REF] Probert | Bacterial biofilms in the human gastrointestinal tract[END_REF].

Symbiotic microbiota as a human organ

After millions of years of co-evolution, trillions of microbes live harmoniously in the mammalian gut [START_REF] Nilsen | Expansion of the eukaryotic proteome by alternative splicing[END_REF], contributing to the mutualistic relationship between the microbe and the host. The microbe-host interactions are found to be essential to the host's physiology, influencing the metabolism, energy utilisation and storage, as well as immune homeostasis [START_REF] Backhed | The gut microbiota as an environmental factor that regulates fat storage[END_REF][START_REF] Guarner | Mechanisms of disease: the hygiene hypothesis revisited[END_REF][START_REF] Martin | A top-down systems biology view of microbiome-mammalian metabolic interactions in a mouse model[END_REF]. For instance, plant polysaccharides that are not digestible by human are the main substrates for microbial growth in the colon, while the fermentation products provide the host with important energy source [START_REF] Flint | Interactions and competition within the microbial community of the human colon: links between diet and health[END_REF]. Therefore, the ability of the commensal to degrade polysaccharides determines the calories the host can extract from its diet, thus influencing the survival of both host and microbiota [START_REF] Sonnenburg | Microbiology: Genetic pot luck[END_REF].

The microbiota of each individual is as unique as fingerprints and has its share of impact on host physiology, ranging from obesity, immune development and even ageing [START_REF] Turnbaugh | The core gut microbiome, energy balance and obesity[END_REF][START_REF] Barrett | The world within--impact of the intestinal micobiota on whole body physiology and pathophysiology[END_REF][START_REF] Backhed | Coordinated regulation of the metabolome and lipidome at the host-microbial interface[END_REF][START_REF] Tiihonen | Human intestinal microbiota and healthy ageing[END_REF]. Moreover, the microbial commensals may have evolved to improve their own fitness by improving the fitness of their host [START_REF] Guarner | Gut flora in health and disease[END_REF][START_REF] Sachs | The evolution of cooperation[END_REF][START_REF] Dethlefsen | An ecological and evolutionary perspective on human-microbe mutualism and disease[END_REF]. For example, they can both compete with pathogens for resources as effective consumers and secrete molecules that inhibit pathogen growth [START_REF] Tilman | Niche tradeoffs, neutrality, and community structure: a stochastic theory of resource competition, invasion, and community assembly[END_REF][START_REF] Reid | Probiotics to prevent urinary tract infections: the rationale and evidence[END_REF]; they could also detoxify compounds harmful to the host, thereby increasing the lifespan of the host [START_REF] Pool-Zobel | Modulation of xenobiotic metabolising enzymes by anticarcinogens --focus on glutathione S-transferases and their role as targets of dietary chemoprevention in colorectal carcinogenesis[END_REF], in turn giving themselves more opportunities to spread. The importance of this mutualism has led to the birth of the Human Microbiome Project, which aims to collect the genomic information of all the microorganisms in the human body. The close and specific contact of the complex microbial ecosystem in our intestines with human cells, exchanging nutrients and metabolic wastes, makes symbiotic bacteria essentially a human organ and their collective genomes our second genome [START_REF] Zhao | Genomics: The tale of our other genome[END_REF][START_REF] Possemiers | The intestinal microbiome: A separate organ inside the body with the metabolic potential to influence the bioactivity of botanicals[END_REF].

Adhesion of the bacteria to the host surface in gastrointestinal tract is essential to maintain members of this normal microflora. Even though anaerobic bacteria largely outnumber E.

coli in the gastrointestinal tract [START_REF] Berg | The indigenous gastrointestinal microflora[END_REF], E. coli is the predominant aerobic microorganism in the intestine and is among the first species to colonize the infant gut [START_REF] Mackie | Developmental microbial ecology of the neonatal gastrointestinal tract[END_REF]. Moreover, E. coli is also critical in all diarrheal infections caused by pathogenic E. coli strains [START_REF] Torres | Adherence of diarrheagenic Escherichia coli strains to epithelial cells[END_REF]. In this respect, the step of E. coli attachment to the overlying mucus gel layer of the intestinal epithelium is fundamental both to the establishment of a stable commensal microbiota and to the intestinal disease development.

The importance of mucin

However, as the French proverb says "La liberté des uns s'arrête là où commence celle des autres", which translates into "the freedom of one exists up to the point where it starts to infringe on that of others", the symbiotic nature of the intestinal host-microbial relationship is relevant only if bacteria penetration of host tissues is effectively limited [START_REF] Duerkop | Immune responses to the microbiota at the intestinal mucosal surface[END_REF]. This limitation is facilitated by the mucus layer, which consists of complex mucin glycoproteins secreted by specialised goblet cells [START_REF] Corfield | Mucins and mucosal protection in the gastrointestinal tract: new prospects for mucins in the pathology of gastrointestinal disease[END_REF].

There are 21 different mucin genes identified and MUC2 is the major mucin produced by the intestinal mucosa [START_REF] Sharma | Molecular modulation of intestinal epithelial barrier: contribution of microbiota[END_REF]. It has been shown that two layers of mucus exist on the epithelial lining: the densely packed inner layer devoid of bacteria that serve as a physical barrier preventing bacteria penetration, and the loose bacteria-containing outer layer. A vivid example illustrating the proverb can be found within this context:

while the commensal E. coli limit themselves within the mucus layer and does not infringe on the host, the diarrheagenic E. coli strains are defined and characterized by their ability to penetrate the mucus layer and efficiently colonize the mucosa to the detriment of the host [START_REF] Torres | Adherence of diarrheagenic Escherichia coli strains to epithelial cells[END_REF].

It has long been thought that mucins only serve to protect and lubricate the epithelial surfaces, until recently researchers found that they participate in many other important The carcass of the animal can also be contaminated during slaughtering. People in direct contact with the animal, including those working in farms or slaughterhouses, may be contaminated by bacteria too (4). Finally, bacterial transmission can occur between human hosts (5). Figure is adopted from the website of Montreal University (http://www.ecl-lab.com/fr/ecoli/pathogenesis.asp).

Fecal excretion + contamination of the environment Contamination of food and water

Transmission between human host

Transmission from animal to human host (farms, slaughterhouse…) Ingestion of pathogenic E. coli functions such as growth, epithelial renewal, differentiation, barrier integrity, metastasis, carcinogenesis and even foetal development [START_REF] Moniaux | Structural organization and classification of the human mucin genes[END_REF][START_REF] Corfield | Mucins in the gastrointestinal tract in health and disease[END_REF][START_REF] Vieten | Impaired mucosal regeneration in neonatal necrotising enterocolitis[END_REF]. In the case of intestinal mucin, it is involved directly and indirectly in the enteric host defence against harmful microorganisms (Lievin-Le [START_REF] Moal | The front line of enteric host defense against unwelcome intrusion of harmful microorganisms: mucins, antimicrobial peptides, and microbiota[END_REF]. As early as 1974, mucin has been reported to bind to cholera toxin and inhibit its action [START_REF] Strombeck | Binding of cholera toxin to mucins and inhibition by gastric mucin[END_REF]. More recent findings demonstrated that probiotics, which are live microorganisms that when administered in adequate amounts confer a health benefit on the host, and mainly represented by Lactic Acid Bacteria, can act by inducing mucin gene expression [START_REF] Mack | Probiotics inhibit enteropathogenic E. coli adherence in vitro by inducing intestinal mucin gene expression[END_REF][START_REF] Lutgendorff | The role of microbiota and probiotics in stress-induced gastro-intestinal damage[END_REF]. It is now suggested, in addition, that the mucus layer acts as a source of nutrients to intestinal commensal bacteria, which may be an advantage for them to efficiently colonise the mucus layer or the epithelial cell surface underneath [START_REF] Hoskins | Mucin degradation in human colon ecosystems. Isolation and properties of fecal strains that degrade ABH blood group antigens and oligosaccharides from mucin glycoproteins[END_REF][START_REF] Yang | 2-DE and MS analysis of interactions between Lactobacillus fermentum I5007 and intestinal epithelial cells[END_REF][START_REF] Ruas-Madiedo | Mucin degradation by Bifidobacterium strains isolated from the human intestinal microbiota[END_REF][START_REF] Fakhry | Characterization of intestinal bacteria tightly bound to the human ileal epithelium[END_REF]. Although mucin degradation has been linked to pathogenesis [START_REF] Pultz | Vancomycin-resistant Enterococci may obtain nutritional support by scavenging carbohydrate fragments generated during mucin degradation by the anaerobic microbiota of the colon[END_REF][START_REF] Vieira | Two atypical enteropathogenic Escherichia coli strains induce the production of secreted and membrane-bound mucins to benefit their own growth at the apical surface of human mucin-secreting intestinal HT29-MTX cells[END_REF] because of the mucus layer's role as a physical barrier to pathogen adhesion to epithelial cells [START_REF] Moncada | Mucin and Toll-like receptors in host defense against intestinal parasites[END_REF], the ability to degrade mucin was also observed in commensal and probiotic bacteria, which are closely associated with the human gut epithelial cells without showing cytotoxicity [START_REF] Fakhry | Characterization of intestinal bacteria tightly bound to the human ileal epithelium[END_REF]. Thus mucin degradation can be beneficial in that it regulates mucin turnover and may stimulate the shedding of mucus layer that helps to eliminate the colonizing pathogens, while affecting commensals less due to their growth advantage in colonising the new mucus layer.

From the bacterial point of view

Bacteria such as E. coli face changing environments, from water, soil, food to mammalian urinary or digestive tract (Figure 1.8). Published data show that enterobacteria such as E. coli owns in its core genome a whole set of adherence structures [START_REF] Beloin | Escherichia coli biofilms[END_REF], and amongst others, gene transfer of the F plasmid enabling the expression of F conjugative pilus adds on to the variety (Ghigo, 2001). The production of theses extracellular structures are under complex regulation in response to external environmental conditions, resulting in biofilm establishment in different locations (environment or gut).

E. coli can sense environmental changes via mainly two-component signalling pathways such as EnvZ-OmpR or CpxA-CpxR [START_REF] Jubelin | CpxR/OmpR interplay regulates curli gene expression in response to osmolarity in Escherichia coli[END_REF] but also via nucleoidassociated protein such as H-NS [START_REF] Dorman | H-NS, the genome sentinel[END_REF]. Noticeably, all these proteins can detect changes in osmolarity, a fluctuant parameter in the environment as in the host, and were shown to regulate adherence structure such as curli and concomitant biofilm formation (Gerstel et al., 2006;[START_REF] Dorel | The Cpx system of Escherichia coli, a strategic signaling pathway for confronting adverse conditions and for settling biofilm communities?[END_REF][START_REF] Dorman | Chapter 2 Nucleoid-Associated Proteins and Bacterial Physiology[END_REF].

In this work, we want to explore the transcriptional response of E. coli when facing new environment constituted by human gut. As the gut epithelial cells are covered by a layer of mucus, a first perception is likely to be bacterial mucin sensing and their interaction with other commensal bacteria. Establishment and optimisation of appropriate experimental models and tools are needed to trace E. coli in mixed communities.

Objective

Given all these facts, we could see the importance of the collaborative effort of mucin and commensal bacteria in maintaining the health of the host at the front line of the hostenvironment interaction, due to the interdependence between mucin and the commensal microbiota. While the effect of mucin and commensal bacteria on pathogen colonisation has been much investigated, the influence of mucin on commensals and the consequent interaction between commensal species has not been addressed. Commensal bacteria are known to antagonise pathogen colonisation by production of antimicrobial substances, pH modification of the luminal content, and competition for nutrients, but would mucin's presence enhance the competitiveness of and/or the interaction between the commensals against pathogens?

In this work, I attempt to examine the effect of mucin on the biofilm formation of the well characterised commensal strain E. coli, which is one of the representative commensal strains in the microbiota of neonate's intestine [START_REF] Fanaro | Intestinal microflora in early infancy: composition and development[END_REF]. E. coli strain MG1655 was utilized here and by other groups [START_REF] Barbas | Altering and Assessing Persistence of Genetically Modified E. coli MG1655 in the Large Bowel[END_REF][START_REF] Bollinger | Secretory IgA and mucin-mediated biofilm formation by environmental strains of Escherichia coli: role of type 1 pili[END_REF] because of its extensive characterization and facility to be genetically modified.

Since curli production is frequent in faecal isolates and that regulatory mutations often tun off curli genes when bacteria are grown outside the host, both MG1655 and its curliproducing ompR234 derivative were used.

Two other representative strains Klebsiella pneumoniae and Enterococcus faecalis are also chosen in this study to investigate the interactive relationship between these commensal strains in a dual-or multi-species context. Three main objectives were defined:

1) Design experimental models and analytical tools (Chapter 2). The process of how I developed experimental models, strains and analytical tools to serve the purpose of the project is described in this chapter. In bacterial co-culture, it is important that one species be discerned from the other. Chromogenic agar was thus employed for certain type of analyses, and E. coli whole cell-labelled with green fluorescence protein (GFP)

-to be distinguished from K. pneumoniae and E. faecalis -was used for other types of analyses. In order to monitor the influence of partner species on E. coli gene expression, both GFP and red fluorescence protein (RFP) as transcriptional reporters were tested for application in my system. The dual fluorescence system (GFP for E.

coli whole cell labelling, RFP for promoter-reporting) developed by the Singapore laboratory [START_REF] Miao | Dual fluorescence system for flow cytometric analysis of Escherichia coli transcriptional response in multispecies context[END_REF] allows profiling of E. coli transcriptional response in co-culture with K. pneumoniae and E. faecalis. For my project, a reversed dual fluorescence labelling (red-green reversed) was attempted. The success encountered and challenges faced in these attempts are presented.

2) E. coli response to mucin (Chapter 3). Various concentrations of mucin were tested with a few commonly used laboratory E. coli strains and low concentrations of mucin were found to promote biofilm formation in all strains tested. Investigation using the fluorescence tagged promoter validated in Chapter 2 suggested curli's involvement in the mucin-induced biofilm formation. Involvement of a few other extracellular adhesion structures was checked. After examining the regulatory pathway of curli synthesis, we speculated on the possible mechanism for the curli-dependent biofilm induction by mucin.

3) Influence of curli on E. coli interaction with two other commensal bacteria (K.

pneumoniae and E. faecalis). In Chapter 4, the population relationship between E. coli, K. pneumoniae and E. faecalis in both planktonic and biofilm states, is presented in the context of curli expression. The presence of curli alters the surface property of E. coli, and may influence the interspecies interactions between E. coli and its commensal partner species. Therefore, curli-expressing E. coli mutants are used for comparison with the wild type in terms of the population relationship being manifested, and the implications are discussed.

With regard to the results and conclusions drawn from the previous chapters, I propose in Chapter 5 the future direction of this work so as to deepen our understanding on the complex interactions between commensal bacteria and the environment as well as between different commensal species.

Chapter 6 describes the materials and methods employed in this project. It is placed at the end of this thesis in order not to interrupt the flow of reading.

Since this work was carried out in two laboratories (one in France and the other in Singapore) with 6 to 8 months alternation, the different equipments with same functions (for example, fluorometer and confocal microscope) used in the two laboratories render it difficult to compare the results at times. However, this does not affect the comparison within each experiment and the trend does not change with different equipments. Thus experiments conducted using the same machine were all presented in the same scale whereas comparison between results obtained from different machines were adjusted to scales that best illustrate the trend of the data.

Chapter 2 Experimental models and analytical tools

Very little research has been carried out on the interspecies interactions between commensal bacteria using multi-species cultures as experimental models, and reviews on this aspect has only started to appear in the last 2 years [START_REF] Little | Rules of engagement: interspecies interactions that regulate microbial communities[END_REF][START_REF] Haruta | Intertwined interspecies relationships: approaches to untangle the microbial network[END_REF][START_REF] Hibbing | Bacterial competition: surviving and thriving in the microbial jungle[END_REF]. Fundamentally, the influence of a partner species can be expected to be reflected in the gene expression profile. Studies on gene expression have been conducted at the transcriptional (such as microarray, differential fluorescence induction, and in vivo expression technology) and post-transcriptional (for example, signature-tagged mutagenesis and proteomic method) level in pure culture models, but the technical difficulties of applying them in multi-species analysis may have been the main cause of the lack of data on this subject.

As such, I cannot rely much on currently available methods, and need to proceed stepwise by assessing established experimental conditions and concurrently developing necessary new tools specifically for this project. These further need to be optimised and validated. In this chapter, the procedures used and constraints encountered are presented.

Growth media and temperature

The BHI is the preferred choice over Luria-Bertani (LB) for the high osmolarity condition in this study because it supports faster growth (shorter generation time) and higher saturating cell density at stationary phase for all three commensal partners used in our study (Miao, unpublished data). For the low osmolarity condition, the use of M63 minimal medium supplemented with glucose followed by 1% LB or 1% BHI has demonstrated favourable expression of curli in the E. coli MG1655ompR234 mutant (Table 1, flocculation phenotype of MG1655ompR234). To further investigate the impact of curli production on the interaction between E. coli and the mucus layer as well as its commensal partner species, the M63 minimal medium supplemented with BHI was found to be more suitable than LB because although both conserve the flocculation property of the strains useful in this project, E. faecalis does not grow in the latter ( 1. Growth and flocculation property of various strains used in this study. All strains (refer to Section 6.1. for detailed description) were incubated at 30°C. "+" denotes the presence of growth or flocculation whereas "-" the absence.

In addition to osmolarity fluxes, E. coli is further subject to changes in temperature as it shuttles between the intestine of the host (37°C) and the external environment (lower temperatures). In particular, the thermoregulation of curli expression in MG1655ompR234 dictates a favourable expression at the lower temperature of 30°C under low osmolarity conditions [START_REF] Vidal | Isolation of an Escherichia coli K-12 mutant strain able to form biofilms on inert surfaces: involvement of a new ompR allele that increases curli expression[END_REF][START_REF] Jubelin | CpxR/OmpR interplay regulates curli gene expression in response to osmolarity in Escherichia coli[END_REF] the most frequently used conditions: BHI at 37°C (abbreviated as B37) and mM63 at 30°C (abbreviated as M30) are shown in Figure 2.1.

Types of culture

Planktonic

Although the planktonic mode of microbial life only represents a small proportion of bacterial existence in their ecosystems, it is an essential stage of the biofilm life cycle [START_REF] Stoodley | Biofilms as complex differentiated communities[END_REF]. The dispersal of biofilm releases planktonic cells to colonise new locations, which is of fundamental importance for the expansion and survival of the species [START_REF] Kaplan | Biofilm dispersal: mechanisms, clinical implications, and potential therapeutic uses[END_REF]. In the human intestine, bacteria do shuttle between planktonic and biofilm modes (Molin, S. personal communication). While those in biofilm state are closely associated with the mucus layer adherent to the surface of epithelial cells, planktonic cells exist primarily in the lumen.

A large collection of data has been generated over the majority of bacteriology's history based on planktonic experimental models. Therefore, before going into biofilm studies, it is more convenient to check the phenotypes of the strains in our study in the planktonic form against those that are documented among known literature. For example, the promoter-reporter model is validated through the exhibition of reported activity of the well-characterised promoters in the appropriate conditions in our system (discussed in Section 2.5).

Biofilm -saturated vs. interface models

Over the decades, the popularization of biofilm research has brought about the blooming of biofilm assay models, ranging from the commonly used static and flow cell biofilm system to fermenters and even sampling from the nature. The continuous-flow methods such as a flow cell system have a number of advantages, such as (i) continuous renewal of biofilm medium, (ii) growth of biofilms under hydrodynamic conditions, (iii) ease of addition of antibiotics or change of the growth medium at a defined time point, and (iv) Fresh culture medium is pumped from the medium reservoir onto a biofilm grown in the bottom of a well in a 6-well plate through silicone tubing (1) and a 20G needle inserted on one side of the plate lid [START_REF] Danese | Biofilm formation as a developmental process[END_REF]. Planktonic bacteria and spent medium are removed through another needle placed on the opposite side of the well (3) and pumped to waste receptacle (4). possibility of nondestructive real-time microscopic analysis [START_REF] Wolfaardt | Multicellular organization in a degradative biofilm community[END_REF][START_REF] Stoodley | The formation of migratory ripples in a mixed species bacterial biofilm growing in turbulent flow[END_REF][START_REF] Sauer | Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm[END_REF]. However, it is a relatively complicated system to engineer and requires more troubleshooting in technical issues like leakage, air bubbles, and medium back-flow [START_REF] Sternberg | Growing and analyzing biofilms in flow cells[END_REF].

On the other hand, the static biofilm models make use of simple equipment and can be easily adapted to study a variety of biofilm formation conditions [START_REF] Merritt | Growing and analyzing static biofilms[END_REF].

Static biofilm systems basically fall into four categories: (i) surface model; (ii) air-liquid interface model; (iii) colony biofilms and (iv) the "low-flow" Kadouri system (Figure 2.2). The surface model is useful for bacteria that form surface-attached biofilms immersed in the medium. In laboratory experiment setups, abiotic surface materials such as polystyrene (PS), polyvinylchloride (PVC), polycarbonate (PC), and glass are the most frequently used. Often, microtiter plate (96-, 24-, or 6-well plate) is used in this model because of its ease in manipulation and high adaptability of the general protocol to different applications. Cells are allowed to grow in the wells for a desired length of time before planktonic cells are washed off and the surface-attached biofilm cells can thus be either visualized by microscope or quantified by crystal violet or safranin staining or even harvested for viable count on agar plates. This model is also suitable for high throughput genetic screening for mutants defective in surface attachment capabilities. Moreover, both aerobic and anaerobic bacteria can be studied in this model [START_REF] O'toole | Genetic approaches to study of biofilms[END_REF][START_REF] Stepanovic | Influence of dynamic conditions on biofilm formation by staphylococci[END_REF][START_REF] Danhorn | Phosphorus limitation enhances biofilm formation of the plant pathogen Agrobacterium tumefaciens through the PhoR-PhoB regulatory system[END_REF]. To test the biofilm formation of facultative anaerobic bacteria in the absence of oxygen, overlaying the wells with mineral oil is sufficient to simulate anaerobic condition [START_REF] O'toole | Genetic approaches to study of biofilms[END_REF].

Even so, many aerobic bacteria tend to form biofilms at the interface between the medium and air. The air-liquid interface model is thus a better choice. The setup of this model can be a simple modification of the surface model by tilting the microtiter plate 30° to 50° relative to horizontal so that the culture surface is positioned at the centre of the well bottom [START_REF] Caiazza | SadB is required for the transition from reversible to irreversible attachment during biofilm formation by Pseudomonas aeruginosa PA14[END_REF]. Alternatively, a glass or plastic coverslip can be placed vertically in the culture so that the culture surface falls around the centre of the coverslip, which then can be sampled at the appropriate time points for microscopic It is then mounted carefully on a flow cell chamber prepared with water in all channels so that the biofilm is suspended in water.

visualization and quantification via staining or direct enumeration. Recently, new methods have gained popularity for air-liquid interface biofilm studies. For example, microbiologists in the University of Calgary have developed a simple batch culture technique to reliably grow 96 equivalent biofilms at a time (the Calgary Biofilm Device) [START_REF] Ceri | The Calgary Biofilm Device: New technology for rapid determination of antibiotic susceptibilities in bacterial biofilms[END_REF]. Now commercially available, this 96-well plate derived MBEC™ assay allows microorganisms to grow on 96 identical pegs protruding down from a plastic lid into the wells of a microtiter plate (Figure 2.3).

Another model for studying biofilms, typically in connection with its antibiotic resistance but may be modified for other purposes, is the colony biofilm model [START_REF] Anderl | Role of antibiotic penetration limitation in Klebsiella pneumoniae biofilm resistance to ampicillin and ciprofloxacin[END_REF][START_REF] Walters | Contributions of antibiotic penetration, oxygen limitation, and low metabolic activity to tolerance of Pseudomonas aeruginosa biofilms to ciprofloxacin and tobramycin[END_REF]. In this model, biofilm is grown on a semi-permeable membrane that sits on an agar plate. An advantage of this model is that the biofilm can be given fresh supply of nutrient by simply relocating the membrane cells to a fresh agar plate. This aspect can be further improved by using the Kadouri system [START_REF] Merritt | Growing and analyzing static biofilms[END_REF], where growth medium is gently pumped into each well of the 6-well plate through a needle that has been inserted through the plate lid. Waste and planktonic cells exit through a second needle and are pumped away from the well (Figure 2.2). Therefore, in this system the shear forces are minimal and may apply less mechanical stress to the biofilms. It can be considered a "low flow" intermediate between the static and standard flow cell system.

In this study, three different biofilm models were used, which belongs to either the surface or the air-liquid interface model. In the first model, 24-well microtiter plate was used to grow abiotic surface-attached biofilm for either crystal violet staining to grossly analyze the biomass of the biofilm portion (biofilm attached to the bottom of the well), or to enumerate both the planktonic and biofilm portion to obtain the adherence property of the bacterial strains. On this basis, I have developed a second surface model to accommodate both inverted (Zeiss) and upright (Nikon) Confocal Laser Scanning Microscope (CLSM) observation. We named it the "saturated model" to differentiate it from the microtiter plate method. The saturated model consists of 3 sterilized glass coverslips placed at the bottom of a Petri-dish containing 20 ml of seeding culture (Figure 2.4 A). After 24 or 48 hours of incubation, coverslips covered with biofilms are taken out of the Petri-dish with the aid of a needle and forceps for immediate CLSM observation or viable count after scraping the biofilm off the coverslips.

All three bacterial species used in this study being facultative anaerobic, an air-liquid interface model (referred to as "interface model" for simplicity) is also employed. In this model, glass coverslips are vertically placed with a slight angle in a tank containing the medium (Figure 2.4 B). For analysis of the biofilm formed at the air-medium interface, the coverslip is withdrawn and washed gently to eliminate the attached planktonic cells.

Then, biofilm cells can either be observed by CLSM or scraped for viable count.

Due to the difference in conditions for biofilm development between the saturated and interface system, information obtained from CLSM observation is different. The saturated biofilm being completely immersed in the medium, with the whole coverslip surface equally exposed to nutrient and oxygen, is more homogeneous on the XY plane.

Heterogeneity however, exists along the Z-axis revealing mushroom structures and microcolonies (Figure 2.5 A). In the interface model, biofilm is subjected to influence from nutrient and oxygen gradient caused by the air/liquid interface, leading to spatial heterogeneity (Figure 2.5 B). For the homogeneity of biofilm (no air-liquid transition)

and ease of manipulation, I chose to use mainly the saturated model for my thesis. The interface model is used only in specific experiments for multi-species population relationship studies, to be consistent and to allow for comparison with previous experiments done in the Singapore laboratory.

Co-culture of E. coli with K. pneumoniae and E. faecalis

Theoretically, one would expect that preparing bacterial co-culture, be it planktonic or biofilm, is a simple matter of mixing cells of two species in equal proportions. In reality, conventional laboratory protocols adequate for bacterial pure cultures were not able to ensure the desired start ratio of 1:1, and a slight deviation from the 1:1 start ratio resulted in huge differences in the end ratio. Therefore, the inoculation step is of vital importance and at each step, precautions were taken to minimise errors.

Firstly, to precisely inoculate different species at a 1:1 cellular ratio, we first determined stringently the linear range of OD 600 measurement of our spectrophotometer for every strain to ensure accurate reading of the optical density. Secondly, the bacterial cell number as determined by counting the colony forming units (CFU) on agar plate has a proportional conversion factor to the OD 600 measurement, which needs to be employed to calculate the equivalent of 1:1 cell ratio (see Section 6.3.1.2). A generalised OD 600 to CFU conversion (e.g. available in standard molecular biology protocols such as in Molecular Cloning (Sambrook & Russell, 2001) was inadequate, as this factor varies between species, even strains of the same species and between culture conditions.

Therefore I empirically determined the CFU/ml at OD 600 =1 conversion factor for every bacterial strain in both BHI (37°C) and mM63 (30°C) (Table 2). Thirdly, when bacterial cells divide, the old pole have slower growth rates, decreased cell division and are more vulnerable to potential stresses, leading to increasing discrepancy between the new and old pole cells at each generation [START_REF] Stewart | Aging and death in an organism that reproduces by morphologically symmetric division[END_REF]. Therefore, the length of seed culture growth (from which the 1:1 mixing would be drawn from) has to be constant and precise, to ensure a consistent state of homogeneity for inoculation, replicable between experiments. Balancing between the homogeneity of cell state in the population and sufficient cell density for further dilutions, the most practical culturing time was determined to be 14 hours for all strains in BHI (37°C), 16 hours for those in mM63 (30°C) with the exception of E. faecalis which requires 24 hours in mM63 due to its slow growth in minimal medium.

Bacterial

Modes of Analysis

Enumeration (Viable Count) of bacterial strains using UTI agar

In multi-species studies involving planktonic or biofilm co-cultures, the members have to be distinguished from each other and enumerated. Two approaches which we have used include: 1) plating on chromogenic UTI agar for direct enumeration of co-cultures by viable count, because it allows differentiation of the three species based on distinct colony morphology, and 2) Fluorescence Activated Cell Sorter (FACS), which allows a variety of cell properties to be analyzed by virtue of its single cell detection and resolution capability. The second approach will be discussed in Section 2.3.2.

For enumeration by plating, it was possible to discern the different species from one another using the UTI agar which allows the three species to be visualized as colonies with different morphology and colours (Figure 2.6). A preliminary check has been performed which assured us that the CFU counts on UTI agar are comparable to those on agars that are recommended by the American Type Culture Collection (ATCC). The UTI agar was henceforth used for such enumeration purpose.

At the final CFU counting stage for a co-culture, it is possible that overcrowding of one species may mask the colonies of the minority species. The small colony size of E.

faecalis was indeed found to present this problem. To resolve, the deMan, Rogosa and Sharpe (MRS) agar that selects for Gram positive Lactobacillus was used to select for E.

faecalis, which is also lactic acid bacteria [START_REF] Vermis | Enumeration of viable anaerobic bacteria by solid phase cytometry under aerobic conditions[END_REF][START_REF] Domig | Methods used for the isolation, enumeration, characterisation and identification of Enterococcus spp. 2. Pheno-and genotypic criteria[END_REF]. The MRS agar not only inhibited growth of E. coli and K. pneumoniae, but more importantly, showed CFU counts of E. faecalis that were consistent between MRS and UTI agar.

Therefore, for CFU counts involving E. faecalis, the co-cultures were plated on both UTI and MRS agar to obtain the most accurate data.

FACS

Due to the heterogeneity in bacteria's physiological and metabolic state [START_REF] Davidson | Individuality in bacteria[END_REF], single cell analysis is better able to reveal the true composition of the cells' state in the culture, compared to methods that quantify an average of the whole population. The fluidic system of FACS can be manipulated so that single cells pass in a stream across a laser detector, by which light scattering properties such as forward scatter (reflects cell size) and side scatter (corresponds to surface complexity for bacterial cells)

of each cell may be recorded. It can also detect fluorescence intensity, thus when coupled with fluorescence-tagging (see Section 2.4.2), FACS not only allows separate analysis of the fluorescent and non-fluorescent populations, but also detects the fluorescence intensity of every single cell, giving an indication of the extent of the specific biological parameters being measured. In this project, for example, the original plan was to use the GFP-tagged E. coli to distinguish it from the non-fluorescent partner species K.

pneumoniae and E. faecalis by FACS, so as to trace the transcription activity of E. coli in co-cultures.

However, FACS is originally designed to deal with eukaryotic cells, which are considerably larger in size than bacterial cells. Some optimization of the system had to be performed to suit our needs of bacterial analysis. With this powerful tool, I have succeeded in analyzing cells in planktonic cultures for the surface properties of E. coli strains differing in curli expression level (Section 4.3.1.2), which will be presented in Chapter 4. I have also attempted to study multi-species population relationships in the biofilm state by FACS, but unfortunately did not succeed because of the exopolysaccharide-associated clumping, which compromises the resolution of this analysis.

CLSM and COMSTAT

Confocal spatial pinhole to eliminate out-of-focus light in specimens that are thicker than the focal plane [START_REF] Smith | Basic confocal microscopy[END_REF][START_REF] Pawley | Handbook of Biological Confocal Microscopy Berlin[END_REF]. The focal plane is called the X-Y plane, and as the focus moves up or down (the Z-axis) the sample, the information of every layer within the predefined range on the Z-axis is collected and this "cube" is termed a "Z stack". For biofilm samples, the fluorescent biomass of a Z stack can be calculated using the programme COMSTAT [START_REF] Heydorn | Quantification of biofilm structures by the novel computer program COMSTAT[END_REF], which can also be used to determine other parameters such as average and maximum thickness, roughness, surface to volume ratio and others.

Confocal microscopes can typically be configured to capture images of two or more fluorophores simultaneously or sequentially. In this work, for example, green and red fluorescence proteins are used, sometimes simultaneously. To avoid overlapping of their spectra, sequential scanning is preferred. Often, tissue or microbiological samples are fixed before observation, but to obtain the most natural state of the sample, our biofilm is untreated and sometimes immersed in water in order not to disturb its structure. Because this project was carried out in two laboratories, two different models of CLSM were used:

the Zeiss inverted confocal microscope (in both France and Singapore), and the upright Nikon microscope (only in Singapore). Due to the intrinsic differences, albeit trivial, between confocal microscopes, only Z stacks from the same model of microscope are compared in COMSTAT analysis. Fluorescence images are superposed with transmitted light images.

Visualisation of E. coli

The work described in this thesis centres around E. coli, in terms of its response to mucin, as well as its interaction with two other commensal bacterial species, K. pneumoniae and E. faecalis. In engaging these biological questions, both CLSM and FACS analyses would be employed, and to clearly distinguish E. coli by means of fluorescence would be necessary in the application of these tools. This section describes the undertaking related to this purpose.

Using fluorescent stain (SYTO)

In the initial attempt of visualising E. coli with the help of fluorescence, I first tried the simplest method: to stain the biofilm with one of the SYTO® Red Fluorescent Nucleic Acid Stain, SYTO 61. First of all, the saturated biofilm system requires immediate microscopic analysis after sampling to prevent drying of the biofilm. The staining procedure involved about 10 minutes' incubation time, at the end of which the biofilm would be dried out. Second, application of the liquid stain on the biofilm caused undesirable distortion of the biofilm structure. Third, the staining was not homogeneous with respect to the centre and the periphery of the stained area. In my hands, perhaps due to some unknown incompatibility with our culture conditions, the staining had proven inconsistent between experiments with respect to stain spread surface area and biofilm distortion level, all of which made inter-experiment quantitative comparison challenging. Moreover, dual-or multi-species co-culture studies require different species to be distinguishable from each other. This cannot be achieved by staining the whole sample with species-unspecific dyes. Therefore, we opted to perform whole-cell fluorescent labelling of E. coli.

Whole-cell tagging of E. coli

Green Fluorescent Protein

The most widely used fluorescent protein (FP) is the green fluorescent protein (GFP), which was originally discovered in the jellyfish Aequorea victoria [START_REF] Shimomura | Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea[END_REF] and subsequently cloned and successfully expressed in other organisms [START_REF] Chalfie | Green fluorescent protein as a marker for gene expression[END_REF]. Since then, GFP has been engineered to produce improved versions that are brighter, display enhanced photostability, pH tolerance and faster maturation rates [START_REF] Shaner | Advances in fluorescent protein technology[END_REF]. Modified FPs emitting blue, cyan and yellow fluorescence has been created, and subsequently enhanced as with the GFP [START_REF] Olenych | The fluorescent protein color palette[END_REF][START_REF] Patterson | Fluorescent proteins for cell biology[END_REF]. For its proven ease in use and stability, we first chose to label our E. coli with GFP.

Construction of G1

The easiest way of whole-cell tagging (the term "tagging" is used interchangeably with "labelling" in this thesis) would be to introduce a plasmid that carries a fluorescence expression cassette. However, the maintenance of selection pressure requires the use of antibiotics, which is not suitable for our co-culture studies involving antibiotic susceptible partner species. In addition, the high expression of plasmid-borne fluorescence proteins, especially in high copy number plasmids, has been shown to exert metabolic cost on bacteria [START_REF] Rang | Fitness cost of the green fluorescent protein in gastrointestinal bacteria[END_REF], and may thus affect the bacteria's physiology.

The Singapore laboratory has previously generated a GFP-expressing E. coli MG1655 strain via chromosomal insertion of the GFP gene. This is named E. coli strain SCC1 in [START_REF] Miao | Dual fluorescence system for flow cytometric analysis of Escherichia coli transcriptional response in multispecies context[END_REF], but will be referred to as strain G1 throughout this thesis. Briefly, a R6K-based suicide plasmid pDM4 [START_REF] Milton | Flagellin A is essential for the virulence of Vibrio anguillarum[END_REF] carrying P A1/04/03 -gfpmut3* [START_REF] Andersen | New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria[END_REF] MG1655, clones which have undergone recombination can be selected via chloramphenicol resistance. Then, a second recombination event was counter-selected for the loss of the sacB gene via survival on 5% sucrose agar. The P A1/04/03 -gfpmut3* cassette was thus inserted into the chromosome while the rest of the plasmid is excised. This constructed E. coli strain G1 was then profiled using different techniques including CLSM and FACS.

GFP-tagging does not interfere with cells' basic physiology

The expression of chromosomally inserted GFPmut3* was checked and found to be high enough for detection by FACS (Figure 2.9) and CLSM (Figure 2.7 B). It was also shown to be stable by at least four rounds of 24 hour sub-culturing (Figure 2.9 A) as well as 4day old cultures (Figure 2.9 B), based on quantification both by fluorometer (data not shown) and FACS (Figure 2.9). Moreover, FACS data showed that GFP is expressed by almost all of the cells, with the "green" population (G+) being 98.70 -99.60% of the whole population [START_REF] Miao | Dual fluorescence system for flow cytometric analysis of Escherichia coli transcriptional response in multispecies context[END_REF].

In [START_REF] Miao | Dual fluorescence system for flow cytometric analysis of Escherichia coli transcriptional response in multispecies context[END_REF], it was further shown that the G1 strain demonstrates no significant difference from the wild type strain MG1655 with respect to basic physiology, including cell morphology, forward-(FSC) and side-scatter (SSC) profile, growth over time, as well as conversion between cell density (CFU/ml) and light absorbance at 600 nm (OD 600 ). In co-culture with other species, G1 shows very similar population ratio with its partner species to the wild type MG1655, suggesting that the population relationship of these species in co-culture is not affected by GFP expression.

GFP-tagging of MG1655ompR234

In order to investigate the impact of surface property on commensal species interactions, the curli-producing MG1655ompR234 [START_REF] Prigent-Combaret | Complex regulatory network controls initial adhesion and biofilm formation in Escherichia coli via regulation of the csgD gene[END_REF] was exploited, which requires this curli-producing strain to be "visible" too. With the successful GFPtagging of E.coli MG1655 strain giving rise to strain G1 and the rigorous demonstration

A. E. coli G1 (30°C), B. E. coli G1 ompR234 (30°C) and C. E. coli G1 ompR234 (37°C)
cultured on cover slips in a Petri dish containing M63 minimal media at the indicated temperatures for 24 hours. Shown in each panel are the x-y plane (horizontal section), x-z and y-z planes (vertical sections), corresponding to the faint red lines indicated in the respective perpendicular sections. Fluorescence images are superimposed with transmitted light images. In the columns below are their corresponding variables obtained via the software COMSTAT [START_REF] Heydorn | Quantification of biofilm structures by the novel computer program COMSTAT[END_REF]. Surface area/ Volume (µm 2 /µm 3 ) 0.43 ± 0.36 0.14 ± 0.04 0.58 ± 0.36

A G1 (30°C) B G1ompR234 (30°C) C G1 ompR234 (37°C)
that the gfp-insertion did not alter the basic physiology of the parent strain [START_REF] Miao | Dual fluorescence system for flow cytometric analysis of Escherichia coli transcriptional response in multispecies context[END_REF], it logically follows that visualization of the curli-producing MG1655ompR234 can be achieved based on the G1 genetic components (Figure 2.10). For simplicity, we used phage P1 transduction to transfer the ompR234 mutation into G1, making use of the genetic linkage (50% co-transduction) with malT54::Tn10, which allows Tetracycline (Tet) antibiotic selection. Curli fibres bind to Congo red (CR), giving rise to red colonies on plates supplemented with CR [START_REF] Collinson | Thin, aggregative fimbriae mediate binding of Salmonella enteritidis to fibronectin[END_REF]. This provides an easy screening method for curli-producing cells. The Tet resistant G1ompR234 colonies were thus further screened on Congo red indicator plates for curli production phenotype.

Curli-related phenotype is conserved in the GFP version

The biofilm of E. coli MG1655ompR234 made "visible" by SYTO 61 staining and GFP whole-cell tagging (G1ompR234) are similar to each other and comparable with regard to biofilm morphology and biomass (compare In low osmolarity condition (mM63), G1ompR234 forms much more biofilm at 30°C than at 37°C (Figure 2.11 compare B and C). The biofilm formed at 30°C is also more confluent as indicated by its lower surface area/volume value (0.14 ± 0.04 vs. 0.58 ± 0.36). This is consistent with the reported temperature regulation of curli production [START_REF] Olsen | Fibronectin binding mediated by a novel class of surface organelles on Escherichia coli[END_REF], indicating that the curli regulation related to ompR234 mutation is conserved in G1ompR234 strain. G1, on the other hand, does not form structured biofilm at 30°C (Figure 2.11 A). This shows again that the GFP-tagged strains mimic the phenotype of their parental wild type strains.

The success in GFP-labelling of the ompR234 mutant and disrupting neither the fluorescence-expressing property nor the curli-related phenotype, led me to construct other GFP-labelled curli mutants, targeting different curli-related components: The (henceforth referred to as G1ompR), which affects the curli synthesis at the regulatory level; the chromosomal csgA null mutation G1ompR234csgA, resulting in a strain defective in producing the curli structural protein CsgA. Thus, we expect G1ompR to produce much less curli (curli-) and G1ompR234csgA not to have curli expression (curli--) even under the condition favourable for curli production. Table 3 shows the expected curli production profile of the curli-related mutants used in this study. When curli is produced, cells form aggregates that can facilitate biofilm formation. In planktonic culture with slow shaking cell aggregation also occurs, and this is shown macroscopically as flocculation. As expected in the flocculation test under curli producing condition (M63 minimal medium at 30°C with slow shaking), the curli producing strain G1ompR234 flocculated, while others did not (Figure 2.12 A). When examined under microscope, G1ompR234 cells form large aggregates which can be dispersed by vortexing (Figure 2.12 B). All these indicate that the curli-related phenotypes are well conserved in the GFP version of the curli-related mutants.

Genotype

It is important that the GFP expression of this mutant series not be compromised by the insertion of the curli-related mutations, because the GFP label of these stains is necessary in CLSM analysis. In FACS analysis the GFP label is also preferred, because small-sized bacteria often cannot be distinguished from noise particles by FACS and the green fluorescence is useful to discern the cells from noise. Similar to G1, all the mutants show GFP-expression population to be more than 95% of the total population by FACS analysis (Table 3), in both exponential and stationary growth phases. Therefore, we can be confident that almost the whole population of each of these strains is represented by GFP-tagging.

Analysis of Promoter Activity

In reporter system, we hope to be able to reach the ultimate objective, that is, to measure the transcriptional gene activity in GFP or RFP tagged-E. coli strain facing the mucin-rich gut environment and commensal bacteria.

Green vs. Red FP as Transcriptional Reporter

Red FPs, like green FPs, are widely used in both whole-cell tagging and in reporting promoter activities [START_REF] Sorensen | Rapidly maturing red fluorescent protein variants with strongly enhanced brightness in bacteria[END_REF][START_REF] Martineau | Whole cell biosensing via recA::mCherry and LED-based flow-through fluorometry[END_REF]. Indeed, the Singapore laboratory has used AsRed2 (BD Clontech) to fuse with several promoters of different functional categories, and it has reflected their expression status in manners that were consistent with those which have been previously reported [START_REF] Miao | Dual fluorescence system for flow cytometric analysis of Escherichia coli transcriptional response in multispecies context[END_REF].

However, despite claims of fast maturation by its commercial supplier, AsRed2 shows general signs of being a slow-maturing fluorescent protein. It has a long half-life, and the promoter-fusion constructions are carried on the high-copy plasmid pBluescript. These properties of the AsRed2 system that was available to me may lead to inaccurate description of gene activities, so I also made use of the French laboratory's short half-life GFP (GFP[LVA]), with an in vivo half-life of approximately 40 minutes [START_REF] Andersen | New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria[END_REF], which helps to reveal the transient gene expression in the bacteria. In addition, the gene gfp[LVA] (all promoter-fused gfp used in this study is gfp [LVA], so they will be abbreviated as "gfp" hereafter) is carried on a low copy plasmid pPROBE [START_REF] Miller | Improved gfp and inaZ broad-host-range promoter-probe vectors[END_REF], so that we can have more confidence in the results being close to the natural condition.

To validate the promoter-gfp and -AsRed2 fusions, promoters of three "life cycle" genes with distinct functions were chosen with the rationale as explained earlier (Section 2.5).

fis (factor for inversion stimulation) translates to a nucleotide-associated protein in E. coli that is abundant during early exponential growth in rich medium but is in short supply during stationary phase [START_REF] Nasser | Transcriptional regulation of fis operon involves a module of multiple coupled promoters[END_REF], hence making it an ideal "active growth" representative gene. The gene chosen to represent "reduced metabolism" -mazEF -is a suicide module specific for a stable toxin (MazF) and a labile antitoxin (MazE). It can mediate programmed cell death induced by virus, H 2 O 2 , DNA damage, antibiotics and starvation [START_REF] Hazan | Escherichia coli mazEF-mediated cell death is triggered by various stressful conditions[END_REF][START_REF] Kolodkin-Gal | Induction of Escherichia coli chromosomal mazEF by stressful conditions causes an irreversible loss of viability[END_REF]. Stressful conditions that inhibit mazEF expression lead to the MazF toxin persistence in absence of de novo synthesis of the MazE antitoxin, eventually causing cell death [START_REF] Aizenman | An Escherichia coli chromosomal "addiction module" regulated by guanosine [corrected] 3',5'-bispyrophosphate: a model for programmed bacterial cell death[END_REF]. The last is csgBA, which code for curlin and curlin nucleator proteins, the building blocks of the extracellular structure curli [START_REF] Hammar | Expression of two csg operons is required for production of fibronectin-and congo red-binding curli polymers in Escherichia coli K-12[END_REF], and is induced in the stationary phase [START_REF] Olsen | The RpoS sigma factor relieves H-NS-mediated transcriptional repression of csgA, the subunit gene of fibronectin-binding curli in Escherichia coli[END_REF] and during biofilm development (Prigent-Combaret et al., 1999). Promoter fusion constructions are described in materials and methods (Section 6.7).

In planktonic culture

The expression of the promoter-GFP fusions were monitored in planktonic culture by fluorometer measurement. As reported by Nasser and colleagues [START_REF] Nasser | Transcriptional regulation of fis operon involves a module of multiple coupled promoters[END_REF], the fis promoter (P fis ) is activated in the exponential phase in rich medium (Figure 2.13 A).

Marianovsky et al found that P mazEF was activated by FIS in exponentially growing cells in rich media [START_REF] Marianovsky | The regulation of the Escherichia coli mazEF promoter involves an unusual alternating palindrome[END_REF]; but Sat and colleagues reported that the cellular level of MazE was significantly lower in minimal medium M9 than in LB medium [START_REF] Sat | Programmed cell death in Escherichia coli: some antibiotics can trigger mazEF lethality[END_REF]. In accordance with their observations, P mazEF -gfp activity is very low when grown in minimal medium, in cells entering stationary phase (Figure 2.13 B).

When it comes to csgBA, more regulatory properties were tested, since the regulation of curli gene expression is extraordinarily complex and is responsive to many environmental cues. First of all, it is induced at the beginning of stationary phase (Figure 2.13 C). One of the first conditions recognized to promote curli gene expression was growth at temperature below 30°C [START_REF] Olsen | Fibronectin binding mediated by a novel class of surface organelles on Escherichia coli[END_REF]. Thus, csgBA gene expression was tested in MG1655ompR234 background at 30 and 37°C, and the results showed that indeed P csgBA activity was very low at 37°C compared to that at 30°C (Figure 2.13 D). It is also reported that curli expression occurs maximally in media without salt [START_REF] Romling | Multicellular and aggregative behaviour of Salmonella typhimurium strains is controlled by mutations in the agfD promoter[END_REF], and nutrient limitation stimulates curli gene expression (Gerstel & Romling, 2001).

Therefore, P csgBA activity was also tested in different media and showed the expected low activity in the rich medium BHI compared to that in the low osmolarity M63 minimal medium (Figure 2.13 D).

All these indicate that gfp-labeled promoters reflect the expression status of the gene, known from published scientific data. We conclude that our GFP constructions allow to properly estimate the transcriptional activity of the genes of interest.

The AsRed2-promoter fusions were also tested. However, fis activity was too low to be detected by both fluorometer (Figure 2.14 B) and FACS (Figure 2.14 A). Likewise with csgBA (Figure 2.14 C), the temperature regulation of which was not reflected (very low activity in both mM63 at 30°C and BHI 37°C) (Figure 2.14 D & E). P mazEF -AsRed2, nevertheless, was detectable by FACS (Figure 2.14 F) and showed high activity in the exponential phase in rich medium BHI (Figure 2.14 G), in agreement with Marianovsky's observation. Hence AsRed2 was found to be reporting only certain promoters properly. In addition, our concern that AsRed2, being a slow-maturing, long half-life fluorescence protein, will not accurately reflect the gene activity may, unfortunately be true, judging by the lack of demonstration of thermo-and osmo-regulation of P csgBA -AsRed2.

In biofilm

In biofilm, the promoter-gfp fusions were examined in the ompR234 background, because of its capability to form biofilm and its relevance to the osmoregulation of csgBA promoter. Cells were incubated in the low osmolarity M63 minimal medium at 30°C to favour biofilm growth. After 24 hours' incubation, P fis activity was observed to be low in biofilm (Figure 2.15 A). Since fis is mostly expressed in the exponential phase in planktonic culture and not in stationary phase, by analogy, we suggest that the bacterial cells in biofilm are not actively growing, or at least, not as actively growing as planktonic cells in exponential phase. P csgBA , on the other hand, shows very high activity in biofilm with spatial heterogeneity (Figure 2.15 B), under the regulation of ompR234. It is reported that csgBA expression is induced in the stationary phase in planktonic culture and during biofilm development. This result suggests that bacterial cells in biofilm could be in a physiological state similar to stationary phase, which was also reported by Beloin and his co-workers [START_REF] Beloin | Global impact of mature biofilm lifestyle on Escherichia coli K-12 gene expression[END_REF]. In conclusion, the behaviour of P fis and P csgBA suggest that when bacteria form biofilm, most of the population is in a slow growth scheme of gene expression.

No P csgBA activity is observed at 37°C (data not shown). This indicates that the thermoregulation of P csgBA is preserved in biofilm. P mazEF activity seems to be between that of P fis -gfp and P csgBA -gfp, and shows activity heterogeneity (Figure 2.15 C). In nutrient starvation, mazEF system responds with decreased mazE antitoxin synthesis, mediating programmed cell death [START_REF] Aizenman | An Escherichia coli chromosomal "addiction module" regulated by guanosine [corrected] 3',5'-bispyrophosphate: a model for programmed bacterial cell death[END_REF][START_REF] Hazan | Escherichia coli mazEF-mediated cell death is triggered by various stressful conditions[END_REF]. In biofilm In view of the fact that our biofilm is static and without continual supply of fresh nutrient, as discussed in section 2.2.2, I suspected that biofilm morphology and gene expression profiles observed in our biofilm system might be different from those with fresh supply of nutrient. To test this, I renewed the medium every 24 hours for biofilms of E. coli

MG1655ompR234 carrying P fis -gfp, P mazEF -gfp or P csgBA -gfp, but did not observe significant difference in biofilm morphology and gene expression pattern between biofilms with and without renewal of medium at 48 and 72 hours of incubation (Figure 2.16). Therefore, I chose not to renew biofilm medium for the ease of manipulation as well as to avoid disturbance of biofilm structure.

The AsRed2-reported promoters were also tested on biofilm. As in planktonic culture, the activity of P fis -AsRed2 and P csgBA -AsRed2 were too weak to be detected by CLSM (data not shown). P mazEF -AsRed2 shows homogeneous expression by cells (Figure 2.15 D), but the biofilm is considerably thinner (~25µm) than that formed by cells carrying the promoter-gfp fusions (~75µm). This may be due to the high metabolic cost exerted on the bacteria to produce large amount of AsRed2 fluorescent protein, since it is carried by the high copy number plasmid pBluescript. The potential cytotoxicity of RFPs may also be a cause of hindered biofilm growth. Indeed, the burden of RFP production may lead to exclusion of the plasmid by the cells, and this may be manifested as low AsRed2 expression and misleadingly interpreted as low promoter activity (discussed in Section 2.6).

After comparing the behaviour of GFP-and AsRed2-reported promoters in both planktonic and biofilm conditions, we decided that the promoter-gfp fusions are more suitable for use in the later experiments, based on their detectability, stability and reliability in reporting gene activity.

Dual fluorescence systems -attempts and disappointments

The Singapore laboratory has previously developed a dual fluorescence scheme [START_REF] Miao | Dual fluorescence system for flow cytometric analysis of Escherichia coli transcriptional response in multispecies context[END_REF] to analyze the promoter activity of E. coli when it is in co-culture with other species. In this system, the GFP is used to distinguish E. coli from other species, while the AsRed2 reports the promoter activity (Figure 2.17).

The dual fluorescence system allows the fluorescently labelled species (in this case E.

coli, tagged green) to be analyzed for promoter activity (reported by red) even when it is in co-culture with another species, by exploiting the single-cell resolution capacity of FACS. The influence of a partner species can be analysed by detecting the green (E. coli)

particles and measuring its red fluorescence level (promoter activity), and even reveal the inherent heterogeneity of gene expression in the population. Since my work deals with E.

coli in interaction with K. pneumoniae and E. faecalis, it will be useful if I can take advantage of this scheme of analysis, to complement the population relationship studies (chapter 4).

In the dual fluorescence scheme, two contrasting colours of fluorescence are required (Figure 2.17). In Section 2.4.2, I have shown successful tagging of E. coli strains with GFP, but the contrasting red FP (AsRed2) (Figure 2.17 A) reporters were not satisfactory, at least for the few promoters I was interested in (Section 2.5.2 & 2.5.3). The alternative, then, is to make use of the green FP promoter-reporter set, shown to be appropriate for transcriptional analysis (Section 2.5.2 & 2.5.3) and at the same time, whole-cell label the E. coli cell with red FP (Figure 2.17 B).

Theoretically, this reversed red-and green-labelling should be workable. An AsRed2 construct with fusion to the constitutive promoter P A01/04/03 -the same promoter used in GFP whole-cell tagging [START_REF] Miao | Dual fluorescence system for flow cytometric analysis of Escherichia coli transcriptional response in multispecies context[END_REF] -was already available (pCCS325, refer to Section 6.1 for detail), and it showed significant red fluorescent activity. Insertion of this construct using the exact same genetic manipulation scheme as described in Section 2.4.2.2 for GFP-tagging has no conceivable reason to fail. To further improve its reporting ability, a mutant version of the AsRed2 (hereafter referred to as mAsRed2) has strain using the same method as G1 construction (Figure 2.18, also refer to Section 2.4.2.2). The detailed molecular work is presented in Chapter 6 Materials and Methods (Section 6.7.3), but what needs to be highlighted here is that even though the construct was genetically correct, the resultant strain of E. coli MG1655 "R1" cells failed to turn red, i.e. did not sufficiently express the red fluorescence.

We suspected that this may be due to the cytotoxicity caused by aggregation of the tetramer protein [START_REF] Tao | Enhanced green fluorescent protein is a nearly ideal long-term expression tracer for hematopoietic stem cells, whereas DsRed-express fluorescent protein is not[END_REF], as anecdotal evidence and published data suggest that the use of RFPs as whole-cell labels has been limited by cytotoxicity [START_REF] Strack | A noncytotoxic DsRed variant for whole-cell labeling[END_REF].

Following this, I constructed another version of RFP-tagged MG1655 (named "R2")

using DsRed-Max-N1 (Addgene), which is claimed to be noncytotoxic [START_REF] Strack | A noncytotoxic DsRed variant for whole-cell labeling[END_REF], hoping that it could give a stable RFP expression. To my disappointment, the genetically correct P A1/04/03 -DsRed-Max-N1 showed severe instability in sub-culturing even on intermediate plasmids before insertion into the chromosome (Figure 2.18). A red colony carrying the intermediate construction was both streaked and inoculated as planktonic culture twice, hence generating 2 sub-cultures and 2 plates (Figure 2.19 A).

After overnight incubation, although seeded from the same red colony, only 1 planktonic culture was red, while the other merely showed a pinkish tint when pelleted (Figure 2.19 B). The puzzling observation was that the re-streaked colonies on both sets of plates were all red (Figure 2.19 B). The plasmid of both cultures were extracted and analyzed by restriction digestion to check the presence of the DsRed-Max insert. The red culture gave an adequate plasmid yield containing the insert, as expected, whereas the non-red culture very low yield and hardly any detectable insert. This suggests that cells tend to exclude the plasmid carrying RFP, perhaps due to the extra burden or cytotoxicity it brought.

To further test the stability of DsRed-Max, 10 red colonies were used to inoculate 10 planktonic cultures. Only 2 cultures turned red and the 8 others were pinkish (Figure 2.19 D). I also tested sub-culturing by re-streaking of the red colonies on agar plate, since there seemed to be a difference between RFP expression in planktonic liquid cultures and in bacterial colonies. Unfortunately, even though the majority of the colonies were red, a subpopulation of white colonies appeared, especially in the high population density area (Figure 2.19 E), indicating some loss in red fluorescence activity. Therefore, DsRed-Max despite its claims [START_REF] Strack | A noncytotoxic DsRed variant for whole-cell labeling[END_REF], was also judged not stable enough for R1 construction.

We then laid our hope on mCherry [START_REF] Shaner | Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein[END_REF], which was reported to be fast maturing [START_REF] Strack | A noncytotoxic DsRed variant for whole-cell labeling[END_REF] and the best general-purpose red monomer owing to its superior photostability [START_REF] Shaner | Advances in fluorescent protein technology[END_REF][START_REF] Shaner | A guide to choosing fluorescent proteins[END_REF]. It is even claimed to have outperformed gfpmut3.1 [START_REF] Cormack | FACS-optimized mutants of the green fluorescent protein (GFP)[END_REF] in fusions with recA promoter by

Martineau and his colleagues [START_REF] Martineau | Whole cell biosensing via recA::mCherry and LED-based flow-through fluorometry[END_REF]. Unfortunately, again, already at the intermediate plasmid stage, mCherry was showing unstable expression. While continuing to work on the RFP whole-cell tagging by making mutants of the available RFP and trying new candidates, we concluded that the whole-cell labelling with red fluorescent protein was too problematic. The objective of transcriptional monitoring in co-culture by the dual fluorescence scheme had to be abandoned. Instead, other aspects of our experimental models are explored using the currently available and validated tools.

Concluding remarks on fluorescent tools design

Over the period of experimental model set-up, two systems were developed in this study, depending on the strains involved and the culture condition (Table 4). For simplicity, we name them after their biofilm setup model: the interface model and the saturated model.

The saturated model has been chosen over the interface model in this study for the homogeneity of its biofilm, as well as the minimal manipulation involved in the sampling process. While the interface system is the basic model employed in the Singapore laboratory aiming to study interspecies interactions, the saturated system was developed to integrate the French laboratory's expertise in regulation and function of curli. Curli are proteinaceous extracellular fibres that are involved in surface and cell-cell contacts. In the natural multi-species context, they are highly likely to influence the interactions between E. coli and its partner species (K. pneumonia and E. faecalis). To explore the possible effects exerted on these interspecies interactions by curli, specific conditions favouring curli expression such as low osmolarity (M63 minimal medium) and low temperature (30°C) were adopted in the system. In the mean time, a series of E. coli mutants targeting different regulatory levels of curli synthesis were generated to further investigate the involvement of curli in interspecies interactions. A short half-life GFP was then tested and shown to accurately report gene activity in both planktonic and biofilm condition. So we attempted to reverse the FP labelling of the dual fluorescence system of the Singapore laboratory, using instead promoter-gfp as the transcriptional reporter, and red FP for whole-cell tagging. Unfortunately, the process of red whole-cell tagging with several different RFPs (AsRed2, DsRed-Max, mCherry) has been problematic possibly due to its toxicity or instability of expression. Although AsRed2 has successfully reported the activity of some promoters [START_REF] Miao | Dual fluorescence system for flow cytometric analysis of Escherichia coli transcriptional response in multispecies context[END_REF], its potential toxicity and instability makes it a less ideal choice as transcriptional reporter or whole-cell tag.

Interface System Saturated System

Base Although accurate gene profiling in the multi-species context cannot be done due to the AsRed2's inability to serve faithfully as a promoter-reporter for many genes of my interest, as well as expression and the unsuccessful construction of RFP-tagged E. coli, the influence of curli on the interactions between E. coli, K. pneumonia and E. faecalis can be examined by comparing the population relationship of E. coli and the partner strains with respect to the curli-related mutants of E. coli. In E. coli single species culture, differential gene expression in response to various external factors can be monitored in the E. coli wild type and mutant background. In the following chapters, gene regulation in response to mucin was investigated using the promoter-gfp fusion and curli-related mutants. Curli's contribution in the population relationship between E. coli, K.

pneumonia and E. faecalis was also evaluated.

Chapter 3 Mucin influences E. coli biofilm formation through modulations of surface adhesion structures

It is well recognized now that biofilm is the most predominant form of bacteria existence in nature and in the intestine [START_REF] Probert | Bacterial biofilms in the human gastrointestinal tract[END_REF]. To study the interspecies interactions of intestinal bacteria in biofilm, the bacterial species chosen must be able to form biofilm. However, the laboratory strains of E. coli, K. pneumonia and E. faecalis used in this work do not readily form biofilm in vitro, under standard laboratory conditions. It has been observed that mucin increased bacterial adherence to surface [START_REF] Zhang | Role of bile mucin in bacterial adherence to biliary stents[END_REF] and promoted biofilm formation [START_REF] Bollinger | Human secretory immunoglobulin A may contribute to biofilm formation in the gut[END_REF] of E. coli [START_REF] Bollinger | Secretory IgA and mucin-mediated biofilm formation by environmental strains of Escherichia coli: role of type 1 pili[END_REF] and P. aeruginosa [START_REF] Landry | Mucin-Pseudomonas aeruginosa interactions promote biofilm formation and antibiotic resistance[END_REF]. The Singapore laboratory has thus supplemented BHI with mucin in the interface biofilm system (refer to section 2.3.5, Table 3). In published models, it can be seen that mucin composition and concentrations vary in different experimental settings, ranging from 0.01% to 10% [START_REF] Zhang | Role of bile mucin in bacterial adherence to biliary stents[END_REF][START_REF] Bollinger | Human secretory immunoglobulin A may contribute to biofilm formation in the gut[END_REF][START_REF] Cole | Characterization of monospecies biofilm formation by Helicobacter pylori[END_REF][START_REF] Macfarlane | Colonization of mucin by human intestinal bacteria and establishment of biofilm communities in a two-stage continuous culture system[END_REF][START_REF] Landry | Mucin-Pseudomonas aeruginosa interactions promote biofilm formation and antibiotic resistance[END_REF]. In the case of the Singapore laboratory, Sigma-Aldrich Type III Mucin from porcine stomach is being used, and BHI supplemented with 2% of this mucin has been appointed the standard medium for the cultivation of multi-species biofilms on glass in the interface system. This condition was based on the laboratory's previous testing of mucin at 0.1%, 0.5% and 2% for MG1655 biofilm formation, which showed that for both 24 and 48 hours growth, 2% mucin resulted in the highest increase of biofilm thickness and formed well-developed mushroom structures. The specific condition (BHI + 2% mucin) has since then been used for biofilm co-cultures of E. coli with the two commensal partners K. pneumoniae and E. faecalis. However, how mucin promoted better E. coli biofilm development has never been investigated. The mechanism behind this may have a role to play in the interspecies relationship of commensals.

As my thesis is part of a larger project aiming to characterize the transcriptional response of E. coli in the presence of other commensal bacteria, we decided to focus on the aspect relating to effect of mucin on E. coli. As a substantial amount of previous experiments performed by others have been conducted on polystyrene plates (e.g. screening of

Low concentrations of mucin promote E. coli biofilm formation

The nature of biofilm development between the interface and saturated system is expected to be fairly different. Hence we could not assume what is optimal for the interface biofilm to be likewise for the saturated biofilm. To determine the optimal mucin concentration in the saturated system, mucin concentrations in M63 minimal medium from 0 to 10% (0, 2, 4, 6, 8 and 10%) were initially tested for both G1 and G1ompR234

in BHI (37°C) and M63+ BHI (30°C) on polystyrene (PS) 24-well plate to have a grasp on the concentration range to focus on. Interestingly, high concentrations of mucin were observed to result in very low biofilm formation (data not shown), so it was narrowed down to 0 -4% (0, 0.75, 1.5, 2, 3 and 4%) in BHI at 37°C, and 0 -2% (0, 0.1, 0.25, 0.5, 0.75 and 2%) in mM63 (M63 supplemented with 2% glucose and 1% BHI, see materials and methods) at 30°C (Table 5). Biofilm formed on PS 24-well plate was grossly quantified by Crystal Violet (CV) staining (Figure 3.1).

Except for the G1 strain at 37°C in BHI medium, Mucin at certain concentration(s)

enhance biofilm formation (Figure 3.1, red rectangles). However, high concentrations of mucin does not systematically guarantee thicker biofilm formation (e.g. G1 and

G1ompR234 in mM63 at 30°C with 2% mucin), and optimal concentration should be determined for each strain and condition. As thicker biofilms were obtained in mM63 at 30°C further investigations were all carried out under this culture condition.

First, I tested if the chemical nature of the colonising surface affected mucin's effect on biofilm formation. Hence biofilm was also grown on glass cover slip in the saturated system and the structure observed under CLSM (Figure 3.2). While the optimal mucin concentration for biofilm growth on PS surface has been determined visually by CV staining intensity (Figure 3.1), that on glass surface was analysed by comparing microscope images and running calculation with COMSTAT with respect to biomass, The optimal mucin concentration for biofilm growth in the saturated system is listed in Table 5. The discrepancy of the optimal mucin concentration between G1 growing on PS and glass may be due to the difference of surface property between these two materials.

Nonetheless, all evidence points to the fact that low concentrations of mucin promote E.

coli biofilm formation.

It has been documented that mucin greatly increased the number of planktonic Helicobacter pylori while not affecting biofilm bacteria, resulting in a decline in adherence [START_REF] Cole | Characterization of monospecies biofilm formation by Helicobacter pylori[END_REF]. We thus wondered if the increased biofilm formation is the result of an overall increase of bacteria population giving a high adherence impression, because mucin can be an important source of carbohydrate to certain bacteria [START_REF] Macfarlane | Colonization of mucin by human intestinal bacteria and establishment of biofilm communities in a two-stage continuous culture system[END_REF][START_REF] Vieira | Two atypical enteropathogenic Escherichia coli strains induce the production of secreted and membrane-bound mucins to benefit their own growth at the apical surface of human mucin-secreting intestinal HT29-MTX cells[END_REF]. To be sure that mucin acts specifically on the biofilm fraction without affecting the total bacterial population, the effect of this intestinal component on E. coli growth was investigated in the next section. actual bacteria OD 600

Strain

Mucin promotes biofilm formation in E. coli strains without affecting bacteria growth

The antimicrobial activity of salivary mucin (MUC7) has been witnessed by several groups against Streptococcus mutans [START_REF] Wei | Effect of MUC7 peptides on the growth of bacteria and on Streptococcus mutans biofilm[END_REF], and clinical fungal strains including Candida albicans [START_REF] Situ | In vitro assessment of antifungal therapeutic potential of salivary histatin-5, two variants of histatin-5, and salivary mucin (MUC7) domain 1[END_REF][START_REF] Wei | Factors affecting antimicrobial activity of MUC7 12-mer, a human salivary mucin-derived peptide[END_REF]Ogasawara et al., 2007a). It has also been reported that antimicrobial agents at sub-inhibitory level induce biofilm in a wide range of bacteria, probably as an adaptive survival strategy employed by the bacteria under stressful conditions. For example, aminoglycoside antibiotics have been demonstrated to stimulate biofilm formation of E. coli and P. aeruginosa [START_REF] Hoffman | Aminoglycoside antibiotics induce bacterial biofilm formation[END_REF]; sub-inhibitory concentrations of ribosome-targeting antibiotics lead to strong E. coli biofilm induction [START_REF] Boehm | Second messenger signalling governs Escherichia coli biofilm induction upon ribosomal stress[END_REF]; strong detergents that disrupt cell membranes also induce biofilm formation in Vibrio cholera [START_REF] Hung | Bile acids stimulate biofilm formation in Vibrio cholerae[END_REF], P.

aeruginosa [START_REF] Klebensberger | Detergent-induced cell aggregation in subpopulations of Pseudomonas aeruginosa as a preadaptive survival strategy[END_REF] and Bacteroides fragilis [START_REF] Pumbwe | Bile salts enhance bacterial co-aggregation, bacterial-intestinal epithelial cell adhesion, biofilm formation and antimicrobial resistance of Bacteroides fragilis[END_REF]; metals toxic to bacteria such as Nickel, has been shown by the French laboratory to promote E. coli biofilm formation [START_REF] Perrin | Nickel promotes biofilm formation by Escherichia coli K-12 strains that produce curli[END_REF]. Thus we suspect that mucin, having antimicrobial activity, may likewise induce biofilm formation at the concentration inferior to the biocidal level. To test this hypothesis, quantification of E. coli in the presence of various concentrations of mucin is necessary.

Bacteria cannot be analysed by optical methods in the presence of mucin.

Mucins are large, abundant, filamentous glycoproteins that cover the epithelial surfaces in the human gastrointestinal tract [START_REF] Dekker | The MUC family: an obituary[END_REF]. Due to its high molecular weight, mucin deflects light. Hence it is not possible to quantify bacteria by optical methods in the presence of mucin. As can be observed in Figure 3.3, it was possible to measure the absorbance due to mucin at 600 nm (OD 600 ). When the same amount of bacteria was added to various concentrations of mucin, the OD 600 reading of bacteria was masked by that of mucin. The difference between "mucin+bacteria" and "mucin" alone could not reflect the actual bacteria OD 600 , especially with higher concentrations of mucin. Also, bacteria grown in cultures containing mucin cannot be analysed by FACS as the mucin clumps cannot be differentiated from the cells based on size. Furthermore, the machine's fluidic channels are prone to be clogged by the mucin clumps. Therefore, single cell profiling of GFP-labelled promoter activity is not possible unless a second fluorescent component on the bacterial cell is available to fine-tune FACS detection. As a consequence, the laborious (but nonetheless effective) methodology of viable count on agar plate was employed to ensure accurate quantification of bacteria.

Mucin neither inhibits nor promotes the growth of E. coli.

To test the hypothesis that the biofilm induction in E. coli by low concentrations of mucin is due to mucin's antimicrobial activity, total viable count of E. coli grown in different concentrations of mucin was performed. Both E. coli MG1655 and MG1655ompR234

were inoculated at a cell density of 6x10 6 cells/ml in mM63 at 30°C in the presence of increasing amount of mucin (0 -2%). After static incubation on 24-well PS plate for 48 hours, the planktonic and biofilm fractions were diluted and plated on LB agar for viable count. Total bacteria population refers to the sum of the planktonic and biofilm portion, while adherence is the percentage of biofilm in the total population.

To our surprise, mucin does not appear to have inhibitory effect on the growth of E. coli.

As is shown in Figure 3.4, despite the change in population of the biofilm portion with respect to increasing mucin concentrations, the total population stays constant for both strains (P = 0.69 by Anova analysis).

Other E. coli K-12 strains such as MC4100 and W3110 and their ompR234 derivatives showed similar pattern to that of MG1655 (data not shown). These results led us to abandon the hypothesis of "sub-inhibitory level of mucin induce biofilm formation", and conclude that mucin does not show antimicrobial activity against E. coli K-12 strains in our experimental setting. well plate provides a satisfactory estimation of the biofilm induction level by mucin.

Curli are involved in mucin-induced biofilm formation

Mutations impairing curli production hamper mucin's induction effect on biofilm

Intriguingly, for all three lineage of E. coli strains used in my study (MG1655, W3110 and MC4100), the biofilm induction effect on PS surface is prominent for the ompR234 mutant, but very minimal for the wild type. Since the most apparent difference between the two strains is in their curli expression, we speculate that curli may be involved in the mucin-induced biofilm formation. In addition, previous observation (refer to section 3.1) that the induction effect is very little in the ompR234 mutant grown under non curliexpressing condition BHI (37°C) supports our speculation. To assess this, the curli null- Since the csgA mutant also carries the allele ompR234, and lacks only the structural gene of curli, this set of data effectively eliminates the possibility that other effects of ompR234 mutation are responsible for the mucin-induced biofilm formation. It therefore strongly supports our hypothesis that curli is the mediator of this effect in E. coli K-12 strains. In view of the highly complex regulation of curli production pathway, we can only speculate with the data that we have at this moment. In the E. coli MG1655 background, while the ompR234 mutation lead to increased biofilm formation and showed higher induction by mucin, the csgA mutation abolished biofilm formation completely, indicating that the mucin-mediated biofilm formation is csgBA-dependent, that is, curlidependent. On the other hand, a null mutation of ompR reduced biofilm formation but did not wipe out mucin's induction effect (Figure 3.6). Since no expression of csgBA could be observed in the absence of a functional ompR gene [START_REF] Prigent-Combaret | Complex regulatory network controls initial adhesion and biofilm formation in Escherichia coli via regulation of the csgD gene[END_REF], we conclude that other factor(s) beside curli are able to promote biofilm formation in a mucin-inducible manner in the ompR mutant of the MG1655 strain.

Low concentrations of mucin up-regulate csgBA gene expression in MG1655 genetic background

We further investigated curli's involvement in mucin-induced biofilm formation at the transcriptional level, making use of our transcriptional reporter fusion P csgBA -gfp (refer to section 2.3.3). Since 0.25 and 0.5% mucin produced the highest biofilm induction effect on both PS and glass surfaces, these two concentrations were tested in comparison with 0% mucin. The activity of P csgBA -gfp was monitored in both planktonic and biofilm mode.

The plasmid P csgBA -gfp, harbouring the short-life GFP[LVA] coding sequence under the control of the csgBA promoter was transformed into both MG1655 and MG1655ompR234, and the resulting strains were used in the following series of experiments.

On agar surface

For a preliminary test, MG1655ompR234 carrying P csgBA -gfp were streaked on BHI, LB and mM63 agar plates with or without 2% mucin (concentration used in the interface biofilm system, refer to the first paragraph of Chapter 3) and incubated for 24 hours at 30°C and 37°C. At 30°C, strong csgBA expression was visible on rich media BHI (Figure 3.7 A "BHI", GFP expression resulted in bright yellow-green coloration within the streaks and colonies) and LB agar (Figure 3.7 A "LB"). However, only very low expression was visible on mM63 agar (Figure 3.7 A "mM63 at 30°C"), which could partially be attributed to the poor growth of colonies. Although the growth on mM63 agar was improved at 37°C, the P csgBA -gfp activity remained low (Figure 3.7 A "mM63 at 37°C"), as was on BHI and LB agar (data not shown). For a more objective comparison, P csgBA -gfp activity was quantified by dissolving a loopful of cells in 1ml sterile water and measuring the fluorescence level, then normalising against the cell density. As could already be visually observed on agar, mucin up-regulated csgBA promoter activity at 30°C on all three media agar (Figure 3.7 B compare blue bar to red bar in blocks of "BHI", "LB" and "mM63 30°C"), while having the highest induction on LB (approximately 6-fold increase in relative fluorescence compared to 2-fold on mM63 and 1.5-fold on BHI), whereas no induction can be observed on mM63 at 37°C (Figure 3.7 B compare blue bar to red bar in the block of "mM63 37°C"). It has been routinely

observed that csgBA expression on agar plate is not affected by the osmolarity of the medium. It is likely that osmolarity influence is not crucial in the agar culture condition, considering that most cells in the colony are not in direct contact with the medium.

However, temperature regulation of csgBA expression remains critical even in the context of agar cultures.

I then tested the induction effect of 0.25% and 0.5% mucin in BHI and LB agar plate using the same method as that for 0% and 2% mucin. As shown in Figure 3.7 C, both 0.25% and 0.5% mucin up-regulated csgBA promoter activity on BHI and LB agar plate, as with 2% mucin.

In planktonic culture

Strains MG1655 and MG1655ompR234 carrying P csgBA -gfp were inoculated at a cell density of 6x10 6 cells/ml into mM63 supplemented with 0, 0.25 and 0.5% mucin. The planktonic culture was gently shaken (60 rpm) and sample aliquots were collected hourly from 3 to 14 hours of culture, followed by 24 and 48 hour time points. GFP expression level was measured by fluorometer whereas cell concentration was determined by viable The average activity (fluorescence/CFU, calculated as described in 6.8.1) of P csgBA -gfp in E. coli MG1655 (A) and MG1655ompR234 (B) in planktonic culture was calculated.

Mean values and SD of 2 experiments are indicated. Mucin did not affect the growth of E. coli but significant up-regulated csgBA expression in MG1655ompR234. Black boxes indicate enlarged regions for better observation. 5-fold (with 0.25% mucin) and 1.5-fold (with 0.5% mucin) up-regulation of csgBA expression can be observed in MG1655, whereas 2.5 fold (with 0.25% mucin) and as high as 13-fold up-regulation can be seen in MG1655ompR234. count on agar plates. The average activity of P csgBA -gfp was calculated as measured fluorescence per CFU. As expected, while the growth of bacteria is not influenced by mucin, csgBA expression in MG1655 shows 5-fold increase in the presence of 0.25% mucin and 1.5-fold increase with 0.5% mucin at 14 hours (Figure 3.8 A). In MG1655ompR234, the "basal activity level" of P csgBA -gfp is much higher than that in MG1655, owing to the strong constitutive positive regulation of the ompR234 mutation.

Despite this, the up-regulation of P csgBA -gfp is evident even at 8 hours of incubation (Figure 3.8 B). 0.25% mucin increases P csgBA -gfp activity up to 2.5 fold while 0.5% mucin leads to as high as 13-fold increase at 24 hours.

The up-regulation of csgBA promoter activity by low concentrations of mucin in the planktonic cultures corroborates that curli is involved in mucin-induced biofilm formation. However, it should be borne in mind that the P csgBA -gfp activity calculated here is an averaged promoter activity of the whole population in the sample.

In recent years, we have become more aware that promoter expression may be heterogeneous even within the same planktonic culture [START_REF] Dubnau | Bistability in bacteria[END_REF][START_REF] Davidson | Individuality in bacteria[END_REF], and it should be noted that the averaged promoter activity will not reflect heterogeneity even if it exists. For example, in the case of csgBA promoter activity, if the average P csgBA -gfp activity without mucin in the heterogeneous population is designated as 1, as shown in Figure 3.9 A, the increased average P csgBA -gfp activity (as reflected by green fluorescence) in the presence of mucin may be a result of three scenarios. First, it could be an increase in the csgBA-active ("green") population (Figure 3.9 B); or the presence of mucin could lead to increased csgBA expression ("green" intensity) of csgBA-active cells (Figure 3.9 C); lastly and most probably, the increased average csgBA activity may be the result of both increased csgBA-active population and csgBA expression (Figure 3.9 D). A. The expression of csgBA gene is heterogeneous with cells of no expression (gray ovals designated "0"), or certain level of expression (light green ovals designated "5"). The average P csgBA -gfp activity for the cluster of 10 cells is therefore 1. An increase in the average P csgBA -gfp activity may be a result of increased csgBA-active population (B), or increased P csgBA -gfp activity level of csgBA-active cells (C), or both (D).

In biofilm

In biofilm, cells are considered to be even more heterogeneous than their counterparts in planktonic culture, because of the existence of spatially restricted microenvironments in biofilms [START_REF] Grantcharova | Bistable expression of CsgD in biofilm development of Salmonella enterica serovar typhimurium[END_REF]. Cells in different microenvironment may thus exhibit heterogeneity, e.g., with respect to motility, antibiotic susceptibility and production of extracellular matrix components [START_REF] Klausen | Biofilm formation by Pseudomonas aeruginosa wild type, flagella and type IV pili mutants[END_REF][START_REF] Haagensen | Differentiation and distribution of colistin-and sodium dodecyl sulfatetolerant cells in Pseudomonas aeruginosa biofilms[END_REF][START_REF] Chai | Bistability and biofilm formation in Bacillus subtilis[END_REF]. It has been demonstrated that CsgD is expressed in a bistable manner during Salmonella biofilm development, that is, both high (CsgD-ON) and low (CsgD-OFF) csgD-expressing subpopulations exist [START_REF] Grantcharova | Bistable expression of CsgD in biofilm development of Salmonella enterica serovar typhimurium[END_REF]. They also suggested direct correlation between expression of the biofilm regulator CsgD and the phenotypic output, i.e., the production of curli and cellulose. This is in agreement with the observation by White and co-workers that the promoters of adrA (encoding a DGC that activates cellulose biosynthesis) and csgBA are primarily activated in the subpopulation of aggregated cells [START_REF] White | Aggregation via the red, dry, and rough morphotype is not a virulence adaptation in Salmonella enterica serovar Typhimurium[END_REF]. Therefore, we can expect that the expression of the CsgD-regulated csgBA to be highly heterogeneous in biofilm. Thanks to CLSM and the advancement of image analysing software like COMSTAT and ImageJ, the heterogeneity of P csgBA -gfp activity can be examined and taken into account for the assessment.

Difficulties in promoter activity measurement using traditional method

Traditionally, in biofilm, the measurement of promoter activity using promoterfluorescence fusion is achieved by calculating the proportion of the fluorescent cells (in which the gene of interest is actively expressed) in the total population [START_REF] Moller | In situ gene expression in mixed-culture biofilms: evidence of metabolic interactions between community members[END_REF][START_REF] Dulla | Quorum size of Pseudomonas syringae is small and dictated by water availability on the leaf surface[END_REF]). An increased promoter activity is thus shown as an increase in the fluorescent population proportion, as explained in Section 3.3.2.2, Figure 3.9 B. However, this method could not be used in determining the promoter activity in our biofilm system due to the nature of our experimental model, in which the heterogeneity exists in several aspects including spatial distribution of cells and mucin, as First of all, the gene expression of biofilm cells appears heterogeneous (see Section 3.3.2.3), as in planktonic cultures. Thus, not all cells carrying P csgBA -gfp have their csgBA promoter activated and turn green (Figure 3.10 D). As explained in Section 3.3.2.2, Figure 3.9 B, the "up-regulation" of the csgBA promoter can be manifested as an increase of the csgBA-expressing subpopulation with respect to the total bacterial population. In microscopic images, it is reflected as an increased proportion of green cells in the whole population (in the observation field). In COMSTAT programme, only fluorescent pixels in the field are recognized as cells, giving us the possibility to compute the biomass of csgBA-expressing population (refer to Section 2.3.3). Unfortunately, the other parameter necessary to complete the analysis -total bacterial population -is difficult to determine, since non-fluorescent cells are indistinguishable from mucin. Therefore we cannot use the traditional method to quantify P csgBA -gfp activity because in our case, the total bacterial population cannot be determined easily.

The possibility of using fluorescent stain (such as SYTO 61) on biofilms mixed with mucin for the determination of total bacterial population was decided against, because of the observed disruption of biofilm during the staining process and its inconsistency in staining even pure bacterial biofilm without mucin (refer to Section 2.4.1).

In the search for an alternative method for total bacterial population measurement, one may consider using E. coli G1 biofilm or its derivative strains in the presence of mucin as a parallel control sample, to serve as an estimation of the total bacterial population because its "green" population is 98.70 -99.60% of the whole population [START_REF] Miao | Dual fluorescence system for flow cytometric analysis of Escherichia coli transcriptional response in multispecies context[END_REF]. The complication is that the spatial distribution of cell clusters and mucin in the saturated system is heterogeneous (and can be highly heterogeneous), thus a generalised estimation of total bacterial population using E. coli G1 derivatives may not be representative for different observation fields. For example, on the same cover slip, derivative biofilms have to be assessed in similar ways in order to determine the "corresponding" total bacterial population, too many factors of subjectiveness will be compounded, and confidence in the final interpretation will be compromised.

Due to the inability to discern mucin from non-fluorescent cells, it may also seem difficult to determine whether a correlation exists between csgBA expression and biofilm development, i.e. we may see more "green cells" but how would we know if all "grey patches" are not just mucin patches? To alleviate this worry, I compared the view of the mucin on its own with other cell-containing samples (Figure 3.11). The increased heterogeneity in morphology (complex 3D structure) brought about by bacterial cell during biofilm formation (Figures 3.11 A -D) is not negligible, compared to the homogeneous distribution of mucin alone (Figure 3.11 E). Hence qualitatively we could still make assessment of the observation fields, but qualitatively, we were unable to deepen the analysis. "Green" population% 40% 40% 70% 40% Specific activity 7.5 7.5 11.4 20

The use of "specific activity"

In view of the above mentioned difficulties in applying the traditional method, I needed to develop a new method to quantitatively determine the P csgBA -gfp activity in biofilm mixed with mucin. It can be observed from the microscopic image that within the csgBAexpressing (i.e. "green") population, the intensity of green fluorescence varies. This fluorescence intensity is another indication of P csgBA -gfp activity, which reflects the transcription level of csgBA expression within the single cell (Figure 3.12, see cells with fluorescence intensities "5", "15" etc). As previously discussed in Section 3.3.2.2 and illustrated in Figure 3.9, other than an increase in active subpopulation (Figure 3.9 B), upregulation of a gene promoter activity can also be a result of increased promoter activity level in the promoter-active cells or a combination of the two (Figure 3.9 C and D). I considered the function of the software ImageJ which measures the fluorescence intensity of every X-Y plane in a Z-stack (refer to Section 2.3.3 on Z-stack). Thus the total intensity (total promoter activity) of the observed population can be obtained by summing the intensity of all X-Y planes in the Z-stack. The measurement of total intensity by averaging the intensity of 10 observation fields has been used to determine promoter-gfp fusion activity by others [START_REF] Lequette | Timing and localization of rhamnolipid synthesis gene expression in Pseudomonas aeruginosa biofilms[END_REF]. However, their method is only applicable for promoter activity measurement in biofilms if the parameter "biomass" is taken into account. This can be appreciated by the illustration shown in Figure 3.12.

Fluorescent biomass is a measurement of the number of total fluorescent pixels in a Zstack, and in our experiments it reflects the quantity of fluorescent bacteria in the observation field. Therefore, an increased total intensity (Figure 3.12, compare the total intensity of B with A) may simply be a result of increased fluorescent biomass, i.e., fluorescent bacterial population (Figure 3.12, compare the biomass of B with A), with neither increased promoter activity in single cells nor increased promoter-active cell population (Figure 3.12, compare the "green" population percentage of B with A).

Moreover, due to the heterogeneity of the promoter activity ("green" intensity) within the same sample, neither the total intensity nor the fluorescent biomass alone is sufficient to reflect the state of the population (Figure 3.12, compare A and D: although the total intensity increases, fluorescent biomass remain the same; compare C and D: the same Therefore, I chose to use the average intensity of only csgBA-expressing ("green") cells, which is calculated as the total fluorescence intensity (processed by ImageJ) per unit fluorescent biomass (processed by COMSTAT), and termed it "specific activity". The activity level of promoter-active cells in a population when presented as specific activity would be undiluted by promoter non-active cells (compare the specific activity of Figure 3.12 C and D), unlike conventional calculation of average activity. In other words, "specific activity" is a measurement describing how green the green cells are. The specific activity measurement is useful when (i) the quantity of total bacterial population is unknown or incomparable and conventional average activity cannot be obtained (as in our case, as discussed in Section 3.3.2.3.1), and (ii) there is a specific interest to focus on the promoter-active subpopulation. Furthermore, the use of both P csgBA -gfp specific activity and the proportion of csgBA-active population in describing a biofilm may be more biologically relevant than any of the two parameters used alone. It is conventionally considered that biofilm formation is an energetically costly process due to the production of extracellular matrix components, therefore only a subpopulation of cells is responsible for the production of these "expensive" structures, which can be shared by the whole population [START_REF] Branda | A major protein component of the Bacillus subtilis biofilm matrix[END_REF]. This strategy maximises the benefits through collaborative effort in biofilm formation while minimising the cost [START_REF] Grantcharova | Bistable expression of CsgD in biofilm development of Salmonella enterica serovar typhimurium[END_REF]. By quantifying these two aspects of the population (specific activity and proportion of promoter-active subpopulation), we can better assess such scenarios and derive more biological information in future studies. The specific activity of P csgBA -gfp is increased in MG1655ompR234 with 0.25% mucin, suggesting that 0.25% mucin up-regulates csgBA expression in csgBA-active MG1655ompR234 cells.

It should be noted however, that the specific activity is also an averaged value of all the csgBA-expressing cells in the sample. The heterogeneity in csgBA expression being very high with 0.5% mucin in MG1655 implies that 0.5 % mucin may be involved in both activation of csgBA in csgBA-inactive cell and augmentation of csgBA gene expression level.

Even though quantification of the csgBA-expressing MG1655 population proportion is impractical due to the difficulty in total bacterial population quantification, it is nonetheless unmistakable from the confocal microscopic image that MG1655 without mucin forms only microcolonies, but develops complex biofilm structure with 0.25% mucin (Figure 3.10, compare A and B). The addition of 0.5% mucin further improves the 3D structure complexity of biofilm (Figure 3.10 C). P. aeruginosa biofilms formed on glass coated with human airway or bovine submaxillary mucins display similar aggregates [START_REF] Landry | Mucin-Pseudomonas aeruginosa interactions promote biofilm formation and antibiotic resistance[END_REF]. Therefore, induction of bacterial biofilms with complex 3D structure could be a general property of mucins. In our case, it is possible that mucin possesses bacterial attachment sites mimicking those on intestinal epithelial cells [START_REF] Blomberg | Piglet ileal mucus contains protein and glycolipid (galactosylceramide) receptors specific for Escherichia coli K88 fimbriae[END_REF][START_REF] Mack | Probiotics inhibit enteropathogenic E. coli adherence in vitro by inducing intestinal mucin gene expression[END_REF][START_REF] Lillehoj | Muc1 mucins on the cell surface are adhesion sites for Pseudomonas aeruginosa[END_REF]. E. coli, belonging to the enterobacteriaceae family, may recognise these sites and attach to mucin. Cell-surface interactions induce remodelling of the bacterial envelope [START_REF] Otto | Adhesion of type 1-fimbriated Escherichia coli to abiotic surfaces leads to altered composition of outer membrane proteins[END_REF], this attachment and change in envelope could also induce a cascade of changes in gene expression that favour biofilm life style, leading to csgBA activation. It is shown that enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC), the major cause of diarrheal disease, produce curli during adhesion to cultured epithelial cells through activation of the regulator csgD [START_REF] Saldana | Synergistic role of curli and cellulose in cell adherence and biofilm formation of attaching and effacing Escherichia coli and identification of Fis as a negative regulator of curli[END_REF]. I suggest that the early molecular event leading to the csgD gene activation is the E. coli attachment to mucin. strains in laboratory, analysis has been done to compare their genome sequences and showed that although they highly resemble each other, 13 sites have an IS element or defective phage in only W3110 or MG1655, as well as 8 sites with differences between them, 7 of which are in open reading frames (orfs) and 1 in an rRNA gene [START_REF] Hayashi | Highly accurate genome sequences of Escherichia coli K-12 strains MG1655 and W3110[END_REF]. Among these differences, four appeared to be relevant to this study. Found in W3110 and not in MG1655 are: non-functional rpoS alleles, one IS insertion downstream of csgC, one upstream of flhD, and one within the rcsC sequence. rpoS is a global regulator of stationary phase expressed genes, and the non-functional rpoS in W3110 did not lead to repressed csgBA expression but quite the contrary. The IS insertions in noncoding regions is unlikely to have a direct effect on the expression of adjacent genes.

However, the disruption of rcsC sequence by IS insertion in W3110 is highly likely to result in a non-functional Rcs regulatory system. The rcs regulatory pathway regulates synthesis of the capsular polysaccharide colanic acid, and is activated at low temperature with glucose as carbon source [START_REF] Majdalani | The Rcs phosphorelay: a complex signal transduction system[END_REF][START_REF] Huang | The role of the Rcs phosphorelay in Enterobacteriaceae[END_REF]. It represses expression of surface appendages such as flagella (through flhDC) (Francez-Charlot et al., 2003), curli (through csgD), and Ag43 (through agn43/flu) [START_REF] Ferrieres | The RcsC sensor kinase is required for normal biofilm formation in Escherichia coli K-12 and controls the expression of a regulon in response to growth on a solid surface[END_REF], to prevent mobility and surface attachment (Figure 1.2 & 1.5). Therefore the down-regulation of csgBA expression in mature biofilm might be due to the repression of csgD expression by RcsB, which is the downstream response regulator of RcsC (Figure 1.2). Thus, the loss of function of Rcs system in W3110 might result in the absence of csgBA repression, which is reflected as higher csgBA expression compared to that in MG1655 in our experiment.

Role of other extracellular structures in mucin induced biofilm formation

The expression of type 1 pili in MG1655, as in fecal isolates, facilitates mucin-mediated biofilm formation in Minimum Essential Medium (MEM) [START_REF] Bollinger | Secretory IgA and mucin-mediated biofilm formation by environmental strains of Escherichia coli: role of type 1 pili[END_REF].

Besides curli, would other extracellular structures that contribute to biofilm formation be involved in mucin-induced biofilm formation? The residual adherence observed in the ompR mutant suggests it. Curli is involved in almost every step in biofilm formation (Figure 1.1) [START_REF] Van Houdt | Role of bacterial cell surface structures in Escherichia coli biofilm formation[END_REF], so I chose to preliminarily assess the role of type 1 pili that is important for initial attachment and Ag43 for early biofilm structure development in biofilm formation in the presence of mucin.

Antigen 43:

It is reported that Ag43 contributes to E. coli biofilm formation in glucose-minimal medium, but not in LB broth (Danese et al., 2000a). When grown in 24-well plate in mM63 at 30°C, deletion of agn43 did not significantly affect mucin-induced biofilm formation of MG1655ompR234, if not slightly increased the overall biofilm quantity (Figure 3.15). This effect was even more prominent in E. coli W3110 (Figure 3.15). This suggests that Ag43 acts negatively in biofilm formation of E. coli MG1655 and W3110 on glass and PS. To further investigate the possible regulation of agn43 by ompR, the GFP tagged Agn43 promoter P agn43 -gfp [START_REF] Zaslaver | A comprehensive library of fluorescent transcriptional reporters for Escherichia coli[END_REF] activity was observed in biofilm of MG1655, MG1655ompR234 as well as W3110 and its ompR234 mutants, in the presence of 0, 0.25 and 0.5% mucin. No P agn43 -gfp activity was detectable in biofilm with these strains (data not shown). Suspecting that the ompR234 mutation might have repressed the expression of Agn43, MG1655ompR background was used but gave the same result. The effect of agn43 on the curli genes transcription has then to be investigated. A repressor effect of Ag43 on the curli would help to understand how E.

coli coordinates the expression of its numerous adherence structures.

Type I pili:

Type 1 pili consist of the structural subunit protein FimA and the mannose-specific adhesin FimH. In E. coli W3110, deletion of fimA or fimH both resulted in impairment of biofilm formation with different concentrations of mucin (Figure 3.16), suggesting that type 1 pili play an important role in mucin-induced biofilm formation in W3110.

While the ompR234 mutation augments biofilm formation of W3110 as on MG1655

regardless of mucin's presence, insertional inactivation of csgA in W3110ompR234 reduced the overall biofilm formation but did not completely eliminate the induction effect (Figure 3.16). This result shows that although curli are the main factor involved in adherence to PS in this strain, another factor, such as type 1 pili, contributes to the biofilm formation in the presence of mucin when curli fibers are missing. Two hypothesis can be proposed: biofilm induction by mucin could be due either to a cooperative effect of curli AND type 1 pili, or to the sole effect of type 1 pili. Deletion of fimA in W3110ompR234 seems to result in loss of the induction effect by mucin (Figure 3.16).

Therefore, it is very tempting to speculate that mucin induces biofilm formation in W3110 only through type 1 pili, while curli greatly improves the biovolume of biofilm.

Regulatory interplay between the two adherence factors has now to be investigated, especially in the presence of mucin.

Interestingly, without FimA, not only the biofilm induction effect of mucin is abolished (no increase in biofilm formation can be observed), the presence of mucin seems to even reduce the biofilm forming ability of W3110 and W3110ompR234 (Figure 3.16, both

W3110fimA and W3110ompR234fimA have the highest biofilm formation at 0% mucin).

Therefore, in E. coli W3110, while overexpression of curli might boost the capability of W3110 to form biofilm, type 1 pili is possibly involved in mucin-induced biofilm formation, without which mucin may even decrease the biofilm forming ability of W3110.

In E. coli MG1655ompR234, on the other hand, deletion of fimA did not disrupt the mucin-induced biofilm formation (Figure 3.16). This suggests that type 1 pili is not involved in mucin-induced biofilm formation in this strain. These differences between E. coli MG1655 and W3110 may be due to the difference in their genome, as described above (refer to Section 3.3.2.3.3). However, why and how type 1 pili regulation or expression interferes with that of curli in the presence of mucin remain to be elucidated.

Nevertheless, this could be another example of the coordinated regulation of surface adhesins besides that between type 1 pili and Ag43 (Section 1.1.1.3) as well as between curli and cellulose (Section 1.1.1.1). Indeed, microscopic observations have revealed that in the biofilm of curli-deficient E. coli mutant flagella and type I pili were present [START_REF] Kikuchi | Curli fibers are required for development of biofilm architecture in Escherichia coli K-12 and enhance bacterial adherence to human uroepithelial cells[END_REF]. In Salmonella, pili-frimbriae were most abundant on curli mutant, suggesting co-regulated expression of pili, curli and other surface structures [START_REF] Jonas | Roles of curli, cellulose and BapA in Salmonella biofilm morphology studied by atomic force microscopy[END_REF]. Crosstalk between the regulation of flagellar, Salmonella pathogenicity island 1 (SPI1), and type 1 fimbrial genes has recently been reported by Saini and co-workers [START_REF] Saini | The role of crosstalk in regulating the dynamic expression of the flagellar, Salmonella pathogenicity island 1 (SPI1), and type 1 fimbrial genes[END_REF]. It has also been suggested that fimC, encoding a PapD-like chaperone, might have a CsgD binding site in its promoter region and that csgD null mutation led to increased fimC mRNA level whereas csgD overexpression resulted in a decrease of the mRNA level of fimC [START_REF] Zakikhany | Unphosphorylated CsgD controls biofilm formation in Salmonella enterica serovar Typhimurium[END_REF]. This indicates that curli genes expression could be dominant on type 1 pili genes expression. Our results suggest that a coexpression could occur in strain such as W3110, but further experiments are needed to shed light on this point.

Chapter 4 Partnership of E. coli with K. pneumoniae and E. faecalis

Biofilms in nature are mostly found to be composed of multispecies consortia [START_REF] Stewart | Spatial Distribution and Coexistence of Klebsiella pneumoniae and Pseudomonas aeruginosa in Biofilms[END_REF][START_REF] Stoodley | Biofilms as complex differentiated communities[END_REF][START_REF] Burmolle | Enhanced Biofilm Formation and Increased Resistance to Antimicrobial Agents and Bacterial Invasion Are Caused by Synergistic Interactions in Multispecies Biofilms[END_REF][START_REF] Klayman | Escherichia coli O157:H7 Requires Colonizing Partner to Adhere and Persist in a Capillary Flow Cell[END_REF]. The coexistence of different species in a biofilm often confers fitness advantages owing to cooperation between these species based on their different metabolic profiles, physical properties and growth preferences [START_REF] Klayman | Escherichia coli O157:H7 Requires Colonizing Partner to Adhere and Persist in a Capillary Flow Cell[END_REF][START_REF] Burmølle | Biofilms in chronic infections -a matter of opportunity -monospecies biofilms in multispecies infections[END_REF]. For example, in biofilms comprising of aerobic and anaerobic species, the aerobic species are mostly found in the surface layer that is exposed to oxygen whereas the anaerobic species are buried in the inner biofilm and shielded from oxygen by the aerobic partners [START_REF] Bradshaw | Role of Fusobacterium nucleatum and coaggregation in anaerobe survival in planktonic and biofilm oral microbial communities during aeration[END_REF]. In a nitrifying wastewater treatment biofilm, nitrite-oxidizing bacteria are found to form clusters around ammonia-oxidizing bacteria in nitriteoxidizing zones [START_REF] Okabe | In situ analysis of nitrifying biofilms as determined by in situ hybridization and the use of microelectrodes[END_REF]. The persistence of the soil-inhabiting bacteria

Pseudomonas putida in a condition where benzyl alcohol is the sole carbon source is dependent on Acinetobacter sp., because Acinetobacter converts benzyl alcohol into benzoate, which can be metabolized by P. putida [START_REF] Christensen | Metabolic commensalism and competition in a two-species microbial consortium[END_REF]. A

Lactobacillus strain has been demonstrated to proliferate in a mixed-species biofilm with up to 20 times higher cell numbers than in its monoculture [START_REF] Filoche | Biofilm growth of Lactobacillus species is promoted by Actinomyces species and Streptococcus mutans[END_REF]. An E.

coli strain which was unable to irreversibly attach to glass surface when cultured alone under continuous flow conditions was able to form biofilms in the presence of P.

aeruginosa. Closer observation showed that the adhered E. coli cells were attached to P. aeruginosa cells, showing cooperativity at the level of physical interaction [START_REF] Klayman | Escherichia coli O157:H7 Requires Colonizing Partner to Adhere and Persist in a Capillary Flow Cell[END_REF]. Naturally, not all interactions are positive and cooperative. For example, Ruminococcus gnavus strain E1 isolated from a human fecal sample was shown to inhibit the growth of various pathogenic clostridia, due to its production of the bacteriocin ruminococcin A [START_REF] Dabard | Ruminococcin A, a new lantibiotic produced by a Ruminococcus gnavus strain isolated from human feces[END_REF]Gomez et al., 2002). A newly discovered oral streptococcal species, Streptococcus oligofermentans was observed to inhibit S. mutans through conversion of lactic acid into inhibitory H 2 O 2 [START_REF] Tong | Streptococcus oligofermentans inhibits Streptococcus mutans through conversion of lactic acid into inhibitory H2O2: a possible counteroffensive strategy for interspecies competition[END_REF].

Interspecies interactions can be studied using different approaches, such as by looking at population relationship, gene expression changes, metabolic interplays, biofilm development (colonization influence and spatial distribution) and interspecies communication (e.g quorum sensing). Various studies have been performed on environmental species such as the trichloroethylene (TCE)-degrading Burkholderia cepacia, the oil/water isolate Klebsiella oxytoca [START_REF] Komlos | Interaction of Klebsiella oxytoca and Burkholderia cepacia in dual-species batch cultures and biofilms as a function of growth rate and substrate concentration[END_REF], marine alga surface isolates [START_REF] Burmolle | Enhanced Biofilm Formation and Increased Resistance to Antimicrobial Agents and Bacterial Invasion Are Caused by Synergistic Interactions in Multispecies Biofilms[END_REF], soil [START_REF] Hansen | Evolution of species interactions in a biofilm community[END_REF], and oral bacteria [START_REF] Ledder | Coaggregation between and among human intestinal and oral bacteria[END_REF][START_REF] Kolenbrander | Oral multispecies biofilm development and the key role of cell-cell distance[END_REF][START_REF] Koo | Exopolysaccharides Produced by Streptococcus mutans Glucosyltransferases Modulate the Establishment of Microcolonies within Multispecies Biofilms[END_REF]. However, quite surprisingly, studies on intestinal bacteria such as K. penumoniae and E. coli were frequently partnered with pathogenic species P. aeruginosa [START_REF] Siebel | Observations of binary population biofilms[END_REF][START_REF] Stewart | Spatial Distribution and Coexistence of Klebsiella pneumoniae and Pseudomonas aeruginosa in Biofilms[END_REF][START_REF] Liu | Role of Pseudomonas aeruginosa biofilm in the initial adhesion, growth and detachment of Escherichia coli in porous media[END_REF][START_REF] Klayman | Escherichia coli O157:H7 Requires Colonizing Partner to Adhere and Persist in a Capillary Flow Cell[END_REF][START_REF] Burmølle | Biofilms in chronic infections -a matter of opportunity -monospecies biofilms in multispecies infections[END_REF], while the interactions between intestinal commensal species themselves have not been investigated much. In my work, I chose to investigate E. coli partnership with K.

pneumonia and E. faecalis with respect to their population relationship, since population study is one of the most global assessments which could provide an overview of the fitness and growth relationship of the co-cultured species.

Population relationship -Synergism, commensalism, parasitism or antagonism?

When two organisms grow together, their relationship could be beneficial, neutral or detrimental to each other's growth. Several scenarios could take place. There is a possibility of (i) synergistic relationship where both partners achieve better growth/fitness together than if they are on their own. Alternatively, they could commit to (ii) commensalism where one partner benefits and the other is not affected, or (iii) parasitic interaction where one organism benefits but the other is harmed or disadvantaged. Finally (iv) competition or antagonism could occur when both organisms are disadvantaged compared to when they are alone.

Population Relationship in our model is not based on antibiotics-mediated inhibition

Intestinal commensal species are generally believed to be interacting positively with each other for mutual benefits, as well as the hosts' benefit (Lievin-Le [START_REF] Moal | The front line of enteric host defense against unwelcome intrusion of harmful microorganisms: mucins, antimicrobial peptides, and microbiota[END_REF].

However, it is ironic that for E. coli, K. pneumoniae and E. faecalis, the very few studies that touch on their "interactions" dealt only with the extreme antagonistic end of the interaction spectrum, i.e. biocidal activity against each other. All three bacterial species have been reported to be able to produce bacteriocins, which exhibit antimicrobial activity (Gillor et al., 2008). Specific strains of E. coli have long been known to produce colicin [START_REF] Gratia | Sur un remarquable exemple d'antagonisme entre deux souches de coilbacille[END_REF] and microcin [START_REF] Duquesne | Microcins, geneencoded antibacterial peptides from enterobacteria[END_REF]. K. pneumoniae strains, on the other hand, are famous for the production of microcin E492 [START_REF] Destoumieux-Garzon | Microcin E492 antibacterial activity: evidence for a TonB-dependent inner membrane permeabilization on Escherichia coli[END_REF][START_REF] Bieler | Bactericidal activity of both secreted and nonsecreted microcin E492 requires the mannose permease[END_REF], whereas strains of E. faecalis have been reported to secrete enterocin As-48 [START_REF] Archimbaud | In vitro adhesive properties and virulence factors of Enterococcus faecalis strains[END_REF][START_REF] Fernandez | Processing of as-48ABC RNA in AS-48 enterocin production by Enterococcus faecalis[END_REF][START_REF] Cebrian | Insights into the functionality of the putative residues involved in enterocin AS-48-maturation[END_REF].

Each of these antibiotics produced by one species has been shown to have biocidal effects on certain strains of the other two species.

To identify or eliminate possible effects of bacteriocins among the strains of our experimental model, a simple growth inhibition test was performed based on the method used by Fleming and colleagues [START_REF] Fleming | Microbial inhibition by an isolate of pediococcus from cucumber brines[END_REF]. In this test, aliquots of E. coli MG1655 or G1 strains were spotted onto lawns of K. pneumoniae or E. faecalis, or vice versa (refer to Section 6.4.6) . A clear halo around the spotted strain, showing inhibition of growth of the test strain in the lawn, would indicate antibiotic production by the spotted strain which is effective against the test strain. The strains in the list shown below were tested as both spot (for antimicrobial production) and lawn (for antimicrobial susceptibility) and no growth inhibition was observed in any of the combinations (data not shown).

physical interactions [START_REF] Burmolle | Enhanced Biofilm Formation and Increased Resistance to Antimicrobial Agents and Bacterial Invasion Are Caused by Synergistic Interactions in Multispecies Biofilms[END_REF]. Furthermore, contact-dependent growth inhibition has been reported in E. coli strains [START_REF] Aoki | Contact-Dependent Inhibition of Growth in Escherichia coli[END_REF], underscoring the idea that physical contacts between cells play significant biological roles. Therefore, adherence structures on the cell surface such as curli which can alter the surface property of the cell may influence the outcome of both intra-species and inter-species interactions.

The population relationship between E. coli and its partner species, K. pneumoniae and E.

faecalis were therefore examined with respect to E. coli's curli expression status.

However, prior to the population analysis, I set out to obtain two sets of reference parameters that could be used to qualify the differences in the surface properties of these strains.

The surface property of the mutants

The initial attachment of bacteria to biotic or abiotic surfaces is the result of the interplays of various fundamental physico-chemical forces [START_REF] Van Oss | Long-range and short-range mechanisms of hydrophobic attraction and hydrophilic repulsion in specific and aspecific interactions[END_REF]. For example, van der Waals forces [START_REF] Mccarthy | Colloid transport in the subsurface: past, present, and future challenges[END_REF], electrostatic and Lewis acid-base interactions [START_REF] Van Loosdrecht | Electrophoretic mobility and hydrophobicity as a measured to predict the initial steps of bacterial adhesion[END_REF], hydration forces, hydration pressure, hydrogen bonding, as well as hydrophobic associations have all been reported to influence bacterial adhesion [START_REF] Camesano | Heterogeneity in bacterial surface polysaccharides, probed on a single-molecule basis[END_REF][START_REF] Abu-Lail | Role of lipopolysaccharides in the adhesion, retention, and transport of Escherichia coli JM109[END_REF]. Since most naturally occurring surfaces carry a net negative charge under physiological conditions, bacterial cells which are generally negatively charged on their surfaces need to overcome this electrostatic repulsion, through the compounded effects of attractive van der Waals, hydrophobic and specific interactive forces, in order to achieve adherence [START_REF] Van Merode | Influence of culture heterogeneity in cell surface charge on adhesion and biofilm formation by Enterococcus faecalis[END_REF]. It has been shown that curli production in some E. coli O157 strains correlated with decreased hydrophobicity and showed higher attachment to hydrophilic glass surface [START_REF] Goulter | Characterisation of curli production, cell surface hydrophobicity, autoaggregation and attachment behaviour of Escherichia coli O157[END_REF]. To obtain a perspective of the cell surface charges with respect to curli expression under our experimental conditions, the zeta potentials of E.

coli strains with different curli expression status were measured (Section 4.3.1.1)

Other than physico-chemical forces, structures such as pili, curli and flagella which are protruding from cell surfaces will have effects at the physical and steric level. First and Zeta potential was measured for bacteria grown over night in mM63 at 30°C (A) and BHI at 37°C (B). E. coli mutants with different genotypes were also present: G1ompR234

(curli++), G1 (curli+/-) and G1 ompR234csgA (curli--). foremost, packing and compaction patterns of cells in a cluster or microcolony will be affected. Hence one can imagine that population relationship may be influenced due to the altered "packing" of cells of different species, especially in biofilms. The physical nature of the cell surfaces will also affect the stability of random cell-cell association in a planktonic setting, e.g. whether the cells will "bounce off" each other more readily or will be "sticky" when they collide in fluids. Although steric properties are difficult to quantify directly, the light scattering properties of cells can be used as an indirect indicator of physical surface complexity. For this reason, FACS would be used to capture side-and forward-light scattering profiles of E. coli cells differing in curli expression to obtain reference paremeters reflecting such physical characteristics (Section 4.3.1.2). Unlike zeta potential measurement which provides an averaged value for the whole population, parameters detected by FACS are captured at single-cell level, giving an extra dimension of information.

Electronegativity decreases with increasing curli expression

In fluid dynamics, the zeta potential is used to describe the colloidal nature of particles in solvents. However, to facilitate discussions in the biological context here, we may view it simplistically as the "surface electronegativity" of bacterial cells. The zeta potentials of E. coli strains with different curli expression profiles: curli++ (G1ompR234), curli+/-(G1) and curli--(G1 ompR234csgA), as well as the two partner species, K. pneumoniae and E. faecalis, were measured. When grown under conditions favouring curli expression (in mM63 at 30°C), the curli++ E. coli strain was observed to have the lowest absolute zeta potential, that is, the lowest electronegativity, whereas the curli--strain exhibited the highest electronegativity (Figure 4.1 A). In contrast, under rich medium (BHI) condition at 37°C where curli expression is very limited even for the curli++ strain (G1ompR234), the electronegativity of the three strains is similar (Figure 4.1 B). This suggests that increased curli expression plays a role in decreasing cell surface electronegativity, which may translate to lower electrostatic repulsion, both for cell-cell as well as cell-surface association. Therefore, one of the ways in which curli expression facilitate aggregation In curli permissive condition (mM63 at 30°C), curli over-expressing strain G1ompR234 harbours subpopulation of greater FSC and SSC values, displayed as a "tail" and gated within a polygon in red (C). Closer look at the wild type G1 also revealed a very small subpopulation falling within the "tail" region, also indicated with a red polygon (B). In BHI (37°C), the curli--, curli+/-and curli++ E. coli strains showed similar profiles. Null mutation in the ompR gene resulted in greater cell size than the other E. coli strains. K. pneumoniae and E. faecalis grown in mM63 at 30°C showed very different zeta potentials, with K. pneumoniae exhibiting a drastic reduction in electronegativity.

However, when grown in BHI 37°C, their surface electronegativity does not appear to differ much from each other or from the E. coli strains.

The data above affirmed that the strains' surface property as indicated by zeta potentials are sufficiently altered (i) due to curli expression of E. coli and (ii) between the differing culture conditions, as well as (iii) between K. pneumoniae and E. faecalis. Hence it will be worthwhile to see if these surface variations under different culture conditions could influence the population relationship between E. coli, K. pneumoniae and E. faecalis, and in what ways.

Distinct size and surface profiles of curli-related mutants by FACS analysis

The alteration in physical surface property with respect to curli expression was also captured, using FACS. The curli-related E. coli G1 mutants were grown over night as planktonic cultures both in the low osmolarity curli-inducing condition of mM63 at 30°C, and the high osmolarity BHI medium at 37°C. Figure 4.2 shows plots from such analysis whereby the x-axis indicates the forward scatter (FSC) reflecting cell or particle size, and the y-axis indicates the side scatter (SSC), reflecting the surface complexity of bacterial cells. In the curli permissive condition mM63 at 30°C, the curli++ (G1ompR234) strain which over expresses curli showed a distinct FSC-SSC profile from the curli--(G1ompR234csgA) strain: a "tail" sector (Figure 4.2 C, red polygon) indicating a subpopulation (13.31%) of bigger cell size and greater light scattering. This seems consistent with its high curli production status since it is expected that curli-producing cells would have the curli fibres protruding from the cell surface and perhaps also wrapping around the cells, which would manifest as higher SSC due to greater light scattering. Curli-producing cells also tend to be bulkier due to the curli layers, and may also form cell aggregates, both of which could be detected as larger FSC. In support of pneumoniae and E. faecalis, the curli over-expressing E. coli strain G1ompR234 (curli++)

and the curli null mutant G1ompR234csgA (curli--) were analysed in parallel to the wild type G1 strain (curli+/-) to examine how the range of curli expression influences interspecies interactions that may lead to changes in population relationship. Since E. coli can survive both in the flux of nutrient rich, high osmolarity conditions in the human gut and in the nutritionally poor, low osmolarity conditions outside the host, and these contrasting conditions affect its curli expression level, the population relationship were examined in both BHI (37°C) and mM63 (30°C), and in the planktonic and the biofilm states.

The population relationship was analysed by comparing the population of partner species in two ways: (i) between pure culture and dual-species (E. coli with either K. pneumoniae or E. faecalis) or multi-species (all three species together) co-cultures, which provides information about the influence of the partner species on the growth of the species of interest and (ii) between the relative population proportions of each species in co-cultures, which reveals the dominance relationship between partner species.

To determine whether a difference (or a change) in the population was significant between a pair of data sets, the p-value was calculated using Student's t-Test and the difference was considered significant when the value of p < 0.05. For multiple comparisons, Bonferroni correction was applied. Only significant differences will be highlighted and considered in the discussion.

In planktonic culture

We were first interested to know how the growth (viable cell count) of one commensal species was affected by the presence of another species. Therefore, dual-and multispecies co-cultures were compared with controls of single species cultures which carried samples. Student's t-Test was performed to determine whether the differences were statistically significant.

1.00E+04 1.00E+05 1.00E+06 1.00E+07 1.00E+08 1.00E+09 1.00E+10 G1 ompR234 ΔcsgA pure culture Ec+Kp Ec+Ef Ec+Kp+Ef G1 ompR234 ΔcsgA G1 ompR234 ΔcsgA 1.00E+04 1.00E+05 1.00E+06 1.00E+07 1.00E+08 1.00E+09 1.00E+10 G1 ompR234 ΔcsgA G1 ompR234 ΔcsgA G1 ompR234 ΔcsgA BHI 37 C mM63 30 C K. pneumoniae E. coli E. faecalis A B C D E F G1 ompR234 ompR234csgA G1 ompR234 ompR234csgA G1 ompR234 ompR234csgA G1 ompR234 ompR234csgA G1 ompR234 ompR234csgA G1 ompR234 ompR234csgA
starting cell densities identical to that of each of the component species in the co-cultures.

This combination was executed for all three E. coli strains varying in curli-expression status. After 24 hours, CFU count was performed for every species in co-cultures as well as in pure cultures, and the absolute CFU/ml of each species were determined for comparison (Figure 4.4). When cultured in BHI (37°C), all three E. coli strains and both partner species showed lower growth in co-culture than in pure culture (Figure 4.4 A, B and C, compare blue bar against other bars within each block). However, the situation is different in mM63 (30°C): (i) although E. coli still showed better growth in pure culture than in co-cultures (Figure 4.4 D), (ii) K. pneumoniae did not seem to be influenced by the presence of G1 or the curli++ mutant E. coli (G1ompR234) (Figure 4.4 E, compare blue against red bars of the respective E. coli strains). Nevertheless, it did display significantly lower growth when co-cultured with the curli--mutant (G1ompR234csgA).

When E. faecalis was also present in the co-culture i.e. in multi-species culture (purple bar), this negative effect on K. pneumoniae by E. coli G1ompR234csgA was abolished (compare red and purple bars for the "ompR234csgA" block). (iii) E. faecalis did not appear to be significantly influenced when co-cultured with the curli+/-and curli++ E. coli (Figure 4.4 F) but grew better in the presence of E. coli curli--G1ompR234csgA mutant, in dual-species co-culture (compare blue and green bars) as well as in multispecies co-culture (compare blue and purple bars).

We note that in three-species co-cultures, the influence of the third member is generally neutral or mildly cumulative in BHI medium (Figures 4.3 A, B, C, compare purple bars against red and green bars). In M63 medium, this observation also applies, with the exception of (i) the negative influence on E. coli G1ompR234 and G1ompR234csgA strains by K. pneumoniae (Figure 4.4 D, compare blue against red bars of "ompR234"

and "ompR234csgA" blocks), which was considerably alleviated by the presence of E.

faecalis (purple bars), (ii) the negative influence on K. pneumoniae by E. coli curli-strain (Figure 4.3 E, compare blue against red bars for the "ompR234csgA" block), which was abolished in the presence of E. faecalis (purple bar) and (iii) the positive influence on E.

faecalis by curli-E. coli (Figure 4.3 F, compare blue and green bars of "ompR234csgA" block), which was dampened in the presence of K. pneumoniae (purple bar). The Interestingly, when co-cultured with the curli--E. coli, the growth of K. pneumoniae also dropped to lower than that of its pure culture (Figure 4.4 E, compare the blue bar with red bar for "ompR234csgA"). When grown in co-culture with E. coli which has a higher expression of curli (curli+/-or curli++), however, the growth of K. pneumoniae was not significantly influenced. This trend -that a lack of curli negatively affects K. pneumoniae growth -opens up a speculation that curli production of E. coli may "protect" the growth of K. pneumoniae in co-culture with E. coli.

Taken together, these results suggest that under the nutrient limiting low osmolarity condition, curli production by E. coli may be beneficial to both E. coli and K.

pneumoniae in co-culture, by providing some level of protection from the direct antagonistic influence of each other in co-culture. This relationship in planktonic coculture is interesting to keep in mind, as we will see later that the protection of E. coli brought about by curli's presence can also be observed in biofilm co-cultures of E. coli and K. pneumoniae (Section 4.3.2.2.2).

When it comes to the relationship between E. coli and E. faecalis, the latter showed significantly better growth in co-culture with E. coli in the complete absence of curli (Figure 4.3 F, "ompR234csgA"), while the curli+/-and curli++ status of E. coli did not seem to exhibit any influence. Therefore, it appears that while curli may not have drastic negative influence on the interaction between E. coli and E. faecalis, in some way(s) the lack of curli frees up E. faecalis for enhanced growth in co-culture with E. coli. Unlike the case of E. coli -K. pneumoniae, however, this trend of E. faecalis in planktonic coculture with E. coli was not observed in biofilms. In section 4.3.2.2, we will look at data that suggest instead that curli's presence increases the number of E. faecalis in biofilm co-cultures.

Curli's influence on population dominance status within planktonic cocultures

So far we have discussed the influence of one bacteria species on the growth of another species in planktonic culture, and the impact of E. coli curli production on this influence by comparing between growth in one species' pure culture against the co-cultures.

However, in a bacterial co-culture, the interplay of various factors -such as differences in growth rate, metabolism, physiology as well as the influence from the partner species and the environment [START_REF] Christensen | Metabolic commensalism and competition in a two-species microbial consortium[END_REF][START_REF] Komlos | Interaction of Klebsiella oxytoca and Burkholderia cepacia in dual-species batch cultures and biofilms as a function of growth rate and substrate concentration[END_REF][START_REF] Hansen | Evolution of species interactions in a biofilm community[END_REF][START_REF] Kolenbrander | Oral multispecies biofilm development and the key role of cell-cell distance[END_REF] -may also lead to changes in the majority or minority status of the species involved. While the analysis in the last section (4.3.2.1.1) using absolute cell counts may reveal how the growth of one species is quantitatively influenced by the partner species, it cannot provide us with the gross perspective regarding their relative occupancy in the co-cultures. We would not immediately know if one species has grown into dominance when co-cultured with a second species, or if it has become the minority in co-culture with a third species. For example, the negative influence of curli null mutation on the growth of E. coli and K. pneumoniae that was highlighted in the last section may equally likely present two scenarios: (i) a change in dominance of E. coli in co-culture with K. pneumoniae, or (ii) no effect on their dominance relationship with only proportionate decreases by both species in the total cell number. Therefore, it is necessary to examine the proportion of each population relative to the whole co-culture, for a fuller picture of their inter-relationship.

In Figure 4.5, the population percentage of co-cultured species in planktonic culture is plotted. Several observations can be made: (i) In BHI (37°C), K. pneumoniae grow into dominance when co-cultured with E. coli and is not influenced by the curli-related mutations in E. coli (Figure 4.5 A, B and C, "Ec+Kp"). The dominance relationship between the two species being invariable among different E. coli curli-related mutant is not surprising since the difference in curli production is very minimal among the three E.

coli strains in this condition (refer to Section 4.3.1.2). The domination trend of K.

pneumoniae is also observed in mM63 (30°C), the condition under which curli expression is induced (Figure 4.5 D, E and F, "Ec+Kp"). In fact, it showed greater dominance in this condition compared to the BHI condition (compare "Ec+Kp" of Figure 4.5 A against D, B against E). Interestingly, when curli is not produced (curli--), E. coli grows to a higher proportion in the E. coli-K. pneumoniae co-culture (Figure 4.5 F), i.e. K. pnuemoniae dominance decreases. In planktonic co-culture, dominance of one species over the other has often been attributed as the result of competition between partner species, and could usually be predicted by the growth rate of the various species [START_REF] Komlos | Interaction of Klebsiella oxytoca and Burkholderia cepacia in dual-species batch cultures and biofilms as a function of growth rate and substrate concentration[END_REF]. I checked this explanation with regards to the dominance of K. pneumoniae over E. coli in our system: In BHI (37°C), the generation time (refer that shown in Figure minutes for E. coli G1, G1ompR234 and K. pneumoniae respectively). These values correlate well with our observation of higher dominance of K. pneumoniae under this condition. In the low osmolarity condition of mM63 (30°C), curli expression induced in the curli++ E. coli probably results in higher energy cost to the cell and hence longer generation time. Therefore, when curli synthesis is inhibited (G1ompR234csgA), it is possible that the reduced energy cost increases the growth rate of this E. coli strain, allowing it to compete better against K. pneumoniae, thus altering the degree of dominance. As discussed in the last section (4.3.2.1.1), curli's presence may provide protection to both species from each other's antagonistic effect. Although curli's presence appeared to be protective towards both species, the combined effect of growth rate and antagonism may somehow have rendered K. pneumonia more sensitive to the lack of "protection" by curli.

(ii) When co-cultured with E. faecalis, E. coli G1 becomes the dominant partner in both culture conditions (Figure 4.5 A and D, "Ec+Ef"). However, unlike E. coli -K.

pneumoniae relationship, this dominance does not seem to have originated from their difference in growth rate: The generation time of E. coli and E. faecalis is similar in BHI (37°C) (41.7 ± 1.6 minutes for E. coli and 42.8 ± 0.2 for E. faecalis), whereas in mM63 (30°C), E. coli even has a slightly longer generation time (168.4 ± 0.5 minutes) than that of E. faecalis (152.6 ± 10.3 minutes). In addition, curli expression does not appear to affect their dominance status (compare "Ec+Ef" of Figure 4.5 D, E and F). However, when cultured in BHI (37°C), the ompR234 mutation seems to allow an increase in the proportion of E. faecalis in co-culture with E. coli compared to its wild type background (compare "Ec+Ef" of Figure 4.5 A against B and C), leading to E. faecalis dominance.

This effect is even more striking when all three species are cultured together ("Ec+Kp+Ef" of Figure 4.5 A against B and C). Therefore, factor(s) other than curli expression, which is/are related to the ompR234 genetic background, may be responsible for the dominance of population in BHI. For example, the ompR234 mutation in E. coli may have triggered changes in its expression of signalling molecules that could be sensed by E. faecalis, resulting in its higher competitiveness. In mM63, although the dominance status of E.

coli over E. faecalis remained regardless of its curli expression status ("Ec+Ef" of Figure faecalis in terms of its co-existence with E. coli.

In biofilm

In the previous section we have examined the inter-species interactions in planktonic state, which deals with the average cell density of the bacterial species in suspension in fluids (culture media). In the most natural form of bacterial existence, biofilm, where surface attachment of the bacteria is the distinguishing feature, the interactions can be completely different from that in the planktonic setting. For example, Klayman and colleagues has observed that although E. coli O157:H7 grew 50% faster than P.

aeruginosa PAO1 in batch cultures, P. aeruginosa developed biofilm faster in flow cells with approximately 100 times the biomass of E. coli. They showed that E. coli alone could not attach irreversibly to glass surface to initiate biofilm development on its own, but could form biofilm when P. aeruginosa was introduced. In this dual-species biofilm, attached E. coli cells were in fact associated with P. aeruginosa cells instead of directly with the surface [START_REF] Klayman | Escherichia coli O157:H7 Requires Colonizing Partner to Adhere and Persist in a Capillary Flow Cell[END_REF]. Student's t-Test was performed to determine whether the differences were statistically significant.

1.00E+04

1.00E+05

1.00E+06

A B C D E F G1 ompR234 ompR234csgA G1 ompR234 ompR234csgA G1 ompR234 ompR234csgA G1 ompR234 ompR234csgA G1 ompR234 ompR234csgA G1 ompR234 ompR234csgA
For E. coli -E. faecalis co-culture biofilms, under the BHI (37°C) condition, E. coli strains do not appear to be affected by E. faecalis (Figure 4.5 A, compare blue against green bars within each block). However, the positive effect of the ompR234 mutation in E.

coli on the growth of E. faecalis is hard to miss (compare the blue and green bars of "G1" group against those of "ompR234" and "ompR234csgA" in Figure 4.6 C). This synergistic effect is also exhibited by the ompR234 strain under the mM63 (30°C) condition (Figure 4.6 D and F, blue and green bars of "ompR234"). While the synergistic effect seems to be caused by ompR234 mutation without the involvement of curli in BHI (37°C), the synergistic effect in mM63 (30°C) was solely observed for the curli++ E. coli

G1ompR234 strain (approximately 16-and 14-fold increase for E. coli and E. faecalis populations respectively, Figure 4.6 D and F) and may therefore be curli-mediated.

Consistent to that notion, co-culturing with the curli--mutant E. coli G1ompR234csgA

showed dramatically decreased growth of both E. coli and E. faecalis. This is in contrast to the planktonic co-culture situation whereby growth with curli-E. coli enhanced the growth of E. faecalis (discussed in Section 4.3.2.1.1). By direct visual assessment of biofilms formed on glass slides, we have observed that in mM63 (30°C), E. faecalis is a poor biofilm former (data not shown). Since E. coli G1ompR234 is an excellent biofilm former, it is possible that the synergistic relationship between E. coli G1ompR234 and E.

faecalis in biofilm is due to the biofilm-enhancing role of curli.

Synergistic relationship in dual-species biofilm formation has been reported previously in other experimental models, such as between Listeria monocytogenes and P. putida [START_REF] Hassan | Behavior of Listeria monocytogenes in a Pseudomonas putida biofilm on a condensate-forming surface[END_REF] and between E. coli and P. putida [START_REF] Castonguay | Biofilm formation by Escherichia coli is stimulated by synergistic interactions and co-adhesion mechanisms with adherence-proficient bacteria[END_REF]. The synergistic effect appears to be species-specific in the experiments of Castonguay and coworkers as no such relationship between S. epidermidis and E. coli was observed. They also demonstrated that direct cell-cell contact may be necessary for biofilm stimulation by E. coli [START_REF] Castonguay | Biofilm formation by Escherichia coli is stimulated by synergistic interactions and co-adhesion mechanisms with adherence-proficient bacteria[END_REF]. Therefore, in the case of synergistic relationship between curli-expressing E. coli and E. faecalis in biofilm, curli may not only promote biofilm formation by mediating E. coli attachment to surface, but also by increasing the coaggregation with E. faecalis, bringing more E. faecalis to settle in the growing biofilm.

Population dominance status within biofilm co-cultures

In biofilm, the dominance of K. pneumoniae over E. coli in both growth conditions, which we have already observed in the corresponding planktonic co-cultures, is conserved (Figure 4.7, "Ec+Kp"). However, unlike planktonic co-cultures, in mM63 (30°C), the expression of curli by G1ompR234 in biofilms appears to counteract the dominance effect of K. pneumoniae (compare "Ec+Kp" of Figure 4.7 E against D and F), increasing the population of E. coli from less than 10% for strains with less curli production (Figure 4.7 D for E. coli curli+/-and Figure 4.7 F for curli--mutant) to approximately 30% of the total population (Figure 4.7 E). It could be that curli production confers higher adherence to surface thus offering E. coli the advantage of faster colonisation in competition with K. pneumoniae. It may also be possible that curli production and/or the exhibition of curlin on E. coli cell surface is sensed by K.

pneumoniae, which changes its physiology in response, resulting in an altered population relationship with E. coli.

Relating this back to the previous results, we could see that somehow curli expression always has some influence on E. coli -K. pneumoniae interaction. For example, in planktonic culture, curli expression seems to be important for the growth of E. coli in coculture with K. pneumoniae. Without curli, the antagonising effect of K. pneumoniae is so strong that the growth of E. coli dropped by 1000 fold (discussed in Section 4.3.2.1, Figure 4.4 D, compare blue and red bars of "ompR234csgA"). Surprisingly, while the null mutation of curli structural gene resulted in such drastic decrease in the cell density of E.

coli in co-culture with K. pneumoniae, it increased E. coli's population percentage (Figure 4.5,Section 4.3.2.1.2). This occurs because the cell density of K. pneumoniae also decreases when co-cultured with curli-defective E. coli (Figure 4.4 E "ompR234csgA"). In biofilm co-cultures, we see the positive effect of curli in favour of E.

coli that affected K. pneumoniae dominance (compare "Ec+Kp" in Figure 4.7 E against D and F). One speculation is that the presence of curli plays a signalling role to both E. coli and K. pneumoniae, which affects the growth of both partners. I present this possibility because it has been reported that microcin E492 secreted by K. pneumoniae could assemble into amyloid fibrils (leading to loss of microcin activity) [START_REF] Bieler | Bactericidal activity of both secreted and nonsecreted microcin E492 requires the mannose permease[END_REF], suggesting it may possess mechanisms to sense the presence of this inactive form of microcin E492. Hence although the K. pneumoniae strain used in this work does not secret bacteriocins (Section 4.2), it may still have such sensing mechanisms. Since curli belongs to the amyloid family, K. pneumoniae may be able to sense curli -as something similar to the assembled microcin from its own kind, or alternatively, discern it as foreign signalling molecules. In either case, K. pneumoniae could respond by increasing or decreasing its own growth, with a consequence on the overall population relationship.

Summary

In this Chapter, the relationship between E. coli and its commensal partners K.

pneumoniae and E. faecalis in dual-and multi-species culture were investigated. Their relationship is not based on antiobiotics-mediated growth inhibition (Section 4.2). In view of the altered surface property of curli-producing E. coli (Section 4.3.1), the possible influence of curli on the inter-species interaction was examined in both planktonic (Section 4.3.2.1) and biofilm (Section 4.3.2.2) conditions. Two comparison methods were employed: (i) comparing the cell density of one species in pure culture with that in co-cultures provides information about the influence of the partner species on the growth of the species of interest and (ii) comparing between the relative population proportions of co-cultured species reveals the dominance relationship between partner species. Analysis based on the second method showed a general trend of dominance: K.

pneumoniae dominates over E. coli, while E. coli often exhibits dominance on E. faecalis.

In BHI (37°C) planktonic culture, co-cultured species mostly exhibit antagonistic relationship (Figure 4.3 A, B and C). In the curli permissive condition mM63 (30°C), curli expression appears to be important for the growth of both partners in E. coli -K.

pneumoniae co-culture. With the limited information on their interactions, we hypothesize that curli may protect E. coli from the strong antagonistic effect of K.

pneumoniae, while curli fibre and/or curli subunit proteins may serve as signalling molecules for K. pneumoniae. On the other hand, when comparing the relative population proportion of E. coli -K. pneumoniae co-culture, the absence of curli seems to increase E.

coli's proportion (or decrease K. pneumoniae's proportion), implying that the loss of curlin protein synthesis may have conferred lower energy cost, leading to higher fitness of E. coli, and higher competitiveness against K. pneumoniae.

In biofilm co-culture, we have two main findings: (i) curli production of E. coli seems to counteract the dominance of K. pneumoniae over E. coli, which may be the result of increased fitness brought about by the presence of curli. (ii) curli production may promote the synergistic relationship between E. coli and E. faecalis in biofilm, possibly by increasing the coaggregation of E. coli with E. faecalis as E. coli attachment to surface is facilitated by curli.

Due to the differing nature of cell-cell interactions in planktonic suspensions (random collision of cells in moving fluids) and biofilms (cells are adherent and may attain closeproximity to each other), the surface properties of cells may lead to different interactions between the three species in the two modes of cultures. Based on my work, it can be seen that the presence of curli on E. coli indeed has varying influence on E. coli interaction with K. pneumoniae and E. faecalis in planktonic and biofilm modes. In addition, although it may be at first assumed that because curli is produced by E. coli, it will serve to benefit E. coli at the expense of the other two interacting species, my data clearly showed that this is not always true. Depending on the situation, curli may in fact assist the interacting species, and even alter the dominance status in favour of the non-E. coli species at times.

Multi-species interactions in co-culture involve complex factors ranging from the intrinsic physiological property of each partner species, the communications between them to their distinct response to the external environment. Our current understanding of inter-species interactions is still very limited and the study of their population provides just one level of information. Further investigation on the influence of one species on the gene expression of its partner species may help us to gain some insights on this, because the manifestation of interactions ultimately boils down to the altered gene expression.

Chapter 5 Conclusions and perspectives

Commensal bacteria have lived in harmony with us since our birth, contributing to a mutualistic relationship. However, much focus has been given to the interaction between pathogen and the host historically. It is not until recently that the importance of commensal-host interactions has come under the spotlight, pushing the scientific community towards the commencement of the Human Microbiome Project [START_REF] Qin | A human gut microbial gene catalogue established by metagenomic sequencing[END_REF]. Yet another important actor in these interactions, the local environment of the commensals, has not been well addressed. In the intestine, commensal bacteria are in direct contact with the mucus layer, which are complex mucin glycoproteins secreted by the epithelial cells [START_REF] Corfield | Mucins and mucosal protection in the gastrointestinal tract: new prospects for mucins in the pathology of gastrointestinal disease[END_REF]. Mucins have been described to impact biofilm development of many bacterial species but in different ways [START_REF] Moniaux | Structural organization and classification of the human mucin genes[END_REF][START_REF] Corfield | Mucins in the gastrointestinal tract in health and disease[END_REF][START_REF] Vieten | Impaired mucosal regeneration in neonatal necrotising enterocolitis[END_REF]; (Lievin-Le [START_REF] Moal | The front line of enteric host defense against unwelcome intrusion of harmful microorganisms: mucins, antimicrobial peptides, and microbiota[END_REF], and have been suggested to mediate biofilm formation by E. coli [START_REF] Zhang | Role of bile mucin in bacterial adherence to biliary stents[END_REF]; [START_REF] Bollinger | Human secretory immunoglobulin A may contribute to biofilm formation in the gut[END_REF]; [START_REF] Bollinger | Secretory IgA and mucin-mediated biofilm formation by environmental strains of Escherichia coli: role of type 1 pili[END_REF]. However, the interaction between mucin and the inhabiting bacteria is poorly investigated. In addition, the interaction between the commensal microbiota species themselves has not been well addressed due to its complex and highly diverse nature. Current methodologies have mostly been developed to meet the demands of pure culture analysis, and may not be adequate to cover the demand of studies involving more than one species.

These gaps in knowledge served as a motivation for me to target for my investigation, the relation of E. coli -one of the most studied bacterial species and an intesintal commensal -with the above-mentioned intestinal components: mucin, and commensal partners (K.

pneumoniae and E. faecalis). The common thread that runs through my study is curli: the amyloid surface structure expressed by certain strains of E. coli, and known to have adhesive, virulence-related and protective roles in E. coli's life. My project has been approached from three angles: the first undertaking aimed to develop and optimise new experimental models and analytical tools that are better suited for the complexity of our system. Once the models were established and the tools validated, I moved on to the second aspect of the project, where the effect of mucin on E. coli biofilm formation was investigated. The last part of this project concentrated on the inter-species interactions between the commensal species E. coli, K. pneumoniae and E. faecalis through the study of their population relationship with regards to curli's expression status.

Through these studies I was able to derive these accomplishments:

1) The development and validation of experimental models and tools that permits (i)

tracking of E. coli within the multispecies context that constitute the human gut commensals, (ii) surveying the expression of genes of interest in E. coli and (iii) reliable co-culture and viability analysis of the three species under study.

2) The demonstration that low concentrations of mucin promotes biofilm formation by inducing expressions of surface adhesion structures in E. coli K-12 strains such as curli in the MG1655 background, and type 1 pili in the W3110 background.

3) The revelation that, due to the differing nature of cell-cell interactions in planktonic suspensions (random collision of cells in moving fluids) and biofilms (cells are adherent and may attain close-proximity to each other), the surface properties of cells may lead to different interactions between the three species in the two modes of cultures. I have shown in particular that, depending on the co-culture situation, E. coli curli may assist the interacting species, and even alter the dominance status in favour of the non-E. coli species at times. However, as with any scientific endeavour, not everything would go according to original plans, and one scientific conclusion would only lead to the realisation of how much more we still do not know. Hence, in this chapter, I would like to raise some of the unresolved questions I have encountered and discuss the perspectives for future exploration.

Fluorescent Proteins

One of the greatest disappointments in my project has been the situation that the dual fluorescence system developed by the Singapore laboratory [START_REF] Miao | Dual fluorescence system for flow cytometric analysis of Escherichia coli transcriptional response in multispecies context[END_REF] could not be fully exploited due to the slow maturation and possibly toxicity of the red fluorescent proteins in the E. coli background I have used.

In the system where E. coli is whole cell-labelled with GFP, the coupled gene transcriptional reporter using promoter-RFP fusion did not accurately report certain genes' expression and its reporter function seemed to be promoter-and strain-specific.

With the promoter-GFP[LVA] fusion, which faithfully reported the gene expression status, we went on to construct a RFP whole cell tagged E. coli. Several attempts have been made using three different RFPs, but unfortunately did not succeed due to the instability of red fluorescence proteins that may be caused by their toxicity.

The necessity of using a fluorescent protein well distinguishable from the green FP pushes us to continue to search for the good candidate. RFP has often been reported, either in actual publications [START_REF] Strack | A noncytotoxic DsRed variant for whole-cell labeling[END_REF] or through anecdotal complaints, to be toxic to cells. Although a number of groups have published successful work using RFP for whole cell-labelling or promoter-fusion, we are now fairly convinced that we are not alone in facing problems with RFP. We will continue to hunt for a better version of RFP such as monomer or dimer, which could have lower toxicity to E. coli cells. It seems that because there is a greater demand for the use of FPs in eukaryotic cell biology, improvements of RFPs are mostly based on eukaryotic cell systems and claims made for their applications are not always applicable to bacterial systems. For example, DsRed-Max-N1 [START_REF] Strack | A noncytotoxic DsRed variant for whole-cell labeling[END_REF] and mCherry [START_REF] Shaner | A guide to choosing fluorescent proteins[END_REF] that we have tried were both chosen based on positive testimonials in eukaryotic cells, but did not work out in our strains. Even within bacterial species, one RFP may work well in one species e.g. DsRed-

Express was reliable in P. putida [START_REF] Nancharaiah | Dual labeling of Pseudomonas putida with fluorescent proteins for in situ monitoring of conjugal transfer of the TOL plasmid[END_REF] but completely lost its expression within one day in E. coli (Miao H, personal communication). There seems to be no rational way to predict the success of RFP in a particular expression system. We will have to empirically try the new claimants of "better RFP" such as the rapidly maturing far-red derivative of DsRed-express2 [START_REF] Strack | A rapidly maturing far-red derivative of DsRed-Express2 for whole-cell labeling[END_REF] in our own system. In the mean time, cyan, yellow or orange FP [START_REF] Shaner | Improving the photostability of bright monomeric orange and red fluorescent proteins[END_REF] could be another option to couple to our GFP, aided by the finer-tuned wavelength discriminatory functions of newer models of CLSM and FACS.

Mucin-induced curli induction -mechanism, and inter-relation

with other regulatory factors?

Mucin up-regulates csgBA expression in E. coli K-12 MG1655

Although it was very encouraging to have found that mucin promotes biofilm formation in E. coli MG1655 through up-regulation of csgBA expression, I am most curious about the pathway by which mucin exerts its positive effect on csgBA expression. Since OmpR is the main positive regulator of curli production and that ompR234 mutant has greatly increased csgBA expression, an ompR mutant was used in my study. The overall adherence of G1ompR decreased, but the induction effect remained. This suggests that other factor(s) beside curli are able to promote biofilm formation in a mucin-inducible manner in the ompR mutant of the MG1655 strain. The csgBA operon is regulated by many known factors with CsgD being the principle positive regulator, which also regulates the production of another cell surface structure, cellulose. Since curli expression is under extremely complex regulation, we do not know for now the pathway through which mucin up-regulates csgBA expression, but could only speculate which factors were possibly involved. Preliminary experiments using a mutant of the main csgBA regulator, csgD have been done to observe the effect of the mutation on biofilm induction by mucin in 24-well plate, but gave contradictory results. More experiments using the two different csgD mutants (refer to Section 6.1) available are needed to assess csgD's involvement in mucin's up-regulation of csgBA expression. CsgD involvement in the response to mucin is however very likely for the following reasons. I observed in my G1 and G1ompR234 incubated in BHI at 30°C that there is pellicle formation at the air-liquid interface, difficult dissolution of biofilm-bound crystal violet and the decreased biofilm level at high mucin concentration, implying possible existence of cellulose in biofilm. In particular, decreased adherence was observed in the presence of high mucin concentration (e.g., 0.75% and 2% in Figure 3.4). Landini's group has reported that in E.

Testing the CV quantification procedure on MG1655 and cellulose overproducing or deficient derivatives would help to solve the problem.

Mucin's effect on other adherence structures

Mucin's biofilm induction effect was also observed in other E. coli K-12 strains such as W3110 and MC4100. However, the induction effect is not necessarily a result of higher curli production. In W3110, type 1 pili seemed to be responsible for mucin's biofilm induction effect, while curli production greatly augments the strain's biofilm formation ability. Further investigations are needed to evaluate the interplay between different cell surface adhesion structures in the presence of mucin. In both strains however, high concentrations of mucin (0.75 -2%) resulted in drastically decreased biofilm formation.

In the intestine where mucin is abundant, the expression of cell surface structures is likely to be influenced. The concentration gradient of mucin from the mucus surface to the epithelial lining of the intestine may thus result in decreased attachment and biofilm forming ability of cells, preventing them from penetrating too deep into the mucus layer in order to protect the underlying epithelial cells.

It should be noted however, we were only focusing on the possible influence of mucin on the regulation of surface adhesion structures. Mucin could possibly influence the expression of many other genes with various functions that may affect the bacteria's physiology as well as their interaction with commensal and pathogen species. More effective ways to gain some insights in mucin's influence may include the microarray method, or the use of FACS to sort out the mucin up-and down-regulated genes from a fluorescence-labelled gene promoter library [START_REF] Zaslaver | A comprehensive library of fluorescent transcriptional reporters for Escherichia coli[END_REF]. However, although FACS is a powerful tool, it needs to be optimised for our bacterial sorting. The presence of mucin, which is insoluble with large molecule size, would add on the complexity and difficulty to FACS analysis and sorting. To differentiate mucin from bacterial cells carrying the fluorescence-labelled gene promoter library, the cells would need to be tagged by fluorescence of another colour that can be effectively distinguished by FACS.

Although I have shown mucin's effect on E. coli biofilm formation, how mucin influences the physiology of K. pneumoniae and E. faecalis is still unknown and certainly deserves attention. In the natural habitat where mucin is present, the interaction between these commensal species may be different compared to in vitro conditions. Therefore, the population relationship in mucin's presence can be examined to shed some light on the possible scenarios in the natural environment of their growth.

Partnership of E. coli with K. pneumoniae and E. faecalis

Microbiota functions are the product of interactions within communities of bacteria, the resultant balance of various synergism and antagonism occuring concurrently between multiple species. The production of curli which changes the surface property of E. coli cells to the point that it can influence interactions with abiotic surfaces as well as with mammalian cells, would probably also exert an effect on their commensal partners.

While a general trend of dominance can be observed that K. pneumoniae dominates over E. coli, which often itself exhibits dominance on E. faecalis, the results showed interspecies interactions are complex. In planktonic culture, curli expression appears to be important for the growth of both partners in the E. coli -K. pneumoniae co-culture. The antagonistic effect observed by K. pneumoniae on E. coli, prompted me to speculate that curli expression on the surface of E. coli cells may serve as a protection against K.

pneumoniae, for which the curli fibre and/or curli subunit protein may be sensed by K.

pneumoniae as signalling molecules. On the other hand, loss of curli seems to increase E.

coli's proportion (or decrease K. pneumoniae's proportion), implying that the loss of curlin protein synthesis conferring lower energy cost may lead to higher fitness of E. coli, thus higher competitiveness against K. pneumoniae. To further investigate curli's role in K. pneumoniae growth, purified curli fibres or curlin proteins could be added to K.

pneumoniae culture to see if a slowed growth of K. pneumoniae could be observed.

Conditioning medium may be used to dissect the role of secreted factors from the role of direct physical contact involving curli. In biofilm co-culture, two main findings must be underlined: (i) curli production may promote the synergistic relationship between E. coli and E. faecalis in biofilm, possibly by mediating E. coli attachment to surface, as well as increasing the coaggregation with E. faecalis. (ii) Curli production of E. coli seems to counteract the dominance of K. pneumoniae over E. coli, which may be the result of increased fitness brought about by the presence of curli.

The different population relationship between commensal species obtained between planktonic and biofilm culture confirmed that cells grown in biofilm are in different physiological state from that in planktonic culture. However, in this project, only the population relationship aspect of the co-cultures was examined. The biofilm formation by dual-or multi-species involves the attachment of cells to surface, with one species under the influence of its partner species, and the further inter-species interactions in the biofilm maturation process all adds on to the complexity of multi-species biofilm analysis. In addition to population studies, the spatial distribution of different species in biofilm may be of particular interest. For example, E. faecalis being a poor biofilm former alone showed greatly increased cell number when co-cultured with curli-expressing E. coli.

Would curli on E. coli cell surface, besides their higher attachment to surface, attract E. faecalis cells and coaggregate with them, dragging them to settle in biofilm? To deepen our understanding of this phenomenon, confocal investigations will help to determine if E.

faecalis cells are attached to E. coli cells in initial biofilm formation and when biofilm forms, whether E. coli would be the one in contact with the surface. Fluorescent-tagging of E. faecalis with a different colored FP will certainly facilitate such analysis.Another approach is to study the gene expression profile of E. coli under the influence of the partner species. The effective way would still be to use FACS to screen for genes that have their expression level changed in the presence of the partner species from a fluorescence-tagged gene library. This approach can also be used to examine mucin's effect on E. coli gene expression, as well as the influence of partner species on E. coli gene expression in the presence of mucin, in a farther future.

Finally, we did not have time to consider in depth the influence of a third member in three-species co-cultures in this study. After establishing more information from dualspecies co-culture, we may be able to put together such information and obtain better insights. As it is now, all we know is that in M63 medium, there are conditions under which the negative or positive influence on one species may be dampened or eliminated by the presence of a third species. It will certainly be of interest to address the mechanism of such neutralizing effects. One approach will be to supplement a dual-species coculture with the conditioned medium of a third member and compare the effects to the corresponding three-species co-culture. The Singapore laboratory has also performed a preliminary profiling of the medium composition of the various co-cultures in comparision to pure cultures using tandem liquid-chromatography-mass spectrometry (LC-MS) technique. We can be certain that much development of data mining and analysis will be required in order to exploit these undertaking and unravel the levels of complexity involved.

In conclusion, I would like to express my amazement at how every component in a tiny bacterium is tightly regulated and highly cooperating with each other, that understanding a fraction of it is not sufficient to grasp the whole picture. Not only that, the interaction with biological partners have aspects that are surprising and beyond what we can fathom.

Such intricacy is involved in order for a "simple" bacterium to survive, to adapt to various conditions, to proliferate, to conserve the species. Standing in awe of the vast mysteries of life, I dedicate this thesis to the delicately composed symphony of nature.

Chapter 6 Materials and Methods

OD 600 -cell density relationship

Colonies of E. coli MG1655, E. coli G1 and their curli-related mutants as well as K.

pneumoniae and E. faecalis cultures were inoculated into BHI and mM63 media and cultured at 37°C and 30°C, respectively. All bacteria were cultured in triplicate. After culturing for 14 hours (in BHI at 37°C) or 16 hours (in mM63 at 30°C), the OD 600 of each culture was recorded and the culture was serially diluted before plating on agar. CFU enumeration determined the cell densities (CFU/ml) of the cultures which were correlated to their corresponding measured OD 600 values. The calculated cell density of each species under the particular growth condition for OD 600 = 1 was then determined (Table 2).

Starting inocula were thereafter adjusted according to these values.

Biofilm single cultures

24-well polystyrene microtiter plate

Single-species E. coli overnight cultures were inoculated in BHI at 37°C or mM63 at 30°C (depending on the conditions of the biofilm culture) for 14 or 16 hours respectively and their cell densities checked by OD 600 measurement. They were then inoculated in 2ml medium each well at the concentration of 6x10 6 cells/ml, according to the OD 600 -cell density relationship (refer to CellPath) were placed at the bottom of each Petri dish. After 24, 48 or 72 hours static incubation at 30°C, biofilm formed on the entire surface of the cover slips.

The interface system

Overnight bacterial culture in BHI at 37°C was used as seeding culture to inoculate 30ml BHI supplemented with 2% mucin (Sigma Aldrich) at 6x10 6 cells/ml concentration, according to the OD 600 -cell density relationship (refer to Table 2 in Section 2.2.3). After 24 hours static incubation at 37°C, biofilms formed at the air-medium interface on glass coverslips (24x50 mm, CellPath) slanted at a slight angle in a glass tank (Figure 2.4).

Co-cultures

For population relationship experiments involving co-cultures, individual strains were first shaken in either BHI at 37°C for 14 hours or mM63 at 30°C for 16 hours (except Enterococcus faecalis in mM63 for 24 hours due to its slow growth in minimal medium)

and their cell densities checked by measuring OD 600 . These cultures were then mixed at 1:1 ratio to achieve a final density of 6x10 6 cells/ml for each species, according to the OD 600 -cell density relationship (refer to Table 2 in Section 2.2.3). The planktonic cocultures were shaken at 250 rpm while the biofilm co-cultures incubated statically in either BHI (37°C) or mM63 (30°C) for 24 hours. Only the saturated and interface systems were used for biofilm co-culture experiments.

Growth monitoring and analysis

Planktonic cultures

Growth of planktonic cultures was monitored via OD 600 on a BioSpec-mini spectrophotometer (Shimadzu). To obtain growth curves, the OD 600 of 14 hours (in BHI at 37°C) or 16 hours (for mM63 at 30°C) cultures were first determined and subsequently diluted to an OD 600 of 0.1 in 100 ml of corresponding fresh medium. Cultures were shaken at 30 or 37°C at 250 rpm. OD 600 was measured every 30 min for the first 4 h and hourly after that. Mean data of the triplicate cultures in exponential phase were used to

Colony Forming Unit (CFU) enumeration

Viable cell numbers were determined by CFU counts. A series of 10-fold dilutions were performed by transferring 200 μl cultures to 1800 μl sterile ddH 2 O in 5 ml snap-cap tubes and colony forming units per ml (CFU/ml) were determined by plating 100 µl of suitably diluted cultures on LB (for E. coli single-species cultures) or UTI (for K. pneumoniae and E. faecalis single species cultures as well as co-cultures) agar plate in triplicates. Cultures with E. faecalis were also spread onto deMan, Rogosa and Sharpe (MRS) agar plates for selective enumeration of E. faecalis. LB and UTI agar plates were routinely incubated for 12~16 h at 37°C, while MRS agar plates were incubated for 24 h under the same conditions. Only plates with the number of colonies in the range of 30~300 were counted.

Biofilms

The 24-well plate

To obtain the test strain's adherence property, the 2ml supernatant was carefully collected without disturbing the biofilm and referred to as "planktonic portion" The well was then gently washed with 1ml mM63 and pooled to the planktonic portion. The biofilm at the bottom of the well was recovered with 1ml of mM63 by scraping with the blue pipette in 3 directions and referred to as "biofilm portion". For OD 600 measurement, both portions were vortexed for 3 min before measurement. For direct enumeration by CFU count in biofilm cultures containing mucin, both the planktonic and biofilm portions were vigorously vortexed for 20 pulses, followed by sonication at 50% power on an ultrasonic bath (Tru-Sweep TM Ultrasonic Cleaners 575, Crest Ultrasonics) for 2 cycles of 15 min, and vigorously vortexted for 20 pulses after each round of sonication. Then both portions were diluted and plated on LB agar plate. Total bacteria population is the sum of the planktonic and biofilm portion, while adherence is the percentage of biofilm in the total population.

The saturated system

For enumeration of the biofilm by CFU count, the glass cover slips were taken from the Petri dish with the aid of a sterile needle and a pair of autoclaved forceps and place in a small Petri dish. The edge of the cover slip was gently dabbed the on tissue paper to remove excess culture before placed in the small Petri dish, with the biofilm growing side facing upward. Two cover slips were place side by side in the small Petri dish and scraped twice by a blue pipette tip with 500µl of sterile water. The total 1ml scraped biofilm was then collected in a 1.5ml Eppendorf tube and sonicated as described in Section 6.4.3.1 before serial-diluted into the appropriate dilution and plated on agar plate.

The interface system

For enumeration of the biofilm by CFU count, the cover slip was withdrawn from the tank and the planktonic cells below the interface biofilm were washed by dipping in sterile water. The interface biofilm on both sides of the cover slip was scraped twice by a blue pipette tip with 500µl of sterile water and the total 1ml scraped biofilm was then collected in a 1.5ml Eppendorf tube and sonicated as described in Section 6.4.3.1 before serial-diluted into the appropriate dilution and plated on agar plate.

Crystal violet staining of biofilm formed on 24-well polystyrene plate

For biofilm formation estimation by crystal violet staining, the 2ml supernatant was gently removed without disturbing the biofilm and discarded. Then each well was washed carefully with 1ml of medium and discarded before fixing the bacteria at 80°C for 1 and a half hours. After the plate was cooled down, each well was stained by 1% crystal violet (CV) for approximately 1 min before being gently washed 3 times to remove excess crystal violet. The plate was then air dried and scanned.

6.4.5 Biofilm observation using CLSM 6.4.5.1 Biofilm formed in the saturated system For biofilm observation using CLSM, the cover slip was taken out from the Petri dish by lifting up one side of the cover slip with a needle and then holding it with a pair of 6. To prepare a phage stock containing the gene fragment to transduce, the bacterial strain carrying this gene was shaken at 37ºC, 250 rpm overnight, and 100μl of which was then inoculated in a glass tube containing 5ml LB supplemented with 50μl of 20% MgSO 4 and 100μl 0.2M CaCl 2 . After shaking at 37ºC for 2 and half hours, 500μl phage P1 that does not contain the selection antibiotic resistance of the gene of interest was added to the culture and incubated statically at room temperature (RT) for 30 mins. The phage-bacterial cell mixture was then shaken at 37ºC, 250 rpm until lysis of bacterial cells was complete (if lysis does not occur after 4 hours of incubation, it is not likely to occur, thus the whole process needed to be repeated). To kill the remaining bacterial cells, 5 drops of chloroform was added to the mixture and vortexed for 30 seconds before left at RT for 30 min and on ice for 10 min. After that, the tube was vortexed for 30 seconds again and left on ice for 10 min before centrifuged at 15ºC 6000 rpm for 10 min. The supernatant containing the phage P1 was carefully collected without touching the pellet and conserved in a sterile glass tube at 4ºC. The stock of phage was prepared and ready to be used.

Transduction

For transduction of the recipient bacterial cells by phage P1, the recipient bacterial strain was incubated in 5ml LB at 37ºC, 250 rpm overnight. 1.5ml of the overnight culture was collected in a sterile Eppendorf tube and centrifuges to remove the supernatant. The pellet was resuspended in 0.9ml transduction solution and 0.1ml of phage stock. Negative controls were set up to check possible contamination of phage stock (0.9ml LB + 0.1ml phage stock) and the recipient strain (1.5ml pelleted overnight culture + 0.9ml transduction solution +0.1ml LB). The mixture of bacterial cells and phage were incubated statically at 37 ºC for precisely 20 min before centrifuged at 10000g for 1min.

Suicide-plasmid based chromosomal insertion for construction of E. coli strain R1 and R2

Insertion of the red fluorescence cassette into the E. coli chromosome (refer to Section 6.7.3) using R6K suicide-plasmid based chromosomal insertion was performed by two rounds of recombination events, as shown in Figure 6.1.

First recombination event

The chloramphenicol resistance gene on pDM4-derived suicide plasmids was utilized for the first round of screening for plasmid integration. After electroporation (refer to Section 6.6.6.2), both static (incubate without shaking in 1ml SOC at 37°C overnight before plating on LA Cm) and patch (drop 5 µl of the newly electroporated cells in 1ml SOC on LA to incubate overnight, then take a scoop to spread on LA Cm) methods are used to obtain colonies containing cells which have undergone first recombination. After 24h incubation, many (more than 700) colonies grew for MG1655 1 st recombinants but none for MG1655ompR234. PCR was performed using PR304 and PR307 to verify the presence of mAsRed2 insert.

Primer sequences are:

PR304: 5' -GCGGATAACAATTTCACACACTGCAG -3' PR307: 5' -CTGAGATGAGTTTTTGTTCTAGAAAGCT -3'

Second recombination event

The sacB gene on the suicide plasmid served as a counter selective marker for the screening of those strains that had recombined a second time, and hence likely to have excised the plasmid backbone out of the chromosome, leaving only P A1/04/03 -mAsRed2 cassette remaining on the chromosome. With the induction by sucrose, the sacB gene encodes an enzyme (levansucrase) that is lethal to Gram negative bacteria in the presence of 5% sucrose in the agar medium (Gay et al., 1985). Only strains that did not carry the sacB gene could survive on 5% sucrose plate. Due to the delay of this lethal effect, strains with sacB gene could, in reality, form colonies on the sucrose-containing agar. However, they would lyse upon longer incubation, and this would be manifested by "sticky" colonies when picked by toothpicks. To screen for second recombination events, the strains screened as described above were pooled together, of which 200µl was used to inoculate to 20ml low salt LB for overnight shaking. The next morning 5% sucrose was added, and shaken for the day at 37°C before being spread on LB 5% sucrose plates to screen for "sticky" colonies. were picked with inoculation loop and suspended in 100 µl sterile water, followed by 3 minutes' vortex. Then 5 µl of the solution was taken as template DNA.

Standard thermocycling reactions involved the initial denaturation at 94ºC for 5 min followed by 30 cycles of 94°C for 30 s (denaturation), Annealing temperatures (T a ) °C for 1min (annealing), and 72°C for 1 min (extension). The reaction was completed by a further 7 min at 70°C. Annealing temperatures was set at 5ºC lower than the higher T m of the two primers. T m refers to the melting temperature of the primer, at which 50% of DNA duplexes become single-stranded. All PCR experiments included a sample without DNA template which served as a negative control to check for the presence of contamination in any of the reagents.

Restriction endonuclease digestion

Restriction endonuclease digestions were usually carried out in 20μl or 100μl reaction volume for verification or DNA extraction purpose respectively. In 20μl reaction volume, 5U of enzyme for 500ng DNA was used and incubated at 37ºC for 2~5 h whereas 20U of enzyme for 2μg DNA was incubated at 37ºC for 12~16 h for 100μl reaction volume.

Digestion with two enzymes was carried out simultaneously in a suitable buffer according to the manufacturer's instruction. All PCR-amplified fragments for cloning was digested with the corresponding endonuclease for which the sites were designed into the primers, unless otherwise stated. Both vectors and inserts for cloning were purified with Nucleospin ® ExtractII Kit (Macherey-Nagel) to remove the restriction enzymes and buffers prior to ligation.

6.6.5 DNA ligation 6.6.5.1 Conventional ligation DNA vectors with compatible ends were prepared by restriction enzyme digestion and followed by treatment with alkaline phosphatase, unless otherwise stated. Vector DNA (about 200 ng) and insert DNA (in 3:1 molar ratio to vector) were ligated using the Rapid DNA Ligation Kit (Roche). A reaction without insert DNA was included as negative control.

In-Fusion TM 2.0 PCR Cloning Kit

Ligation using In-Fusion TM 2.0 PCR Cloning Kit (Clontech) was also used. The DNA insert was PCR-amplified with the primers containing at least 15 bases of homology with the sequence that flank the site of insertion in the linearized vector. The PCR-amplified DNA insert was then fused with the linearized vector via single stranded regions generated by In-Fusion enzyme. PCR fragment and vector at a molar ratio of 2:1 were mixed in deionized H 2 O in a total volume of 10μl and added into the tube provided, mixed by pipetting up and down. The tube was incubated at 37ºC for 15 min, then at 50ºC for 15 min, and then transfered on ice. Mastercycler ® ep gradient thermal cycler (Eppendorf) was used for the required incubations.

6.6.6 E. coli competent cell preparation and transformation 6.6.6.1 Chemically competent cells Fresh colonies were prepared by streaking out cells from the -80C frozen stock onto LB agar plate and incubating them overnight at 37°C. On the second day, a single colony of E. coli strain was inoculated into 3 ml LB medium and grown overnight at 37°C. On the third day, 2 ml of the overnight E. coli culture was transferred into flask containing 100ml LB medium and incubated at 250 rpm at 37°C until the OD 600 reached 0.4~0.6.

The flask was put on ice for 10 min to cool the cells. The cells were then transferred to 250 ml centrifuge bottles and centrifuged at 4ºC for 10 min at 6000rpm with Avanti TM J-25 centrifuge (Beckman Coulter TM ). The pellet was resuspended in 50 ml ice-cold 100mM CaCl 2 and incubated on ice for 20 min. After centrifugation as before, the cells were resuspended in 10 ml 100 mM CaCl 2 with 15% glycerol. The cells were then aliquoted by 400μl into sterile Eppendorf tubes and stored at -80°C.

For Transformation of DNA into chemically competent cells, 200 μl competent cells were thawed quickly and stored on ice immediately after thawing. After the addition of DNA (200 ng~300 ng), the mixture was mixed by tapping gently and then incubating on ice for 30 min. A negative control was performed in parallel without addition of DNA to detect any possible contamination of the competent cells. Then the mixture was heat shocked at 42ºC for 90 seconds and immediately cooled on ice for 5 min. Then 800μl LB medium was added into the tube, followed by incubation at 37ºC for 45 min with shaking.

The cells were then spread onto agar plates containing the appropriate antibiotics. To ensure that single colonies can be obtained with samples of unknown transformation efficiency, 100 μl cells were spread on one agar plate and the remaining 900 μl spun down and resuspended in 100 μl LB and spread on another agar plate.

Electrocompetent cells

Similar to that of the preparation of chemically competent cells, cells of the strain of interest were cultured in 100 ml LB medium at 250 rpm at 37ºC until the OD 600 reached 0.7~0.9. The cells were harvested by centrifugation at 4355g for 10 min at 4°C. The pellet was rinsed in sterile ice-cold ddH 2 O twice (100 ml and 50 ml respectively) and finally resuspended in 5 ml sterile ice-cold 10% glycerol solution. The cells were then aliquoted into 100 μl/tube and stored at -80°C.

For each electroporation, electrocompetent cells were thawed and 45 μl of which were transferred to ice-cold 0.2 cm electropration cuvette (Bio-Rad) and approximately 1 μg DNA (maximum volume 10 μl) was added. Another 45μl of cells were used as negative control with no DNA added. The cuvettes were gently tapped to mix and to ensure that there were no air bubbles. Gene Pulser (Bio-Rad) apparatus was set at 25 μF, 200 ohm and a voltage of 2.5 kV. Time constant would be displayed as >4.5 for high electroporation efficiency. Immediately after electroporation, 1ml of SOC medium was added into the cuvette and transferred to a sterile Eppendorf tube to be shaken at 37ºC for 1 hour. The mixture was spread onto plates containing the appropriate antibiotics. To ensure that single colonies can be obtained for samples with unknown transformation efficiency, 100 μl cells were spread on one agar plate and the rest 900 μl spun down and resuspended in 100 μl LB and spread on another agar plate.

DNA extraction

For small scale purification of plasmid DNA, 3 ml (for high copy plasmids) or 5ml (for low copy plasmids) of LB medium with appropriate antibiotics was inoculated with a single colony of the required strain and incubated at 37ºC, for 12~16 h, with shaking at 250 rpm. A Nucleospin Plasmid Kit (Macherey-Nagel) was used to extract DNA. For large scale purification of plasmid DNA, a QIAfilter Midi (in France) or NucleoBond PC100 Kit (in Singapore) was used for high copy plasmids and QIAfilter Maxi Kits (in France) or NucleoBond PC500 (in Singapore) was used for low copy plasmids. All kits were used according to the manufacturer's instructions. Previously, the high copy plasmid pBluescript carrying mAsRed2 gene under the promoter P A1/04/03 (pCCS326) was constructed in the Singapore laboratory. To construct R1 following the same principle as that of G1 (refer to section 2.4.2.2), the P A1/04/03 -mAsRed2 cassette was digested from pCCS326 and inserted into the backbone containing the regions homologous to the chromosome of E. coli MG1655 (pCCS124) for recombination (Figure 6.1), giving rise to pCCS343. The whole Region A-P A1/04/03 -mAsRed2-Region B cassette needed to be then digested from pCCS343 and inserted into the R6K-based suicide plasmid pDM4 with the restriction digestion sites SpeI and XhoI.

However, there was an unexpected additional XhoI site (in green in Figure 6.1) just upstream of the P A1/04/03 -mAsRed2 cassette. Therefore, this additional XhoI was eliminated by partially digesting pCCS343 with XhoI restriction enzyme and bluntending the digested site with Klenow Fragment (Fermentas) followed by ligation. The Region A-P A1/04/03 -mAsRed2-Region B cassette was then digested from the resulting plasmid pCCS443 and ligated to the pDM4 backbone pre-digested with SpeI and XhoI.

The purified ligation mixture was then transformed into the permissive strain E. coli S17 λ-pir. The highest transformation yield was given by vector:insert ratio of 1:5 with excess ligation buffer. The plasmid was then extracted, verified for insertion by restriction digestion and purified before being electroporated into E. coli MG1655 and MG1655ompR234.

Using DsRed-Max for construction of E. coli R2

The general procedure for construction of E. coli strain R2 using P A1/04/03 -DsRed-Max was the same as that with P A1/04/03 -mAsRed2 except for the first step of plasmid construction. DsRed-Max was PCR amplified from the plasmid pDsRed-Max-N1 [START_REF] Strack | A noncytotoxic DsRed variant for whole-cell labeling[END_REF] to replace the mAsRed2 sequence in the plasmid pCCS443 (Figure 6.2). This was done with In-Fusion® Dry-Down PCR Cloning Kits (BD Clontech), which can clone a PCR fragment into a linearized vector with the aid of the In-Fusion Enzyme that can fuse PCR-generated sequences to linearized vectors efficiently and precisely by

Fluorescence activated cell sorter

Analysis of cell size, surface properties and fluorescence at the single cell level was achieved by using the FACSAria TM Cell Sorter (Becton Dickinson), which is equipped with air-cooled argon lasers at 488 nm. The detection of green fluorescence GFPmut3* was through the FITC filter (530/30 nm) and for the red fluorescence proteins, the PE filter (585/42 nm). Since FACSAria TM was designed for the use of mammalian cells, we have optimised its settings to suit our analysis of bacterial cells. The flow rate for the FACSAria TM was kept at the minimum value of 1. The optimised settings are as follows: threshold voltage: SSC 200; PMT voltages: FSC 300, SSC 300, FITC 500, PE 650.

To achieve analysis and sorting resolution approximating 1 event = 1 cell, the samples were diluted to a cell density of 10 7 cells/ml (see section 4.4.1), and kept on ice prior to analysis. 10,000 events of each sample were analysed in duplicates.

The gates separating green fluorescent or red fluorescent cells from non-green or non-red population (G+/G-or R+/R-) were defined using control strains that are green negative (E. coli MG1655) or red negative (E. coli MG1655 and G1) in fluorescence. Fluorescence levels higher than those of the negative controls were defined as green positive (G+) or red positive (R+).

Figure 1. 1 2 FigureFigure

 12 Figure 1.1 Schematic representation of biofilm development stages and the extracellular structures involved in each stage…………………………....2 Figure 1.2 Model of the Rcs phosphorelay in Enterobacteriaceae…………………...3 Figure 1.3 Model describing the coordination of the phase-variable Fim and Ag43 phenotypes via OxyR-relayed thiol-disulfide signal transduction……… 5 Figure 1.4 A schematic representation of type 1 pili………………………………... 6 Figure 1.5 A schematic representation of the two curli gene operons……………….7 Figure 1.6 The secretion and assembly machinery for curli formation in E. coli….…7 Figure 1.7 Regulatory network of curli genes via DGC and PDE proteins………...

Figure 1. 8 Figure 2

 82 Figure 1.8 Various environment encountered by bacteria such as E. coli………….15 Figure 2.1 Growth curves of the various bacteria………………………………..… Figure 2.2 The Kadouri system……………………………………………………..

Figure 2 . 3

 23 Figure 2.3 Side view of the MBEC™ High-throughput (HTP) Assay…………….

Figure 2 . 4

 24 Figure 2.4 The biofilm systems used in this study………………………………….

Figure 2 . 5

 25 Figure 2.5 Confocal microscopic images of biofilms formed by E. coli selfexpressing green fluorescent protein (GFP)…………………………….

Figure 2

 2 Figure 2.6 UTI agar allows distinction between different species………………….

Figure 2

 2 Figure 2.7 CLSM photos of 24 hour biofilm……………………………………….

Figure 2 . 8 Figure 2

 282 Figure 2.8 Schematic representation of chromosomal recombination of P A1/04/03 -gfpmut3* in E. coli MG1655…………………………………………… Figure 2.9 Green fluorescence of E. coli G1………………………………………..

Figure 2 .

 2 Figure 2.10 A schematic representation of A. E. coli strain G1 and B. E. coli strain G1ompR234……………………………………………………………..

Figure 2

 2 Figure 2.11 E. coli biofilms and their corresponding variables……………………..

Figure 2 .Figure 2 .

 22 Figure 2.12 Flocculation test of curli-related mutant strains………………………… Figure 2.13 Kinetics of gene promoter-gfp fusions in MG1655 ompR234…………..

Figure 2 .Figure 2 .Figure 2 .Figure 2 .Figure 2 .Figure 2 .FigureFigure

 222222 Figure 2.14 Expression of promoter-AsRed2 fusions in MG1655ompR234………… Figure 2.15 Expression of promoters in 24h biofilm by MG1655 ompR234 in mM63 …………………………………………………………………...

Figure 1 . 1

 11 Figure 1.1 Schematic representation of biofilm development stages and the extracellular structures involved in each stage. Biofilm development can be divided into five steps: (i) initial reversible attachment of cells to surface; (ii) production of exopolysaccharide (EPS) leading to irreversible attachment; (iii) early development of biofilm structure; (iv) maturation of biofilm and (v) biofilm dispersal giving rise to planktonic cells to colonise new sites. The main extracellular structures known to be

  Figure modified from Biofilm hypertextbook, Montana State University Center for Biofilm Engineering.

Figure 1 . 2

 12 Figure 1.2 Model of the Rcs phosphorelay in Enterobacteriaceae. The phosphorylation of RcsB involves a complex phosphorelay mediated by the RcsC and RcsD proteins. Phospho-RcsB binds to DNA and regulates the expression of many genes. The Rcs phosphorelay is activated by a variety of signals and the perception of many of these signals is mediated by the RcsF lipoprotein. The cytoplasmic membrane protein

Figure 1 . 3

 13 Figure 1.3 Model describing the coordination of the phase-variable Fim and Ag43 phenotypes via OxyR-relayed thiol-disulfide signal transduction. In the fimbrial phase on state, disulfide bond formation of fimbrial subunit proteins takes place in the periplasm catalysed by DsbA, -B and -C. The thiol-disulfide status of glutathione is monitored by OxyR, which under these conditions is driven to the reduced state and acts as an active repressor of the agn43/flu gene. Other auxiliary enzymes are indicated. In the fimbrial phase off state, OxyR can exist as both a reduced and oxidized form, with the result that repression of Ag43 synthesis is relieved. Figure from Schembri & Klemm, 2001.

Figure 1

 1 Figure 1.4 A schematic representation of type 1 pili. The FimH adhesin is shown in green while the chaperone FimC attached to the last subunit to be incorporated into the pilus in yellow. Numbers indicate the number of copies of each subunit in the pilus. The usher dimers are indicated in purple and blue. E, extracellular space; P, periplasm. Figure modified from Waksman et al. 2009.

Figure 1 .

 1 Figure 1.5 A schematic representation of the two curli gene operons. The csg operon is organised in two clusters, csgBA and csgDEFG. CsgD is a transcriptional activator of the csgBAC operon.

Figure 1 . 6

 16 Figure 1.6 The secretion and assembly machinery for curli formation in E. coli. Products of both csgBA and csgDEFG operons are required for curli biosynthesis. CsgD activates of the csgBA transcription. CsgB, CsgA, CsgE, CsgF and CsgG are transported across the inner membrane (IM) with the aid of their SEC signal. CsgG, CsgE and CsgF interacts at the outer membrane (OM), and CsgA and CsgB are secreted across the outer membrane in a CsgG-dependent manner. CsgB interacts with the outer membrane and presents an amyloid-like template to soluble CsgA (red triangles). CsgA adopts the amyloid conformation (red ovals) and becomes anchored to the cell surface and fold onto unpolymerized CsgA monomers. Figure from Epstein & Chapman, 2008.

Figure 1 . 7

 17 Figure 1.7 Regulatory network of curli genes via DGC (open ellipses) and PDE (dark squares) proteins. In E. coli, YdaM and YciR represent the c-di-GMP control

  The dispersal step of biofilm development (Figure1.1, stage 5) has recently gained increasing attention because of its important role in bacteria transmissions including those from environment to human hosts, in horizontal and vertical transmission, as well as the exacerbation and spread of infection within the host (Hall-[START_REF] Hall-Stoodley | Biofilm formation and dispersal and the transmission of human pathogens[END_REF][START_REF] Kaplan | Biofilm dispersal: mechanisms, clinical implications, and potential therapeutic uses[END_REF]. In this "final" stage of biofilm development, single cells are released from the biofilm, and temporarily exist as planktonic cells, where flagella are again employed. The bacteria are then ready to migrate to new colonization sites to start the biofilm life cycle all over again. This phenomenon, as the sequential production of fimbriae and exopolysaccharides (stages 2 and 3), illustrates the regulatory dynamics occurring through biofilm development and points at the necessity to develop appropriate tools allowing dynamic gene expression survey.

Figure 1 . 8

 18 Figure 1.8 Various environment encountered by bacteria such as E. coli. The potentially pathogenic E. coli are ingested by cows and other ruminants (1). They colonise the intestinal tract but not always cause disease. The bacteria are excreted as feces and contaminate the environment including drinking water and streams[START_REF] Danese | Biofilm formation as a developmental process[END_REF]. There can also be contamination of various foods such as fruits, vegetables and raw milk (3).

Figure 2 . 2

 22 Figure 2.2 The Kadouri system.Fresh culture medium is pumped from the medium reservoir onto a biofilm grown in the bottom of a well in a 6-well plate through silicone tubing (1) and a 20G needle inserted on one side of the plate lid[START_REF] Danese | Biofilm formation as a developmental process[END_REF]. Planktonic bacteria and spent medium are removed through another needle placed on the opposite side of the well (3) and pumped to waste receptacle (4).

Figure 2 . 3

 23 Figure 2.3 Side view of the MBEC™ High-throughput (HTP) Assay. Biofilms form on the polystyrene pegs of the MBEC™ device when planktonic bacteria adsorb to the surface. 96 identical plastic pegs are attached to the peg lid, which fits into a standard 96well microtiter. Figure from the website of Innovotech Inc. (http://www.innovotech.ca/).

Figure 2 . 4

 24 Figure 2.4 The biofilm systems used in this study. A. The saturated system. Biofilm is grown on cover slips in a Petri-dish containing media. The cover slips are observed directly under confocal microscope. B. The interface system. Biofilm forms at the airmedium interface on coverslips vertically placed in the biofilm tank. The coverslip is withdrawn from the tank and the attached cells at the bottom of the coverslip are removed by dipping the coverslip several times in water. It is then mounted carefully on a flow cell

Figure 2 . 5

 25 Figure 2.5 Confocal microscopic images of biofilms formed by E. coli self-expressing green fluorescent protein (GFP). A. Biofilm in saturated model and B. Biofilm in interface model. In saturated model, biofilm forms a "carpet" covering the entire surface of the coverslip. The "patches" reflect the heterogeneity on Z axis, but not on the XY plane. In interface model, the air-liquid transition creates a gradient for nutrient and oxygen, leading to spatial heterogeneity.

Figure 2 .

 2 Figure 2.6 UTI agar allows distinction between different species. The three species used in this project have different colony morphology on UTI agar plate. E. coli colonies are large and purple coloured while those of E. faecalis are small and dark blue with white tips on top. K. pneumoniae have large, dark green colonies.

  microscopy is a powerful tool for visualizing fluorescent specimens. It was invented by Marvin Minsky for the purpose of improving the traditional widefield fluorescence microscopes. The most important feature of confocal microscopy is its acquisition of in-focus images from selected depths, thus allowing reconstruction of three-dimensional structures from the obtained images. There are several types of confocal microscope, the most common of which is the laser scanning confocal microscope. It captures images by scanning the specimen with a focused beam of laser and collecting the emitted fluorescence signals with a photodetector. The optical resolution and contrast of images is increased by employing point illumination and a

Figure 2 .

 2 Figure 2.7 CLSM photos of 24 hour biofilm. A. E. coli MG1655ompR234 stained with fluorescent dye SYTO61. B. Whole cell GFP-tagged MG1655ompR234 (named G1ompR234). The two "visualisation" methods give similar biofilm morphology.

Figure 2 .

 2 7 A shows the SYTO 61-stained biofilm of E. coli MG1655ompR234. Although the staining concentration and duration have been optimized, several technical challenges have restrained us from using the staining method for later experiments.

Figure 2 . 8

 28 Figure 2.8 Schematic representation of chromosomal recombination of P A1/04/03 -gfpmut3* in E. coli MG1655. RBS: Ribosomal Binding Site; T 0 and T 1 : transcriptional terminators; cat: chloramphenicol resistance gene; sacB: gene encoding the enzyme levansucrase, which confers lethality in the presence of sucrose. Dotted lines represent homologous recombination (double crossover).

  was introduced into E. coli MG1655 (Figure 2.8), for constitutive GFP expression. The R6K-based suicide plasmid requires the pir gene encoded protein π (Germino & Bastia, 1983) to replicate. In the non-permissive E. coli

Figure 2 . 9

 29 Figure 2.9 Green fluorescence of E. coli G1. (A) Fluorescence profiles of E. coli G1 and E. coli MG1655 with 4 rounds of sub-culturing. (B) Fluorescence profiles of E. coli G1 and E. coli MG1655 cultures continuously shaken for 4 days. All data from are representative of three independent experiments.

Figure 2 .

 2 Figure 2.10 A schematic representation of A. E. coli strain G1 and B. E. coli strain G1ompR234. The P A1/04/03 -gfpmut3* cassette was inserted to the chromosome by double recombination of a R6K-based suicide plasmid carrying the cassette. The ompR234 malT54::Tn10 mutation was introduced to the chromosome by phage P1 transduction.

Figure 2 .

 2 Figure 2.11 E. coli biofilms and their corresponding variables. CLSM images of biofilms of A. E. coli G1 (30°C), B. E. coli G1 ompR234 (30°C) and C. E. coli G1 ompR234 (37°C)

Figure 2 .

 2 7 A and B). While giving similar results, the GFP-tagging method is more advantageous, since it does not involve additional staining step and the consistent fluorescent signals allow inter-experiment comparison.

Figure 2 .

 2 Figure 2.12 Flocculation test of curli-related mutant strains. A. G1 (curli +/-), G1ompR234 (curli ++), G1ompR (curli -), G1ompR234csgA (curli --) are shaken at 60 rpm overnight in M63+LB at 30°C. Curli production leads to the clumping of cells, giving the flocculation phenotype (clusters and clear medium). B. Cell aggregates produced by curli-expression strain G1ompR234. When curli is produced, cells form aggregates that can mediate biofilm formation. These aggregates can be dispersed by vortexing.

  addition to analysing bacterial cells and biofilm structures (made possible by wholecell fluorescence tagging), I intended to have additional tools that could glean information at another level -the transcriptional level. Transcriptional gene fusions with fluorescent reporter allow in situ detection of promoter activity in living cells. Moreover, transcriptional activity can be assayed in individual cell by using flow cytometry. I therefore constructed a set of transcriptional fusion either with GFP or RFP reporter genes. Three genes were chosen as representative of active growth (fis gene), fitness indicator (mazEF gene) or biofilm development (csgB gene). To compare their transcriptional activity in planktonic and biofilm cells, they were assayed in E. coli strain with the MG1655ompR234 genetic background. With this fluorescent transcriptional

Figure 2 .

 2 Figure 2.13 Kinetics of gene promoter-gfp fusions in MG1655 ompR234. A. maximal expression of P fis -gfp in rich medium is at exponential growth phase. B. P mazEF -gfp expression is low in cells entering stationary phase in minimal medium. C. P csgBA -gfp is expressed at early stationary phase in minimal medium. D. P csgBA -gfp expression is very low in minimal medium at 37°C compared to that at 30°C. E. P csgBA -gfp expression is much lower in rich medium (BHI) than in minimal medium (mM63). In A. B. and C., yellow curves show growth by OD 600 measurement whereas green curves indicate fluorescence/OD 600 . In D. and E., fluorescence/OD 600 of P csgBA -gfp in different conditions is shown. Two fluorometers were used to measure the fluorescence level, thus the scale in different graphs can be different (see Section 6.8.1).

Figure 2 .

 2 Figure 2.14 Expression of promoter-AsRed2 fusions in MG1655ompR234. Genepromoter-AsRed2 activity was measured using both FACS (24 hours culture, represented in histogram) and fluorometer (kinetics). P fis -AsRed2 and P mazEF -AsRed2 were cultured in BHI (37°C) for optimal expression, whereas P csgBA -AsRed2 in both mM63 at 30°C (D) and BHI at 37°C (E). Expression is very low for P fis -AsRed2 (A & B) and P csgBA -AsRed2 (C), whose thermo-and osmo-regulation are not reflected (D & E). The expression of P mazEF -AsRed2, on the other hand, was detectable by both FACS (F) and fluorometer (G). It also showed high expression in cells grown in rich medium (BHI) in exponential phase (G) as reported[START_REF] Marianovsky | The regulation of the Escherichia coli mazEF promoter involves an unusual alternating palindrome[END_REF]. In FACS histograms, black curves represent the promoter-less negative control, whereas red curves indicate P lac -AsRed2 positive control. Blue curves show the fluorescence level of the promoters being tested. Two fluorometers were used to measure the fluorescence level, thus the scale in different graphs can be different (see Section 6.8.1).

Figure 2 .

 2 Figure 2.15 Expression of promoters in 24h biofilm by MG1655ompR234 in mM63. A. P fis -gfp B. P csgBA -gfp C. P mazEF -gfp and D. P mazEF -AsRed2. Having their highest expression is in the exponential phase, P fis -gfp and P mazEF -gfp activities are relatively low in biofilm. On the contrary, P csgBA -gfp exhibits high and heterogeneous activity in biofilm. P mazEF -AsRed2 shows homogeneous activity, but the biofilm formed is very thin, possibly due to the toxicity of RFP. Fluorescence images are superimposed with transmitted light images.

Figure 2 .

 2 Figure 2.16 Differential expression of genes in biofilm corresponding to different growth conditions. E. coli MG1655ompR234 carrying P fis -gfp (A&B), P mazEF -gfp (C&D) or P csgBA -gfp (E -I) were incubated in saturated biofilm system for 24, 48 and 72hours (only for P csgBA -gfp). Biofilms formed without renewal of medium (upper row) are compared to those with renewal (lower row). Fluorescence images are superposed with transmitted light images.

Figure 2 .

 2 Figure 2.17 Schematic representation of the dual fluorescence system. A. Whole cell GFP-tagged E. coli (G1) carrying RFP-labelled promoter. B. Whole cell RFP-tagged E. coli (R1) carrying GRP-labelled promoter.

Figure 2 .

 2 Figure 2.18 Schematic representation of the molecular cloning for RFP-tagged E. coli (R1) construction. The P A1/04/03 -mAsRed2 cassette is excised from the high copy plasmid and inserted to the suicide plasmid pDM4 by restriction digestion and ligation.The resulting suicide plasmid carrying the P A1/04/03 -mAsRed2 cassette is then allowed to recombine with the chromosome of E. coli MG1655.

Figure 2 .

 2 Figure 2.19 Schematic representation of the RFP instability. Refer to text for details

Figure 3 . 1

 31 Figure 3.1 Biofilm formation on polystyrene 24-well plate visualised by crystal violet staining. E. coli G1 and G1ompR234 were incubated in BHI (37°C) with 0-4% mucin and mM63 (30°C) with 0-2% mucin. Biofilms were stained with crystal violet. Red rectangles indicate mucin concentrations at which enhanced biofilm formation can be observed.

Figure 3 . 2

 32 Figure 3.2 Biofilm formation on glass cover slip observed under CLSM. Biofilms of E. coli G1 (left panel) and G1ompR234 (right panel) were grown in the saturated system with 0% (A & D), 0.25% (B & E) and 2% (C & F) mucin.Confocal images were superimposed with fluorescence and transmitted light photos. Analysis of biomass using COMSTAT was done for both G1 (G) and G1ompR234 (H).

Figure 3 . 3

 33 Figure 3.3 Masking effect of mucin on bacteria OD 600 measurement. The absorbance (OD 600 ) of medium containing various concentrations of mucin was measured (orange curve). A constant amount of bacterial culture was then added into the medium containing the same series of concentrations of mucin and the final OD 600 measured again (blue curve). The black horizontal line indicates the actual bacteria OD 600 in the absence of mucin. The absorbance due to mucin masks that of the bacteria.

Figure 3 . 4

 34 Figure 3.4 Analysis of biofilm formation on polystyrene 24-well plate by CFU and CV staining. Viable count was performed for both the planktonic and biofilm portion of E. coli MG1655 (A) and MG1655ompR234 (C). Only adherence (curves) but not total biomass (bars) was affected by the presence of mucin. CV staining of biofilm (B & D) therefore reflects the adherence of bacteria.

Figure 3 . 5

 35 Figure 3.5 Curli producing status influences E. coli adherence without affecting growth. The growth of E. coli MG1655ompR234csgA (curli--), MG1655 (curli+/-) andMG1655ompR234 (curli++) on 24-well plate in the presence of 0-2% mucin was calculated based on CFU (A) and showed no significant difference. However, the curli++ strain showed highly induced biofilm formation by low concentrations of mucin (B) whereas the curli+/-and curli--strains did not.

Figure 3 . 6

 36 Figure 3.6 Adherence of E. coli MG1655ompR on 24-well plate. The biofilm formed by MG1655ompR at 0 -2% mucin was stained with CV. The ompR null mutation decreased the overall adherence but did not eliminate the induction effect of mucin.

mutant

  MG1655ompR234csgA was subjected to the same adherence test. Results show that while the growth of the MG1655 (curli+/-), MG1655ompR234 (curli++) and MG1655ompR234csgA (curli--) mutants are not affected by the presence of mucin (Figure 3.5 A), both strains deficient in curli expression (MG1655 and MG1655ompR234csgA) have lost the biofilm induction effect by mucin (Figure 3.5 B).

Figure 3 . 7

 37 Figure 3.7 Mucin's effect on P csgBA -gfp activity in E. coli MG1655ompR234 colonies on agar plates. 2% mucin in BHI, LB and mM63 agar up-regulates P csgBA -gfp activity at30°C, but very low activity was observed at 37°C. This up-regulation can be observed as increased fluorescence intensity by direct visual assessment (A) and quantification of fluorescence/OD 600 (B) using fluorometer (refer to Section 6.8.1). Due to the poor growth of colonies on mM63 agar plate, 0.25% and 0.5% mucin were only tested on BHI and LB plate for induction effect of P csgBA -gfp activity and both showed up-regulation of P csgBAgfp activity (C). All measurements were based on at least three repeated experiments.

Figure 3 . 8

 38 Figure 3.8 The expression of csgBA is up-regulated by low concentrations of mucin.

Figure 3 . 9

 39 Figure 3.9 Heterogeneity of gene expression in a population. Ovals represent cells and the colour of and numbers on the ovals show their gene expression (fluorescence) level.

Figure 3 .

 3 Figure 3.10 P csgBA -gfp activity in biofilm by confocal microscopy. The expression of csgBA in biofilm was observed under CLSM using P csgBA -gfp fusion. csgBA expression in the MG1655 (A, B and C) and MG1655ompR234 (D, E and F) background were tested in the presence of 0, 0.25 and 0.5% mucin. Photos shown are superimposed with transmitted light images.

Figure 3 .

 3 Figure 3.11 Heterogeneity in biofilm morphology and csgBA expression pattern. Confocal microscopic observations of E. coli W3110 carrying P csgBA -gfp biofilms in the presence of mucin revealed heterogeneous biofilm morphology and csgBA expression patterns (A -D). Two fields on the same coverslip are shown for each concentration of mucin (A&B: 0.25% of mucin, C&D: 0.5% of mucin). Homogeneous structure can be observed in sample containing mucin alone (without bacteria) (E). Confocal photos shown are superposed with transmitted light images.

  various biofilm morphology and P csgBA -gfp activity pattern can be observed in different observation fields (Figure 3.11): some with highly developed 3D structure and high csgBA-expressing population (Figure 3.11 A & D), some with flat biofilm and high csgBA-expressing population (Figure 3.11 B), while examples also exist when the biofilm structure is well developed but csgBA expression is sparse (Figure 3.11 C). Therefore, for my experiments, at least 6 observation fields have been taken for each sample, and the ones with the biofilm morphology and promoter activity pattern most representative of the majority of the 6 have been chosen for presentation or discussion. If the parallel G1

Figure 3 .

 3 Figure 3.12 Specific activity in an activity-heterogeneous population. In biofilm, the fluorescence intensity measurement only deals with fluorescent cells. So the total fluorescence intensity (summation of fluorescent intensity in the whole cluster of cells) and fluorescent biomass (number of fluorescent cells) may not reflect the state of gene expression (promoter activity) as well as the specific activity (total intensity divided by the number of green cells only) does. A. An activity-heterogeneous population with various promoter activity levels. B. An activity-heterogeneous population with the same composition as A, but doubles its total population. Although the total intensity doubles, the fluorescent biomass, proportion of fluorescent population and specific activity remain the same. C & D. An increase in the total intensity may be able to reflect the increased overall promoter activity but fails to reveal the how active the promoter-active cells are, due to heterogeneous gene expression of the total population.

Figure 3 .

 3 Figure 3.13 The specific activity of P csgBA -gfp in the MG1655 (left) and MG1655ompR234 (right) background. The specific activity of P csgBA -gfp in biofilm with 0, 0.25 and 0.5% mucin was calculated with the aid of softwares ImageJ and COMSTAT. 0.25% mucin up-regulates the expression of csgBA in MG1655ompR234 but has no significant effect on MG1655.

Figure 3 .

 3 Figure 3.13 shows the calculated specific activity of P csgBA -gfp in MG1655 and MG1655ompR234 biofilms. As expected, the specific activity, indicating the expression level in single cells, is higher in MG1655ompR234 compared to MG1655 without mucin, because the ompR234 mutation increases the expression level of csgBA in single cells. No induction of csgBA expression by mucin is observed in MG1655, hinting that mucin's induction effect may not be through elevating csgBA gene expression level in single cells.

Figure 3 .

 3 Figure 3.14 P csgBA -gfp activity in biofilm of W3110 and W3110ompR234 in the presence of various concentrations of mucin. Confocal microscopic photos of W3110 (A, B and C) and W3110ompR234 (D, E and F) are shown as superposed fluorescence and transmitted light images. Mucin concentrations of 0% (A & D), 0.25% (B & E) and 0.5% (C & F) were tested. COMSTAT analysis of fluorescent biomass was done for W3110 (G), W3110ompR234 (H), MG1655 (I) and MG1655ompR234 (J). The W3110 strain and its ompR234 mutants showed much higher fluorescent biomass than their MG1655 counterparts.

Figure 3 .

 3 Figure 3.15 The effect of Ag43 on biofilm formation. G1ompR234 and W3110ompR234 as well as their agn43 mutants were tested for biofilm formation ability on polystyrene 24-well plate in the presence of 0 -2% mucin. The agn43 null mutation appeared to increase the overall biofilm formation ability in both G1ompR234 and W3110ompR234 strains while conserving the biofilm induction effect of mucin.

Figure 3 .

 3 Figure 3.16 The interplay between type 1 pili and curli on biofilm formation. The effect of fimA (coding for type 1 pili major subunit) null mutation of W3110 and W3110ompR234and W3110ompR234 were tested for biofilm formation ability on polystyrene 24-well plate in the presence of 0-2% mucin by cristal violet staining. The deletion of fimA seems to abolish the biofilm formation ability of W3110. However, although the ompR234 mutation increases the overall biofilm formation ability of W3110, it does not seem responsible for the biofilm induction effect of mucin, because the deletion of curli structural subunit encoding gene csgA did not remove the induction effect of mucin. On the other hand, deletion of fimA in the ompR234 mutant of W3110 abolished the induction effect of mucin while maintaining a relatively high biofilm formation. These results suggest that type 1 pili was involved in the mucin-induced biofilm formation in W3110, while curli production enhances the overall biofilm formation ability. This is different from MG1655 where curli is responsible for both the enhanced biofilm forming ability and mucin-induced biofilm formation. Deletion of fimA neither significantly affected the biofilm formation nor impairs the biofilm induction effect of mucin.

Figure 4 . 1

 41 Figure 4.1 Zeta potentials of E. coli and K. pneumoniae (Kp) and E. faecalis (Ef).

Figure 4 . 2

 42 Figure 4.2 The FSC (forward scatter) and SSC (side scatter) profile of E. coli strains varying in curli expression level. E. coli strains were cultured overnight in mM63 at 30°C (left column) or BHI at 37°C (right column). In curli permissive condition (mM63 at 30°C), curli

  cell attachment to surface in a M63-based environment may be through this property.

Figure 4 . 3

 43 Figure 4.3 The FSC (forward scatter) and SSC (side scatter) profile of E. coli G1ompR234 along the growth. The profile of the curli++ E. coli strains was monitored at 3, 6, 9 and 24 hours of growth in both mM63 (30°C) and BHI (37°C). While little changes in FACS profiles was observed along the growth in curli repressing condition (right column, BHI at 37°C), development of the "tail" can be seen along the growth in mM63 at 30°C (left column).

2

 2 The population relationship of E. coli with K. pneumoniae and E. faecalisIn the study of population relationship between E. coli and its commensal partner K.

Figure 4 . 4

 44 Figure 4.4 The influence of partner species on E. coli, K. pneumoniae and E. faecalis in planktonic co-cultures. The influence was investigated in both BHI (37°C) (first row) and curli permissive condition mM63 (30°C) (second row). Absolute CFU/ml values were plotted in order to compare the growth of one species in pure culture (blue bars) with that in co-culture (other colored bars). Curli expression-related mutants of E. coli were compared: G1ompR234 curli++ (ompR234) and G1ompR234csgA curli--(ompR234csgA), along with the wild type strain G1. The boxes indicate the species for which the cell density is presented in the plot. At least three independent experiments were performed. Data shown are mean ± SD of triplicate

Figure 4 . 5

 45 Figure 4.5 The population percentage of E. coli (Ec), K. pneumoniae (Kp) and E. faecalis (Ef) when co-cultured in planktonic condition. Data from Figure 4.4 are presented as population percentages here. The upper row shows their population relationship in BHI (37°C) and the lower in mM63 (30°C). The boxes in the middle indicate the genotype of E. coli and their curli expression status in mM63 (30°C). The

  4.5 D, E and F), the degree of E. coli dominance appears to decrease as curli expression increases (from Fig 4.4 F -> D -> E). Hence curli seems to have positive effect on E.

Figure 4 . 6

 46 Figure 4.6 The influence of partner species on E. coli, K. pneumoniae, and E. faecalis in biofilm co-cultures. The influence was investigated in both BHI (37°C) (first row) and curli permissive condition mM63 (30°C) (second row). In BHI (37°C) biofilms are formed at the air-medium interface with 2% mucin supplemented in the medium while in mM63 (30°C) the biofilms are totally immersed. Absolute CFU/ml values were plotted in order to compare the growth of one species in pure culture (blue bars) with that in co-culture (other colored bars).Curli expression-related mutants of E. coli were compared: G1ompR234 curli++ (ompR234) and G1ompR234csgA curli--(ompR234csgA), along with the wild type strain G1. The boxes indicate the species for which the cell density is presented in the plot. At least three independent experiments were performed. Data shown are mean ± SD of triplicate samples.

Figure 4 . 7

 47 Figure 4.7 The population percentage of E. coli (Ec), K. pneumoniae (Kp) and E. faecalis (Ef) when co-cultured in biofilm. Data from Figure 4.6 are presented as population percentage here. The upper row shows their population relationship in BHI (37°C) and the lower in mM63 (30°C). The boxes in the middle indicate the genotype of E. coli and their curli expression status in mM63 (30°C). The broken lines separate

E

  . coli interacts considerably differently with E. faecalis in biofilm co-cultures compared to planktonic ones. The dominance of E. coli over E. faecalis observed in mM63 (30°C) in the planktonic condition (Figure4.5 D, E, F, "Ec+Ef") is replaced by almost equal growth of both partners in biofilms, regardless of the E. coli strains' curli-expression status (Figure4.7 D, E, F "Ec+Ef"). However, at the cell number level, we could see that curli expression in fact resulted in increased biofilm cell number in both partners (Figure4.5 D and F, "ompR234", blue and green bars) while deletion of curli led to decreased biofilm of both species (Figure4.6 D and F, "ompR234csgA", blue and green bars). Both changes led to the similar net result of dominance patterns in co-cultures(Figure 4.7 D, E and F). The equal growth of E. coli and E. faecalis in biofilm in mM63 (30°C) (Figure4.6 D, E, F, "Ec+Ef"), as contrasted to the highly shifted equilibrium in BHI (37°C) (Figure4.7 A, B, C, "Ec+Ef") shows that population relationship of commensal species can shift greatly based on changes in environmental conditions. Hence we can perceive that dietary changes of hosts will impact the microbiota equilibrium of intestinal commensals.

From

  calculate generation time (G) using the formula: G = t / [3.3 x lg(b/B)]. This is derived from: (i): G (generation time) = t/n; (ii): b = B x 2 n ; where t: time interval in minutes; b: OD 600 of culture at the end of the time interval; B: OD 600 of culture at the start of the time interval; n: number of generations (cell population doubling) during the time interval. The time interval is taken to be the linear range of the growth curve (early exponential to late exponential phases) ) G = t/n therefore G = t / [3.3 x lg(b/B)].

  stock preparation in liquid culture Bacterial phage P1 vir was used for transduction in this project. The E. coli strains constructed by transduction are listed in Table

  Amplification of DNA fragments was carried out using PCR. PCR primers were designed with the aid of Oligo Calc, an online oligonucleotide properties calculator to maintain a value of %GC between 40 -60 and to ensure the absence of significant secondary structures. PCR was routinely performed in a 50 μl reaction volume. A typical PCR reaction contained less than 5 ng/μl template of chromosomal DNA, 200 μM of each oligonucleotide primer, 200μM of each dNTP, 1.5 mM MgCl 2 , 1x buffer (provided in 10x concentration by the manufacturers) and 1~1.5U DNA polymerase. Taq DNA polymerase (Fermentas) was used for colony PCR screening. For colony PCR, colonies

a

  Promoter fusion refers to promoters of interest, fused with fluorescence protein gene of either GFP[LVA] (gfp) or AsRed2. b Pr refers to primers used to PCR-ampilfy each promoter of interest. The numbering follows the French laboratory primer collection number. c Limit refers to limits of promoters cloned upstream of AsRed2 gene as defined by the primers. The numbers shown are with respect to the start codon of each gene. d Ref refers to the references that documented the transcriptional start site of each promoter.

Figure 6 . 2

 62 Figure 6.2 Construction of R2 using DsRed-Max. See text for detailed descriptions.

  mAsRed2 for construction of E. coli R1

  et E. faecalis. Les implications basées sur ces données ont été examinées. messenger RNA

	ml	milliliter
	MOPS	3-(N-Morpholino)propanesulfonic acid
	MRS	deMan, Rogosa and Sharpe
	ng	nanogram
	nt	nucleotide(s)
	OD 600	optical density at 600 nm
	PCR	polymerase chain reaction
	QS	quorum sensing
	RBS	ribosomal binding site
	rcf	relative centrifugal force
	rpm	revolutions per minute
	rRNA	ribosomal RNA
	RT	room temperature
	s	second
	SD	standard deviation
	Sp r	spectinomycin resistance
	T	thymine
	Tc r	tetracycline resistance
	UTI agar	HiCrome UTI agar plate
	v/v	volume per volume
	w/v	weight per volume
	μg	microgram
	μl	microliter
	min	minute
		v vi

Table of Contents

 of

	Abstract…………………………………………………………………………………………….i xi

Table E

 E

	Table	Growth and flocculation property of various strains used in this study. 20
	Table	The CFU/ml values of cultures at OD 600 =1…………………………… 25
	Table	Green fluorescence level of GFP-tagged E. coli and its curli-related
		mutants measured by FACS……………………………………………. 33
	Table	Comparison between the interface system and saturated system. ……... 42
	Table	Concentrations of mucin that gave the maximum biofilm formation (the
		"optimal" concentration) in different conditions……………………….. 46

. coli strains constructed by transduction in this work..……………... 108 Table Primers used for construction of promoter-gfp and promoter-AsRed2 fusions.………………………………………………………………… 116

Table 1

 1

	). The

Table

Figure 2.1 Growth curves of the various bacteria.

	A.
	B.
	100
	10
	1
	0	6	12	18	24
	0.1				Kp B37 Kp M30	35.3 ± 0.1 104.0 ± 1.3
					Ef B37	42..8 ± 0.2
					Ef M30	152.6 ± 10.3
	0.01
					E. coli G1, E. coli G1ompR234 (A),
	K. pneumoniae (Kp) and E. faecalis (Ef) (B) cultured under the various conditions (M63
	minimal medium at 30°C (M30) and BHI medium at 37°C (B37) with OD measurements
	taken at intervals between 0 -24h. Data shown are the mean ± SD of 3 or more
	independent experiments. Generation times (see Materials and Methods section 6.4.1 for
	formula) are shown on the right.			. Hence both
		temperatures, 37°C and 30°C, would be assessed in this study based on the specific
		questions posed for each experiment. For general reference, the growth curves of the 4
		primary strains (E. coli G1, E. coli G1ompR234, K. pneumoniae and E. faecalis) under

Generation time (min) OD at 600 nm Hours

	100
	OD at 600 nm	1 10
		0	6	12	18	24	Generation time
				Hours			(min)
		0.1				G1 B37	41.7 ± 1.6
						G1 M30	168.4 ± 0.5
						G1 ompR234 B37	40.1 ± 0.2
						G1 ompR234 M30	177.5 ± 1.4
	0.01

strain CFU/ml at OD 600 =1

		BHI (37°C)	mM63 (30°C)
	E. coli MG1655	4.92 × 10 8	7.17 × 10 8
	E. coli G1	5.39 × 10 8	1.01 × 10 9
	E. coli MG1655ompR234	4.53 × 10 8	7.85 × 10 8
	E. coli G1ompR234	5.52 × 10 8	8.20 × 10 8
	E. coli G1ompR234 csgA	7.61 × 10 8	6.94 × 10 8
	E. coli G1ompR234	1.57 × 10 8	4.53 × 10 8
	K. pneumoniae	1.28 × 10 8	1.06 × 10 9
	E. faecalis	1.17 × 10 8	9.05 × 10 8

Table 2 . The CFU/ml values of cultures at OD 600 =1.

 2 Different bacterial species, and even mutants of the same species have different CFU/ml values at OD 600 =1, that is, OD 600 to CFU conversion. Growth at different conditions results in different OD 600 to CFU conversion too.

Table 3 . Green fluorescence level of

 3

		Curli profile %G+ exponentional	%G+ stationary
	G1	curli +/-	99.60 ± 0.50	98.70 ± 0.28
	G1ompR234	curli ++	98.10 ± 0.82	98.65 ± 1.17
	G1ompR	curli -	99.30 ± 0.14	97.78 ± 2.13
	G1ompR234csgA	curli --	99.55 ± 0.19	99.00 ± 0.50

GFP-tagged E. coli and its curli-related mutants measured by FACS. "%G+" indicates the percentage of cells falling within the Green positive (G+) gate from the total of 10 000 events analysed. Cells in both exponential and stationary phase were sampled. Data are expressed as mean ± SD of three or more independent experiments.

Table 4 . Comparison between the interface system and saturated system.

 4

	strain	MG1655	MG1655ompR234,
	Mutant strains	G1	G1ompR234; G1ompR; G1ompR234csgA
	Reporter FP	AsRed2	GFP[LVA]
	Reporter plasmid	High copy pBlueScript Low copy pPROBE
	Planktonic condition BHI, 37°C	mM63, 30°C
	Biofilm condition	BHI+mucin, 37°C	mM63, 30°C
	Biofilm formation	Air-liquid interface	Surface immersed in medium

Table 5 . Concentrations of mucin that gave the maximum biofilm formation (the "optimal" concentration) in different conditions

 5

		Surface	BHI 37°C	mM63 30°C
	G1	PS glass	not obvious 3%	0.50% 0.25%
	G1ompR234	PS glass	not obvious 2%	0.25% 0.25%

. The optimal mucin concentration for biofilm formation on PS surface is determined by direct visual observation of CV staining intensity. On glass surface, it is determined by COMSTAT analysis on biomass, average thickness and maximum thickness of CLSM Z-stacks (in bold).

  2.1) of E. coli G1 is 41.7±1.6 minutes and 35.3±0.1 minutes for K. pneumoniae. The ompR234 mutation does not significantly influence the growth rate of E. coli, with a generation time of 40.1±0.2 minutes. In mM63 (30°C), the growth rate difference between E. coli and K. pneumoniae is greater (168.4±0.5, 177.5±1.4 and 104.0±1.3

  ). The broken lines separate different E. coli mutants.

	A	100%		B			C			Ef
		80%								Kp
	BHI	60%								Ec
	37 C	40%
		20%
		0%
		Ec+Kp	Ec+Ef	Ec+Kp+Ef	Ec+Kp	Ec+Ef	Ec+Kp+Ef	Ec+Kp	Ec+Ef	Ec+Kp+Ef
			G1			G1ompR234		G1ompR234csgA
			curli+/-			curli++			curli--
	D	100%		E			F
		80%
	mM63	60%
	30 C	40%
		20%
		0%
		Ec+Kp	Ec+Ef	Ec+Kp+Ef	Ec+Kp	Ec+Ef	Ec+Kp+Ef	Ec+Kp	Ec+Ef	Ec+Kp+Ef

6.1 Strains and plasmids

	Strains Strains Plasmids Plasmids 6.3 Culture conditions Relevant description Relevant description Promoter fusion Relevant description Ab a PHL b ECS c Source Ab a PHL b ECS c Source Source Promoter fusion Relevant description Source autoclaving, add:
	Strains E. coli strains TOP10 S17 λ-pir G1ompR234csgA G1ompR W3110 Non-E. coli strains Klebsiella pDsRed-Max-N1 pCCS445 P A1/04/03 -DsRed-Max Ap r ; pCCS124 carrying the DsRed-Relevant description Ab a PHL b ECS c Source Chemically competent cells Nil -20 Invitrogen provides Pir protein, which is essential for replication of R6K-based plasmids Tp r Sm r -22 (Miller & G1 ompR234 malT::Tn10 csgA::aadA7 Cm r Tc r Sp r 1624 565 This work d G1 ompR331::Tn10 Cm r Tc r 1623 564 This work d An E. coli K-12 strain closely related to MG1655 Nil 1104 enteric, Gram negative rod Nil -NES3 ATCC 13883 Kan r ; ori; 4.7 kb; used for cloning of DsRed-Max gene for R1 Addgene plasmid Max-N1 gene under the control of This work 6.3.1 Single-species cultures 1ml of 1M MgSO 4 pneumoniae NCTC 9633 Enterococcus faecalis OG1RF enteric, Gram positive coccus Nil -NES4 ATCC 47077 construction 21718 (Strack P A1/04/03 promoter 0.0005% (w/v) vitamin B1 6.3.1.1 Planktonic cultures et al., 2008) pCCS401 p173 P csgBA -gfp Kan r ; pPROBE-gfp[LVA] carrying the csgBA promoter; 8.5 kb This work pCCS446 P A1/04/03 -DsRed-Max Cm r ; pDM4 carrying the XhoI-Region A-P A1/04/03 -DsRed-Max-This work 0.2% (w/v) glucose Single-species planktonic cultures of E. coli MG1655 and G1, as well as their curli-765 (Danese et al., 2001) W3110fimA W3110 fimA::Cm Cm r 1106 766 Gift from P. a Antibiotic resistance of the bacterial strains pCCS402 p171 Kan r ; pPROBE-gfp[LVA] carrying This work Region B-SpeI fragment; 9.97 kb 1% (w/v) autoclaved BHI or related mutants were routinely inoculated in LB (for general propagation and cloning P fis -gfp the fis promoter; 8.2 kb 1% (w/v) autoclaved LB purposes) or BHI medium (for experiments involving co-cultures) and incubated at 37°C, Mekalanos, 1988) MG1655 λ -F -prototroph Nil 1256 43 ATCC 700926 MG1655csgA MG1655 csgA::aadA7 Sp r 1295 -C. Beloin, Institut Pasteur MG1655ompR234 MG1655 ompR234 malT::Tn10 Tc r 818 561 (Prigent-Combaret et al., 2001) MG1655ompR234 csgA MG1655 ompR234 malT::Tn10 csgA::uidA-Km Tc r Km r 1137 761 (Perrin et al., 2009) Danese W3110fimH W3110 fimH::Cm Cm r 1107 767 Gift from P. Danese W3110Ag43 W3110 (argF-lac)U169 agn::Cm Cm r 1166 768 gift from R. Kolter W3110csgA W3110 csgA::uidA-Km Km r 1144 2630 This work d W3110ompR234 W3110 ompR234 malT::Tn10 Tc r collection W3110ompR234 W3110 ompR234 Tc r 1145 2631 This work d Vectors Relevant description Source pDM4 Cm r ; R6K ori, low copy; 7.1 kb (Milton et al., pCCS411 p181 P mazEF -AsRed2 Ap r ; pBluescript carrying the mazEF promoter; 5.2 kb Laboratory stock solutions filtration through 0.22 μm filter This work 100 mg/ml ampicillin Prepared in ddH 2 O and sterilized by 1131 2628 Laboratory b The collection number in the French laboratory c The collection number in the Singapore laboratory d E. coli strains constructed by bacterial phage P1 transduction (refer to Table 6 in Section 6.5.1) Plasmids pCCS405 p175 P mazEF -gfp Kan r ; pPROBE-gfp[LVA] carrying This work a p number refers to the collection number in the French laboratory b pCCS number refers to the collection number in the Singapore laboratory Transduction solution with shaking at 250 rpm unless otherwise stated. When gene transcriptional activity with 9.8ml autoclaved ddH 2 O respect to curli-related genetic background was concerned, E. coli strains were shaken at the mazEF promoter; 8.4 kb pCCS407 p177 P csgBA -AsRed2 c Region A is a XhoI-ClaI fragment corresponding to base-pairs 312037-312754 and Region 100μl of 1M CaCl 2 60 rpm in mM63 medium at 30°C. Where appropriate, media were supplemented with Ap r ; pBluescript carrying the csgBA promoter; 5.2 kb This work pCCS408 p178 P fis -AsRed2 promoter; 4.9 kb This work 6.2 General reagents, kits and media Media supplements Ap r ; pBluescript carrying the fis B a BamHI-SpeI fragment corresponding to base-pairs 312771-313495 of E. coli MG1655 100μl of 1M MgSO 4 antibiotics. Non-E. coli strains were grown without antibiotics in BHI at 37°C or mM63 genome (GenBank accession no.U00096) medium at 30°C.
	MG1655ompR234 csgD csgA pCCS325 P A1/04/03 -AsRed2 MG1655 ompR234 malT::Tn10 csgD::uidA-Km malT::Tn10 csgA::uidA-Km Ap r ; pBluescript carrying the Tc r Km r 1087 Km r 10x TBE 30 mg/ml chloramphenicol Prepared in ethanol 0.9 M Tris, 0.9 M boric acid, 20 mM 760 (Prigent-1996) Laboratory Combaret et al., 2001) MG1655ompR234 agn43 MG1655 ompR234 malT::Tn10 agn43::Cm Tc r Cm r 1563 762 (Perrin et al., 2009) MG1655ompR234 fimA MG1655 ompR234 malT::Tn10 fimA::Cm Tc r Cm r 1675 2634 This work d MG1655ompR MG1655 ompR331::Tn10 Tc r 1171 763 Laboratory collection MG1655ompR csgD MG1655 ompR331::Tn10 csgD::uidA-Km Tc r Km 1089 -(Prigent-Combaret et al., 2001) G1 MG1655 with chromosomal insertion of PA1/04/03-G1ompR234 G1 ompR234 malT::Tn10 Cm r Tc r 1581 562 This work d from BD Clontech Region B-SpeI fragment; 9.97 kb Nucleospin Plasmid From Macherey-Nagel Region A-P A1/04/03 -mAsRed2-gfpmut3* Cm r 1414 354 (Miao et al., (=pCCS127) RBS-AsRed2-T 1 -T 2 ; 4.1 kb; AsRed2 2009) pCCS444 P A1/04/03 -mAsRed2 Cm r ; pDM4 carrying the XhoI-This work Rapid DNA Ligation Kit From Roche 2009) W3110ompR234 fimA W3110 ompR234 malT::Tn10 fimA::Cm Tc r Cm r 1673 2632 This work d W3110ompR234 fimH W3110 ompR234 malT::Tn10 fimH::Cm Tc r Cm r 1674 2633 This work d MC4100 araD139 Δ(argF-lac)U169 rpsL150 relA1 flbB5301 deoC1 ptsF25 rbsR Nil 645 2624 (Vidal et al., 1998) MC4100ompR234 MC4100 ompR234 malT::Tn10 Tc r 744 pPROBE-gfp[LVA] (=p116 a ) Kan r ; pBBR1 replicon; GFP-destabilized variant LVA; low copy; 7.4 kb (Miller et al., 2000) pBluescript SK(+) Ap r ; pUC ori, high copy; 3.0 kb Stratagene pGEM ® -T Ap r ; pUC ori, high copy; 3.0 kb Promega pBS-EcoRV/NotI pBluescript SK(+) derivative; 3 kb V. Shingler mAsRed2 gene under the control of collection Na 2 EDTA (pH 8.0), 100 mg/ml kanamycin Prepared in ddH 2 O and sterilised by P A1/04/03 promoter; 4.3 kb pCCS326 P A1/04/03 -mAsRed2 TE filtration through 0.22 μm filter 10 mM Tris/HCl (pH 8.0), 1 mM Ap r ; pBluescript carrying the This work Na 2 EDTA (pH 8.0) 10% mucin (Sigma-Aldrich, Mucin Prepared in either Brain-Heart Infusion mAsRed2 gene under the control of P A1/04/03 promoter; 4.3 kb pCCS343 P A1/04/03 -mAsRed2 Ap r ; pCCS124 carrying the ClaI-P A1/04/03 -mAsRed-BamHI fragment; Restriction enzymes from porcine stomach Type III) broth (for BHI supplement) or M63 From Fermentas, New England Biolabs supplemented with glucose (for mM63), and Roche and sterilised by autoclaving at 121ºC This work Alkaline phosphatase (ClAP) for 20 min From Fermentas 2625 (Vidal et al., 1998) MC4100ompR234 csgA MC4100 ompR234 malT::Tn10 csgA::uidA-Km Tc r Km r 819 pBluescript-AsRed2 Ap r , pBluescript SK(+) derivative with (Miao et al., promoter P A1/04/03 ; 5.8 kb Commercial kits 1999) MG1655 chromosome; 4.4 kb extra XhoI site just upstream the 2627 (Dorel et al., pCCS124 b pBS-EcoRV/NotI derivative with Region A and B c homologous to 5.8 kb Laboratory collection pCCS443 P A1/04/03 -mAsRed2 pCCS343 derivative, removed the This work Taq DNA polymerase From Fermentas a Prepared as recommended by the manufacturer.
	100

Table 2 in

 2 Section 2.2.3) as determined in Section 6.3.1.2.

	With biofilm cultures containing various concentrations of mucin, mucin was diluted
	from 10% stock to the desired concentrations prior to bacterial inoculation. The biofilm
	cultures were then incubated at 37°C (in BHI) or 30°C (in mM63) for 48 hours.
	6.3.1.3.2 The saturated system
	Overnight bacterial culture in mM63 at 30°C was used as seeding culture to inoculate
	20ml mM63 in the presence of increasing amount of mucin at 6x10 6 cells/ml
	concentration in each Petri dish, according to the OD 600 -cell density relationship (refer
	to

Table 2 in

 2 Section 2.2.3). Three well separated sterile glass cover slips (22x22 mm,

Table 7 .

 7 Primers used for construction of promoter-gfp and promoter-AsRed2 fusions.

	Promoter fusion a Pr b	Sequence (5'->3')	Limit c	Ref d
	P csgB -gfp	CGGGATCC(EcoRI)CGGCCGTTGATATGAGGCCAG GGAATTC(BamHI)CGTTGTCACCCTGGACCTG	-1072 -1	(Hammar et al., 1995)
	P fis -gfp	CGGGATCC(EcoRI)CGCCCATCCAACCACCTCTCTG GGAATTC(BamHI)CGAGTTAAGAAATGACCATACTGTGACTGC	-789 -1	(Nasser et al., 2002)
	P mazEF -gfp	CGGGATCC(EcoRI)CGCGCTTACCACATCCACAGTG GGAATTC(BamHI)CCAACGCTTTACGCTACTGTG	-1032 (Marianovsky et al., 2001) +26
	P csgB -AsRed2	GCCGTTGATATGAGGCCAG GAAGATCT(BglII)TCGTTGTCACCCTGGACCTG	-1072 -1	(Hammar et al., 1995)
	P fis -AsRed2	CCCATCCAACCACCTCTCTG GGAAGATCT(BglII)TTCGAGTTAAGAAATGACCATACTGTGACTGC	-789 -1	(Nasser et al., 2002)
	P mazEF -AsRed2	CGCTTACCACATCCACAGTG CGTAGATCT(BglII)TCCAACGCTTTACGCTACTGTG	-1032 (Marianovsky et al., 2001) +26

December 2008 Nan YANG, Corinne DOREL and Chun Chau SZE. Biosensors for pathogen detection: Mapping E. coli gene expression in biofilm. (Poster presentation) 13 th International Biotechnology Symposium and Exhibition, Dalian, China, October 2008 (Journal of Biotechnology, Volume 136, Supplement 1, October 2008, Pages S103-S104)

Thanks to the Merlion PhD Scholarship offered by the French Embassy in Singapore, my

Abbreviations

A adenine Ap r

List 1. Antimicrobial activity test between E. coli and its partner species.

Hence the relationship of the strains of E. coli, K. pnuemoniae and E. faecalis which we would study is independent of antibiotics-based growth inhibition. As interactions among intestinal commensals are likely to be more moderate than extreme in nature, we hope that our study using this combination of strains will be more biologically relevant than those that were focused on antibiotics production (Gillor et al., 2008).

Influence of curli on population relationship

The Singapore laboratory has previously reported the dual-and multi-species population relationship of the wild type E. coli MG1655 (and its GFP-expressing derivative strain G1) with K. pneumoniae NCTC 9633 and/or E. faecalis OG1RF in rich media BHI at 37°C [START_REF] Miao | Dual fluorescence system for flow cytometric analysis of Escherichia coli transcriptional response in multispecies context[END_REF]. Since in the natural intestinal environment, one of the consequences of mucin's presence is the induction of curli synthesis in certain strains of E. coli (as shown in Chapter 3), we were interested to see the possible impact of curli expression on the inter-species interaction between E. coli and its commensal partners.

Although the most often reported mode of bacterial cell-cell interaction is quorum sensing (QS), which involves secreting and sensing signalling molecules such as n-acyl homoserine lactones, peptides and furanones [START_REF] Balestrino | Characterization of Type 2 Quorum Sensing in Klebsiella pneumoniae and Relationship with Biofilm Formation[END_REF][START_REF] Brenner | Engineered bidirectional communication mediates a consensus in a microbial biofilm consortium[END_REF][START_REF] Federle | Autoinducer-2-based chemical communication in bacteria: complexities of interspecies signaling[END_REF][START_REF] De Araujo | Quorum sensing affects biofilm formation through lipopolysaccharide synthesis in Klebsiella pneumoniae[END_REF][START_REF] Lopez | Biofilms[END_REF][START_REF] Dickschat | Quorum sensing and bacterial biofilms[END_REF], the physical interactions between cells are recognized to be important for bacterial co-metabolism and cooperative association, especially in the biofilm context [START_REF] Drago | Inhibition of in vitro growth of enteropathogens by new Lactobacillus isolates of human intestinal origin[END_REF][START_REF] Kolenbrander | Oral microbial communities: biofilms, interactions, and genetic systems[END_REF]Foster et al., 2003;[START_REF] Ledder | Coaggregation between and among human intestinal and oral bacteria[END_REF]. By replacing co-cultured strains with their cell-free culture supernatant, it has been possible to demonstrate that synergistic influences depend on both extracellular secreted factors and species-specific this interpretation, both the curli--and curli++ E. coli strains were cultured in BHI at 37°C, where curli expression is generally repressed, and they both showed FSC/SSC profiles lacking the "tail" sector (Figure 4.

E & G).

Samples of the curli++ strain in both mM63 (30°C) and BHI (37°C) were also taken at 3, 6, 9 and 24 hours of growth to be analysed by FACS (Figure 4.2 B). In mM63 (30°C), the profiles reveal that the "tail" only starts to form at 6 hours of culture and becomes more and more prominent (Figure 4.3 "mM63 30°C" column). This observation is in agreement with the fact that curli is not produced in the exponential phase but only when cells enter the stationary phase [START_REF] Olsen | The RpoS sigma factor relieves H-NS-mediated transcriptional repression of csgA, the subunit gene of fibronectin-binding curli in Escherichia coli[END_REF]. When cultured in BHI (37°C) where curli expression is repressed, E. coli can be observed to exhibit fairly unchanging profiles at different growth phases (Figure 4.3 "BHI 37°C" column).

Incidentally, a more thorough examination of the profiles of the curli +/-(G1) strain (which is not completely null in curli production) revealed a very small subpopulation (2.14%) corresponding to the "tail" region of the curli ++ strain (Fig 4 .2 B, red polygon).

Hence the "intermediate" curli expression level associated with this strain [START_REF] Vidal | Isolation of an Escherichia coli K-12 mutant strain able to form biofilms on inert surfaces: involvement of a new ompR allele that increases curli expression[END_REF] is due to a small subpopulation of curli-expressing cells, and not a result of an intermediate expression of curli by the entire population of cells.

Out of curiosity, I also checked the FACS profile of the curli-(G1ompR) strain with the null mutation at the regulatory level of curli synthesis. The FSC/SSC profile shows no "tail", suggesting that curli is not present (Fig 4.2 D), similarly to other non-curliexpressing strains. However, it appears that the G1ompR cells are generally bigger than the other E. coli strains (refer position on the FSC scale). Since the osmolarity-and pHinduced global regulator OmpR is not only involved in curli regulation, but also in porin regulation [START_REF] Pratt | From acids to osmZ: multiple factors influence synthesis of the OmpF and OmpC porins in Escherichia coli[END_REF] and other physiological functions, this increase in cell size in our experimental condition may be due to factors related to these functions .

Having affirmed that the expression of curli on E. coli indeed translates to detectable physical properties, which may influence the interaction between cells of the same and/or different species, I went on to explore the growth relationship between E. coli and its commensal partner species K. pneumoniae and E. faecalis. complexity involved in these phenomena will be difficult to unravel with this study, but will certainly be of interest to pursue in the future (Section 5.3).

The observation in BHI (37°C), whereby E. coli and the partner species seem to interact in an antagonistic or competitive manner (Figure 4.4 A, B and C), is fairly easy to rationalize. In co-cultures, the inoculation cell density of each species was kept constant, hence the total starting bacteria number in a dual-or multi-species culture is two or three times of that in the single species culture respectively, and competition for nutrients under higher overall cell density can be expected. However, surprisingly, this overall trend of negative influence was not observed in nutrient-limited mM63 (30°C) situation, which instead, showed varied influences including neutral or even positive ones.

Intuitively, we would imagine that a more drastic competition would occur in mM63 due to its greater nutrient limitation. However, if we consider the fact that differential curli expression and variation in surface properties between the three E. coli strains are more pronounced under the mM63 than the BHI condition (refer sections 4.3.1.1 and 4.3.1.2), it becomes apparent that there are more factors at play in interspecies interactions than competition for nutrients. I will therefore attempt to discuss the data with curli expression in perspective, in the following section.

Curli's influence on bacteria growth in co-culture

From Figure 4.4 D we can see that although K. pneumoniae and E. faecalis both exert negative effects on the growth of E. coli (compare blue against red and green bars within each block), the influence of K. pneumoniae is more prominent than E. faecalis (compare red against green bars). When we compare the effects on G1ompR234 against its csgA derivative, we see that K. pneumoniae's negative influence is especially drastic when E. coli cannot produce curli (compare red bars between "ompR234" and "ompR234csgA").

Since the two E. coli strains are genetically identical except for the null mutation of the curlin structural gene csgA, this observation suggests that curli's presence may serve to counteract some of K. pneumoniae's antagonistic effect on E. coli.

In this thesis, the population relationship of the three commensals in biofilm co-cultures was also analysed and presented in the two formats (Figures 4.6 and 4.7) as shown for the planktonic co-cultures (Figures 4.4 and 4.5). However, since biofilms have been collected from a fixed surface area (refer to Chapter 6 Materials and Methods for details), the absolute CFU/ml analysis method allows an estimation of the surface attached cell number, instead of average cell density in suspension as in planktonic cultures. Thus changes in surface attached biomass, in addition to the growth of one species under the influence of its partner species in biofilm, would be reflected. The analysis of relative population percentage, on the other hand, provides a snapshot of the biofilm's composition as a result of growth and interaction between the co-cultured species.

Growth on the surface

When co-cultured in BHI (37°C), K. pneumoniae in biofilms displayed antagonistic effect on all strains of E. coli (Figure 4.6 A, compare blue and red bars within each block) just as observed in the planktonic counterparts (Figure 4.4 A). The degree of negative influence on the three E. coli strains differs (ompR234csgA > ompR234 > G1). Since we know from several lines of evidence (section 4.3.1.1 & 4.3.1.2) that the curli expression status of these three strains are similar under the BHI (37°C) condition, the difference is most probably conferred by other unknown factors. On the other hand, the influence of E. coli on K. pneunomiae (Figure 4.6 B), seems to be governed by the genotype of ompR: while the wild type E. coli G1 exhibited statistically significant negative influence, the ompR234 mutation of E. coli removed this negative influence on K. pneumoniae (Figure 4.6 B, compare the blue and red bars of "G1" against "ompR234" and "ompR234csgA").

Under the mM63 condition, the K. pneumoniae influence on E. coli was also negative across all three E. coli strains (Figure 4.6 D, compare blue and red bars within each block). We further noted that in general, the additional presence of E. faecalis in multispecies biofilms (purple bars) does not alleviate the negative effect of K. pneumoniae in both culture conditions (Figures 4.6 A and D). As for E. coli influence on K. pneumoniae, regardless of their differing curli-expressing status under the mM63 condition, E. coli strains do not affect the growth of K. pneumoniae in biofilm co-cultures (Figure 4.6 E). coli MG1655, cellulose may impair biofilm formation even in strong curli producing strains probably by physically blocking curli from contacting the surface [START_REF] Gualdi | Cellulose modulates biofilm formation by counteracting curli-mediated colonization of solid surfaces in Escherichia coli[END_REF]. This model seems to support the hypothesized increase in CsgD and concurrent cellulose production in response to increased mucin concentration.

To test whether mucin's presence would favour cellulose production, calcofluor plate with and without mucin can be used to assess cellulose production in the presence and absence of mucin. Alternatively, cellulose's presence can be directly observed using electron microscopy or quantified [START_REF] Gualdi | Cellulose modulates biofilm formation by counteracting curli-mediated colonization of solid surfaces in Escherichia coli[END_REF]. To determine whether csgBA upregulation by mucin was through up-regulation of csgD, the induction experiment in 24well plate of csgD mutant can be repeated. A more direct method would be to monitor csgD gene activity with and without mucin, with the aid of a csgD-gfp fusion.

Another interesting observation that led me to the above hypothesis is that crystal violet (CV) failed to dissolve in ethanol for quantification as described by Kikuchi [START_REF] Kikuchi | Curli fibers are required for development of biofilm architecture in Escherichia coli K-12 and enhance bacterial adherence to human uroepithelial cells[END_REF] nor in ethanol-acetone (80:20 [vol/vol]) [START_REF] Ferrieres | The RcsC sensor kinase is required for normal biofilm formation in Escherichia coli K-12 and controls the expression of a regulon in response to growth on a solid surface[END_REF][START_REF] Valle | UpaG, a new member of the trimeric autotransporter family of adhesins in uropathogenic Escherichia coli[END_REF][START_REF] Korea | Escherichia coli K-12 possesses multiple cryptic but functional chaperone-usher fimbriae with distinct surface specificities[END_REF], in experiments involving mucin. This is not directly due to mucin itself binding CV, because no CV staining was observed in control wells containing the mucin media without bacteria (data not shown). When the same strain was used in the same experimental settings with metals instead of mucin, CV could be completely dissolved. This led me to wonder if mucin is indirectly holding the CV dye within the biofilm. After some literature search, I realized that there is no exact answer as to what crystal violet binds, and no study was done on what component(s) are actually stained by CV in the bacteria cell wall, and why the amount of cell-bound CV should be assumed to be an indication of cell number. Bacterial cell wall is made of peptidoglycan, which are linear glycan chains interlinked by short peptides. The glycan chains are composed of alternating units of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), with all linkages between sugars being β,1→4 [START_REF] Heijenoort | Formation of the glycan chains in the synthesis of bacterial peptidoglycan[END_REF]. Cellulose is a homopolysaccharide consisting of D-glucopyranose units linked by β,14 glycosidic bonds [START_REF] Romling | Molecular biology of cellulose production in bacteria[END_REF]. Would crystal violet, staining the cell wall, possibly stain cellulose, which is compositionally and structurally similar to the cell wall? Dissolve the above in ddH2O adjusting the pH to 7 using KOH. After forceps. The Petri dish-contacting side of the cover slip was wiped with ethanol, and the whole cover slip was place directly on the observation stage of Zeiss inverted microscope with its biofilm side facing up. For observation using the Nikon up-right microscope, the cover slip was placed on the flow cell chamber filled with water with the biofilm side facing down in contact with water and the Petri dish-contacting side facing up.

Biofilm formed in the interface system

For biofilm observation using Nikon up-right CLSM, the cover slip was withdrawn from the tank and the biofilm on the side facing the medium was wiped away with ethanol.

Then the planktonic cells below the interface biofilm were washed in sterile water and the cover slip mounted on flow cell chamber (Bjarke Bak Christensen, Technical University of Denmark) containing water.

Growth inhibition test

To test whether E. coli, K. pneumoniae and E. faecalis have antimicrobial effect against each other, growth inhibition test was performed on soft agar of BHI and mM63. E. coli MG1655, G1 and their curli-related mutants, as well as E. coli, K. pneumoniae and E.

faecalis were inoculated in BHI and mM63 and shaken overnight at 37ºC and 30ºC

respectively. 50 µl of the overnight culture of each strain was added into 5 ml of 0.75% molten soft agar of either BHI or mM63, shaken gently to mix well and immediately poured onto the appropriate plate. The resulting soft agar plate was then swirled to even out the surface and left to solidify. Then they are dried for at least half an hour before drops of 3 µl of strains for testing antibiotic production were aliquoted, and incubated for 24 hours at 37ºC (for BHI plates) or 30ºC (for mM63 plates). Inhibitory antimicrobials against the test strain in the lawn after overnight growth will result in a clear halo around the "drop" strain's patch.

The pellet was washed three times with 1ml LB containing 0.1% citrate (w/v) to eliminate phages that were attached to the bacterial cells. The bacterial cells were then incubated in the last wash solution at 37 ºC for 45 min before spread on LB agar plates containing citrate and appropriate antibiotics. 6 to 12 colonies with antibiotic resistance were re-streaked on a fresh agar plate containing the appropriate antibiotics before phenotypic analysis.

Agarose gel electrophoresis

Agarose gel of 0.7% agarose in 1x TBE was routinely used for electrophoresis. Agarose was casted in gel apparatus (Bio-Rad) and set at RT for at least 30 min. Electrophoresis was carried out in the same apparatus with the gel submerged 1~2 mm below the surface in 1x TBE. DNA samples were loaded into the wells with 1x loading dye. Electric field of about 10 volt/cm was usually applied for the separation of DNA fragments. After electrophoresis, the gel was transferred into 1 μg/ml ethidium bromide (EB) solution for staining. DNA fragments were then visualized by fluorescence over a UV light (302 nm, UV transluminator TM-20, UVP), under which DNA/EB complexes fluoresce, and the image was recorded with a Mitsubishi video copy processor.

DNA quantification and purification

DNA concentration was determined either with agarose gel electrophoresis or by the NanoDrop™ 1000 UV-Vis Spectrophotometer (NanoDrop Technologies). For agarose gel electrophoresis, the intensity of DNA was compared against that of known concentrations on a 100 bp or 1 kb DNA ladder (GeneRuler™, Fermentas) and the sample concentration was estimated. A more precise quantification was achieved with the NanoDrop™ which requires molecular grade water as a blank reference and only 1μl of sample for measurement.

Agarose gel electrophoresis was also used to isolate a particular DNA fragment in a mixture of fragments, for ligation. For optimal separation, the gel was run with 8 volt/cm. Gel containing DNA fragments of interest was excised under 70% power of UV light (Vilber Lourmat) and DNA was extracted using Nucleospin ® Extract II Kit (Macherey-Nagel) according to the manufacturer's instructions.

Plasmid Construction

Construction of promoter-gfp fusions

The promoters of the genes of interest were PCR-amplified from purified E. coli MG1655 genomic DNA and inserted into the plasmid pPROBE-gfp between its EcoRI and BamHI sites. Approximately 1kb upstream of the transcription start site of the gene was amplified with reference to the promoter regions documented ("Ref" column in Table 7).

Construction of promoter-AsRed2 fusions

The promoters of the genes of interest were PCR-amplified from purified E. coli MG1655 genomic DNA and inserted into the pGEM ® -T vector. Approximately 1kb upstream of the transcription start site of the gene was amplified with reference to the promoter regions documented ("Ref" column in Table 7). The orientation of the inserted promoters was verified. The promoters were subsequently digested from the pGEM ® -T vector and inserted into the plasmid pBluescript-AsRed2 between its SphI and BglII sites.

Equipment settings

Fluorometric microplate reader / Fluorometer

Fluorescence levels of bacterial cultures were measured using different fluorometer in the Singapore and French laboratory. The Singapore laboratory possesses an Infinite TM M200 multi-mode monochromator-based microplate reader (Tecan) (referred to as "microplate reader"). The excitation/emission wavelength settings were 488 nm/530 nm for samples with Gfpmut3* expression, and 570 nm/600 nm for samples with AsRed2 expression.

The French laboratory on the other hand, is equipped with a Kontron SMF25 fluorometer (referred to as "fluorometer"), whose excitation/emission wavelength were set to 490 nm/510 nm for samples with Gfp[LVA] expression, and 570 nm/600 nm for samples with AsRed2 expression.

For measurement using the microplate reader, duplicate aliquots of 200 μl of each appropriately diluted culture were dispensed into a 96-well black-wall clear-bottom microplate (Greiner). When using the fluorometer, samples were aliquoted in a 3.5 ml 4-side fluorometer cuvette without dilution. The green and red fluorescence levels (in Fluorescence Unit) and spectrophotometric absorbance at 600 nm (in Absorbance Unit) were sequentially measured and corrected by the medium control (blank). The fluorescence level was calculated as measured fluorescence unit divided by OD 600 .

Zeta potential measurement

E. coli MG1655, G1 and their curli-related mutants, as well as E. coli, K. pneumoniae and E. faecalis were inoculated in BHI and mM63 and shaken at 37ºC for 14 hours and 30ºC for 16 hours (except E. faecalis for 24 hours) respectively. Each strain was then adjusted to a density of 10 7 cells/ml in ddH 2 O (according to the OD 600 -cell density relationship listed in Table 2 in Section 2.2.3) before running on Nano Zetasizer (Malvern Instrument, UK) at 150 V, room temperature.

Confocal laser scanning microscope

Biofilms were observed under either an Eclipse 90i inverted CLSM (Nikon) equipped with 488 nm and 543 nm lasers or Zeiss LSM 510 upright CLSM (Carl Zeiss).

For the Eclipse 90i inverted CLSM, green fluorescence was observed using the channel set at 488 nm excitation and 515/30 nm emission; for red fluorescence, 543 nm excitation and 605/25 nm emission. The confocal images of the two fluorescences were taken sequentially to minimize the crosstalk between them. Controls of the microscope were manipulated by iControl software (Nikon). Images were processed by FreeViewer software (Nikon).