Christopher J Marx
email: cmarx@oeb.harvard.edu

Françoise Bringel
email: francoise.bringel@unistra.fr.

Ludmila Chistoserdova

Lionel Moulin

Muhammad Farhan

Ul Haque

Darrell E Fleischman

Christelle Gruffaz

Philippe Jourand

Claudia Knief

Ming-Chun Lee

Emilie E L Muller

Thierry Nadalig
email: nadalig@unistra.fr

Rémi Peyraud

Sandro Roselli

Lina Russ

Lynne A Goodwin

Natalia Ivanova

Nikos Kyrpides

Aurélie Lajus

Miriam L Land

Claudine Médigue

Natalia Mikhailova

Matt Nolan

Tanja Woyke

Sergey Stolyar

Julia A Vorholt

Stéphane Vuilleumier
email: vuilleumier@unistra.fr.

Ste ´phane Vuilleumier

Ghislaine Magdelenat

´lie Aure

Lajus

Vale ´rie Barbe

Claudine Me

Franc ¸oise Bringel

Hubert Schaller

& St Éphane Vuilleumier

Complete Genome Sequences of Six Strains of the Genus Methylobacterium

Keywords: chloromethane, methylotrophic bacteria, Methylobacterium extorquens, Arabidopsis thaliana, bacterial genomics, bioreporter, HOL1, cmuA chloromethane, phyllosphere, cmu genes, methyl halide metabolism

Chloromethane is the most abundant halocarbon in the environment, and responsible for substantial ozone destruction in the stratosphere. Sources and sinks of chloromethane are still poorly constrained. Although synthesized and used industrially, chloromethane is mainly produced naturally, with major emissions from vegetation and especially the phyllosphere, i.e. the aerial parts of plants. Some phyllosphere epiphytes are methylotrophic bacteria which can use single carbon compounds such as methanol and chloromethane as the sole source of carbon and energy for growth. Most chloromethane-degrading strains isolated so far utilize the cmu pathway for growth with chloromethane which was characterized by the team. The main objective of this work was to investigate whether epiphytes may act as filters for plant emissions of chloromethane, by using a laboratory bipartite system consisting of the model plant Arabidopsis thaliana, known to produce chloromethane mainly by way of the HOL1 gene, and the reference chloromethane-degrading bacterial strain Methylobacterium extorquens CM4, possessing the cmu pathway and of known genome sequence. Three A. thaliana Col-0 variants with different levels of expression of HOL1, i.e. the wild-type strain, its homozygous HOL1 knockout mutant hol1 and an HOL1-OX HOL1 overexpressor, were selected using PCR and qRT-PCR. Chloromethanedegrading strains were isolated from the A. thaliana phyllosphere, and shown to contain the cmu pathway. A plasmid-based bacterial bioreporter for chloromethane was constructed which exploits the promoter region of the conserved chloromethane dehalogenase gene cmuA of strain CM4. It yields rapid, highly sensitive, specific and methyl halide concentration-dependent fluorescence. Application of the bioreporter to the three A. thaliana variants differing in expression of HOL1 investigated in this work suggested that they indeed synthesize different levels of chloromethane. Analysis by qPCR and qRT-PCR of metagenomic DNA from the leaf surface of these variants showed that the relative proportion and expression of cmuA in this environment paralleled HOL1 gene expression. Taken together, the results obtained indicate that even minor amounts of chloromethane produced by A. thaliana in the face of large emissions of methanol may provide a selective advantage for chloromethane-degrading methylotrophic bacteria in the phyllosphere environment. This suggests that chloromethane-degrading epiphytes may indeed act as filters for emissions of chloromethane from plants. Further experiments are envisaged to further assess the adaptation mechanisms of chloromethane-degrading bacteria in the phyllosphere, building upon the comparative genomic analysis of chloromethane-degrading strains which was also performed in this work, and on the preliminary investigations using high-throughput sequencing that were initiated.

Résumé

Le chlorométhane est le plus abondant des composés organo-halogénés dans l'atmosphère et il est impliqué dans la destruction de l'ozone dans la stratosphère. Les sources et les puits de chlorométhane restent mal évalués. Bien que synthétisé et utilisé de manière industrielle, il est principalement produit naturellement, avec comme sources majeures les émissions provenant des végétaux et plus particulièrement de la phyllosphère, qui correspond aux parties aériennes des plantes. Certaines bactéries épiphytes de la phyllosphère sont des méthylotrophes capables d'utiliser des composés organiques sans liaison carbone-carbone comme le méthanol et le chlorométhane comme unique source de carbone et d'énergie pour leur croissance. La plupart des bactéries chlorométhane-dégradantes isolées jusqu'à présent utilisent une voie métabolique pour leur croissance sur chlorométhane appelée voie cmu (pour chloromethane utilisation), caractérisée par l'équipe. L'objectif principal de cette thèse a été de déterminer si des bactéries de la phyllosphère peuvent jouer le rôle de filtre pour l'émission de chlorométhane par les plantes. Dans ce but, un modèle de laboratoire a été mis en place, constitué de la plante Arabidopsis thaliana connue pour produire du chlorométhane par une réaction impliquant le gène HOL1, et la bactérie Methylobacterium extorquens CM4, souche de référence pour l'étude du métabolisme de dégradation du chlorométhane, qui possède la voie cmu et dont le génome complet a été séquencé et analysé. Des variants d'A. thaliana avec différents niveaux d'expression du gène HOL1 (le type sauvage, le mutant homozygote « knockout » hol1 et un variant HOL1-OX avec surexpression) ont été sélectionnés par PCR et qPCR. Des souches bactériennes chlorométhane-dégradantes ont été isolées à partir de la phyllosphère d'A. thaliana, dont il a été montré qu'elles possèdent la voie cmu. Un bio-rapporteur bactérien pour le chlorométhane a été construit à l'aide d'un plasmide exploitant la région promotrice du gène conservé de la déshalogénase (cmuA) de la souche M. extorquens CM4. Il présente une réponse fluorescente rapide, sensible, et spécifique aux méthyl-halogénés de manière concentration-dépendante. L'application du bio-rapporteur aux trois variants d'A. thaliana étudiés suggère des niveaux d'émissions de chlorométhane différents. L'analyse, par qPCR et qRT-PCR, de l'ADN métagénomique extrait de la surface des feuilles a montré une corrélation entre la proportion relative de bactéries portant le gène cmuA et l'exprimant dans cet environnement, et l'expression du gène HOL1. Ces résultats indiquent qu'une production de chlorométhane, même très modeste par rapport aux fortes émissions de méthanol par A. thaliana, confère un avantage sélectif pour les bactéries épiphytes chlorométhane-dégradantes. Ces dernières pourraient ainsi bien jouer un rôle de filtre pour les émissions de chlorométhane de la phyllosphère vers l'atmosphère. En perspective, de nouvelles expériences complémentaires, basées sur l'analyse par génomique comparative des souches chlorométhane-dégradantes également effectuée dans le cadre du projet et sur une analyse par séquençage à haut-débit initiée dans ce travail, sont proposées pour améliorer la compréhension des mécanismes d'adaptation des bactéries chlorométhanedégradantes dans la phyllosphère. Mots-clés : chlorométhane, bactéries méthylotrophes, Methylobacterium extorquens, Arabidopsis thaliana, génomique bactérienne, bio-rapporteur, HOL1, cmuA. period. You have been very supportive, encouraging, kind and a good instructor. I owe you a great deal of appreciation for all of your help not only in lab but also in other matters.

I would also like to thank Dr. Hubert Schaller, co-supervisor of my thesis for all his advices and suggestions. Thanks for all the things I learned from you.

A lot of thanks to Françoise. Your nice ideas, creative suggestions and valuable criticism were always handy and beneficial for me towards the progressive directions of my work.

Merci beaucoup à Thierry for all his help and guidance in experimental work, writing and help in French language.

I wish to thank Christelle and Yousra for all their technical help and maintaining a proper environment for work. I would also like to thank the former PhD students of the group: Christian, Sandro and Emilie for all their assistance and help during my early days in the lab.

Thanks also to the new PhD (Pauline, Omniea) and master students for maintaining a good environment in the lab. Thanks to Eidth, Vincent (from Hubert's lab) and Marta for their help while working with plants. I also appreciate Nathalie and Sandrine for all the administration work they did for me. III I wouldn't miss the opportunity to thank all my friends Umair, Khurram, Ishtiaq, Usman, Jawad, Qadeer, Afzaal, Umer, Mubashir, Atif, Sajid, Naeem, Faheem, Adnan, Imran, Mehboob, Madha, Azeem bhai, and Sarfraz who had been always with me in different times.

I sincerely appreciate Higher Education Commission Pakistan for providing me opportunity for studying in Europe and financially supporting me, otherwise it would have been only an unfulfilled dream.

I hardly can express my affection and gratitude to my family: Ammi, Abbu, Adnan, Naili, Shamaila and Baji and my dear wife (meri moti aalli) Sandly and her parents for their extreme care, love and selfless devotion for me. I would never have been reached to this level without you. In the end, I would like to thank all the persons who have been supportive and well-wisher in my life. The gene coding for YFP TD-GC-MS Thermal Desorption GC-MS VOCs

IV

Abbreviations

Résumé

Le chlorométhane (CH 3 Cl) est un composé organo-halogéné volatil, le plus abondant dans l'atmosphère. Il est responsable d'au moins 15% de la destruction de l'ozone stratosphérique causée par les composés halogénés. Les émissions mondiales de chlorométhane sont estimées entre 4,1 à 4,4 Tg par an, dont moins de 10% sont dues aux activités industrielles. Les sources naturelles de chlorométhane sont notamment les plantes, en particulier les parties aériennes des plantes (la phyllosphère), les marais salants, la combustion de la biomasse et les océans. La contribution du compartiment végétal aux émissions de chlorométhane n'a cependant pas encore été complètement évaluée. Toutefois, une méthyltransférase impliquée dans la production de chlorométhane a été isolée à partir des feuilles de Brassica oleracea, et chez Arabidopsis thaliana, l'implication d'un gène HOL1 (harmless to ozone layer) dans la production de chlorométhane a été mise en évidence. Les puits les plus importants pour le chlorométhane incluent l'oxydation par les radicaux hydroxyles dans la troposphère, les pertes dans les eaux océaniques polaires et vers la stratosphère, ainsi que l'absorption dans le sol et la dégradation bactérienne.

Certaines bactéries méthylotrophes sont capables d'utiliser le chlorométhane comme seule source de carbone et d'énergie. Ainsi, des bactéries chlorométhane-dégradantes ont été isolées à partir de différents environnements tels que les sols, les boues activées et l'environnement marin. Ces bactéries sont phylogénétiquement variées et appartiennent à un large éventail de genres dont Methylobacterium, Hyphomicrobium, Aminobacter, Roseovarius et Acetobacterium. Des études biochimiques ont permis, dans un premier temps, d'élucider la voie de dégradation du chlorométhane chez la souche Methylobacterium extorquens CM4. Dans un deuxième temps, un ensemble des gènes essentiels à la croissance avec le chlorométhane, appelés cmu (chloromethane utilisation), a été identifié. L'expression spécifique de cmuA, induite par le chlorométhane, a été mise en évidence. Les gènes cmu sont très répandus parmi les bactéries chlorométhane-dégradantes, et le gène cmuA est le plus conservé au sein des différentes souches. De ce fait, le gène cmuA est utilisé comme un gène marqueur de la dégradation du chlorométhane dans différents environnements.

L'objectif général de la thèse a été d'explorer le rôle possible des bactéries chlorométhanedégradantes de la phyllosphère comme filtre des émissions végétales de chlorométhane, en utilisant un modèle comprenant M. extorquens CM4 et A. thaliana.

Halomethanes

Halomethanes are derivatives of methane (CH 4 ) with one or more hydrogen atoms replaced by halogens (X): fluorine (F), chlorine (Cl), bromine (Br) or iodine (I) (Table 1.1). They are produced naturally or industrially and represent major pollutants of soil, water and air (http://www.epa.gov). Halomethanes and the chlorofluorocarbons (CFCs) have been amongst the commercially most important halocarbons, and have found widespread applications as cleaning agents; solvents, reactants, and coolants for refrigerators and as propellants for aerosols [START_REF] Benhadid-Dib | Search for an adequate refrigerant[END_REF]. These inexpensive and chemically inert compounds were deemed ideal for industrial processes [START_REF] Benhadid-Dib | Search for an adequate refrigerant[END_REF] and references cited therein). In particular, the chlorinated methanes chloromethane (CH 3 Cl), dichloromethane (CH 2 Cl 2 ), trichloromethane (chloroform, CHCl 3 ) and tetrachloromethane (CCl 4 ) have been produced and used extensively in industry throughout the 20 th century.

Nevertheless; the use of tetrachloromethane is now very strictly regulated, and significant efforts to reduce the use of dichloromethane and chloroform are ongoing (http://www.eurochlor.org).

The extensive use and the high volatility of these compounds (chloromethane is a gas while the three other chlorinated methanes exist in the liquid state under standard conditions of temperature and pressure, Table 1.1) have led to their immoderate entry into the atmosphere.

Concerns that such emissions may cause environmental problems, particularly with respect to their detrimental effects on stratospheric ozone, have given these chemicals scientific attention from the 1970s onwards [START_REF] Prather | Stratospheric ozone depletion and future levels of atmospheric chlorine and bromine[END_REF]. Chlorinated methanes in the stratosphere are activated by ultraviolet (UV) light and the resulting halogen radicals react with ozone to form oxygen (O 2 ) (Fig. 1.1). This catalytic process (it is estimated that one chlorine atom can destroy over 100,000 ozone molecules, (http://www.epa.gov/ozone/science/process.html) is responsible for the formation of the ozone hole over Antarctica each spring (Clerbaux et al., 2007).

Chapter 1

6

Chloromethane in the atmosphere

Chloromethane is the most abundant chlorine-containing organic compound in the atmosphere and contributes to 16% of chlorine catalyzed ozone layer destruction in the stratosphere (Table 1.1) [START_REF] Montzka | Scientific assessment of ozone depletion: 2010. Global ozone research and monitoring project[END_REF]. Global average concentrations of chloromethane in the atmosphere are 550 ± 30 pptv (parts per trillion by volume), with an annual increase of 2. 3 -2.7 ± 1.2 pptv from 2004-2008. The World Meteorological Organization has classified it among the long-lived gases involved in ozone depletion with a half-life of over one year [START_REF] Montzka | Scientific assessment of ozone depletion: 2010. Global ozone research and monitoring project[END_REF]. Chloromethane is also important in the environment due to its global warming potential that is 17 -25 times higher than that of CO 2 (on a mass basis) [START_REF] Grossman | Radiative forcing calculations for CH 3 Cl and CH 3[END_REF]Schäfer et al., 2007).

Global chloromethane budget

Evaluation of the global budget of chloromethane has been investigated in many studies, and involve in particular the use of isotopic signatures (Keppler et al., 2005;Saito and Yokouchi, 2008) and 3D simulation models [START_REF] Xiao | Optimal estimation of the surface fluxes of methyl chloride using a 3-D global chemical transport model[END_REF]. The estimated total atmospheric burden anthropogenic source s such as industries and burning (Table 1.2) [START_REF] Xiao | Optimal estimation of the surface fluxes of methyl chloride using a 3-D global chemical transport model[END_REF]. Natural emissions of chloromethane originate mainly from living vegetation (Saito and Yokouchi, 2008), dead plant material and senescent leaves [START_REF] Hamilton | Chloride methylation by plant pectin: An efficient environmentally significant process[END_REF], wood rot fungi [START_REF] Moore | A pilot study of methyl chloride emissions from tropical woodrot fungi[END_REF] biomass burning [START_REF] Xiao | Optimal estimation of the surface fluxes of methyl chloride using a 3-D global chemical transport model[END_REF] and oceans and coastal waters [START_REF] Hu | Coastal emissions of methyl bromide and methyl chloride along the eastern Gulf of Mexico and the east coast of the United States[END_REF][START_REF] Xiao | Optimal estimation of the surface fluxes of methyl chloride using a 3-D global chemical transport model[END_REF].

With regard to sinks for atmospheric chloromethane, the dominant process for loss of atmospheric chloromethane is via reaction with photochemically produced OH -radicals (Table 1.2). The reaction of chloromethane with chlorine radicals in the marine boundary layer, loss to the stratosphere and polar cold oceans, and degradation by soil microorganisms are other sinks of chloromethane (Table 1.2) [START_REF] Montzka | Scientific assessment of ozone depletion: 2010. Global ozone research and monitoring project[END_REF].

Current estimates of sources (1.7 to 13.5 Tg y -1 ) and sinks (4.2 to 6.6 Tg y -1 ) vary widely (Table 1.2). Additional sources and sinks of chloromethane may exist but remain to be identified. Currently, there is a significant shortage of sources (>1000 Gg y -1 , Table 1.2).

While chloromethane emissions from tropical and subtropical plants, potentially estimated to contribute the major part (approx. 30 -50%) of global emissions of chloromethane, may in fact balance or even possibly outweigh current best estimates of total sinks (Keppler et al., 2005;Saito and Yokouchi, 2008). Evidence is now emerging from microbial studies (Miller et al., 2004;Schäfer et al., 2007) for a much larger bacterial sink for chloromethane (Keppler et al., 2005). Following the large reductions of anthropogenic emissions of many ozonedepleting gases resulting from the Montreal protocol [START_REF] Montzka | Scientific assessment of ozone depletion: 2010. Global ozone research and monitoring project[END_REF], halogenated gases of uncontrolled emissions from natural sources such a chloromethane have now become the major sources of active chlorine in the stratosphere. It therefore appears important to analyze natural sources and sinks in the environment more thoroughly to obtain reliable predictions of the global budget of chloromethane and its implications for stratospheric ozone.

Chapter 1 a Best estimates of sources and sinks (Keppler et al., 2005) b Range of strengths of sources and sinks (Clerbaux et al., 2007) Chapter 1 10

Vegetation as an important source of C-1 compounds

Plants release a considerable proportion of the carbon fixed by photosynthesis back into the atmosphere or into the soil. Terrestrial plants are an important source of reactive volatile organic compounds (VOCs) in the environment such as e.g. methanol, isoprene, monoterpenes, methane (Table 1. 3). The source strength of these biogenic VOCs exceeds that of organic volatile emissions due to human activities by an order of magnitude (Guenther et al., 2012 and references cited therein). Increasing evidence indicates that these compounds may have an important role in the adaptation of plants to changing climate, and these VOCs significantly influence atmospheric processes [START_REF] Hartikainen | Impact of elevated temperature and ozone on the emission of volatile organic compounds and gas exchange of silver birch (Betula pendula Roth)[END_REF]. Therefore, possible changes in the emissions of these volatile compounds from plants are of high importance for predicting future climate. The mechanisms by which chloromethane and methanol are produced by higher plants in leaf tissues in particular are described in the following.

Biosynthesis of chloromethane in plants

Considering the global importance of emissions of chloromethane from living vegetation, surprisingly few investigations aiming at identifying specific plant sources have been reported so far. [START_REF] Yokouchi | Identification of methyl chloride-emitting plants and atmospheric measurements on a subtropical island[END_REF] conducted a survey on an island covered with subtropical forest, and identified 33 species from a variety of families as strong chloromethane-emitting plants (0.01 -2.8 µg g -1 dry weight h - Vitex rotundifolia, Vitex trifolia, and Excoecaria agalloch, each with chloromethane emission rates exceeding 1 µg g -1 dry weight h -1 [START_REF] Yokouchi | Identification of methyl chloride-emitting plants and atmospheric measurements on a subtropical island[END_REF]. The capacity to emit chloromethane was shared at the genus level but not always at the higher family level [START_REF] Yokouchi | Identification of methyl chloride-emitting plants and atmospheric measurements on a subtropical island[END_REF]. Another study (Saito and Yokouchi, 2008) reported 11 glasshousegrown tropical plant species emitting chloromethane, and measured their stable carbon isotopic composition of chloromethane. Saito et al. (2008) also investigated chloromethane fluxes at leaf and canopy scale in a tropical forest and reported that 25 out of 117 plant species were chloromethane emitters.

The mechanism of chloromethane production was not investigated in these studies. It is generally assumed, however, that it may be similar to that characterized in model plants, which is described in the following.

HOL1: a gene involved in chloromethane production in Arabidopsis thaliana

The biochemical basis of chloromethane emissions was first described by [START_REF] Wuosmaa | Methyl-chloride transferase -a carbocation route for biosynthesis of halometabolites[END_REF] who reported a chloride methyltransferase capable of methylating chloride (Cl -) in Mesembryanthemum crystallinum. Involvement of this enzyme suggested that it could be a part of salt tolerance mechanism in halophytes. Another methyltransferase purified from cabbage plant (Brassicaceae family) was able to catalyze the methylation of halides (X -), sulphide (HS -), and thiocyanate (SCN -) on the same active site, and named halide/sulphide/thiol methyltransferase [START_REF] Saini | Biosynthesis of halomethanes and methanethiol by higher-plants via a novel methyltransferase reaction[END_REF]Attieh et al., 2000). It was suggested that this enzyme might help clearance of toxic sulfur compounds resulting from glucosinolate breakdown in plants upon tissue damage (Fig. 1.2A) (Attieh et al., 2000).

Finally, Rhew et al. (2003) characterized the genetic mechanism of production of chloromethane and other methyl halides (bromomethane and iodomethane) in the small flowering plant Arabidopsis thaliana (mouse-ear cress), the best studied model plant at the molecular level (http://www.arabidopsis.org/index.jsp, The Arabidopsis Information Resource). These authors identified the gene encoding the enzyme primarily responsible for the production of chloromethane, designated HOL for HARMLESS TO OZONE LAYER, one of the consequences of loss of gene function in a HOL mutant. Of the three variants HOL1, HOL2 and HOL3 (also denoted as AtHOL1, AtHOL2 and AtHOL3) found in A. thaliana Chapter 1 13 (Rhew et al., 2003;[START_REF] Nagatoshi | Characterization of three halide methyltransferases in Arabidopsis thaliana[END_REF], HOL1 is primarily responsible for more than 85% of methyl halide production (Rhew et al., 2003). The HOL1 gene appears quite conserved in plants, suggesting that chloromethane production by vegetation may be a quite widespread property (Rhew et al., 2003;[START_REF] Nagatoshi | Characterization of three halide methyltransferases in Arabidopsis thaliana[END_REF]. Rhew et al. (2003) showed that the HOL1 protein belonged to a group of methyltransferases capable of catalyzing the S-adenosyl-L-methionine (SAM)-dependent methylation of chloride (Cl -), bromide (Br -), and (I -) to produce methyl halides (Fig 1 .2B). They also showed that chloromethane production was largely eliminated in mutant plants with HOL1 gene disrupted. However, the physiological advantage of chloromethane production by HOL1, if any, remains to be clarified. Recent investigations strongly suggested a physiological role for HOL1, by showing that the enzyme was highly active with thiocyanate SCN -(a pseudohalide), resulting in methylthiocyanate (CH 3 SCN) synthesis (Fig 1 .2A) (Nagatoshi and Nakamura, 2009). The authors observed that substantial amounts of SCN -were released by A. thaliana upon tissue damage, and that SCN -was largely derived from myrosinasemediated hydrolysis of glucosinolates (Fig. 1.2A). Bacterial growth assays indicated that conversion of SCN -into CH 3 SCN dramatically increased antibacterial activities against A. thaliana pathogens which invade damaged tissue at the wound site (Nagatoshi and Nakamura, 2009).

Biosynthesis of methanol in plants

Compared to methanol, however, chloromethane only represents a very minor emission from the aerial parts of plants (Table 1.3). The remodelling of cell walls during plant growth is at the origin of plant emissions of methanol (Galbally and Kirstine, 2002, and references cited therein). The main framework of the cell wall is formed by a cellulose/hemicellulose network, along with small amounts of structural proteins [START_REF] Cosgrove | Comparative structure and biomechanics of plant primary and secondary cell walls[END_REF]. A pectin matrix acts to hold the structure together [START_REF] Carpita | Structural models of primary cell walls in flowering plants -consistency of molecular structure with the physical properties of the walls during growth[END_REF]. Plasticity, rigidity and pore size of cell walls are controlled by the nature of the bonds between adjacent pectin polymers [START_REF] Galbally | The production of methanol by flowering plants and the global cycle of methanol[END_REF]. Polygalacturonic acid (PGA), a homopolymer of α-D- [1][2][3][4]-galactosyluronic acid units with different degrees of methyl esterification, mainly constitutes the basic structure of most pectins (Fig. 1.3) [START_REF] Galbally | The production of methanol by flowering plants and the global cycle of methanol[END_REF].

Along the formation of new cellulose microfibrils during cell wall elongation resulting in cell

Chapter 1 14 growth, continuous adaptations of the pectin matrix are also required to increase cell wall plasticity [START_REF] Galbally | The production of methanol by flowering plants and the global cycle of methanol[END_REF]. This is mediated by differential methylation of PGA carboxylic acid moieties [START_REF] Galbally | The production of methanol by flowering plants and the global cycle of methanol[END_REF]. Once the process of cell wall extension is complete, demethylation of PGA by the action of the enzyme pectin methylesterase (PME) yields galacturonic acid residues and methanol (Fig. 1.3) [START_REF] Galbally | The production of methanol by flowering plants and the global cycle of methanol[END_REF]. (A) Polygalacturonic acid is demethylated by pectin methylesterase (PME) to produce methanol and galacturonic acid residues during elongation of the primary cell wall. (B) General structure of the backbone of pectin consisting of polygalacturonic acid (a homopolymer of α-D- [1][2][3][4]-galactosyluronic acid units) with different degrees of methyl esterification (adapted from [START_REF] Galbally | The production of methanol by flowering plants and the global cycle of methanol[END_REF].

A

Chapter 1

15

Methanol and other carbonaceous compounds such as chloromethane which are released from plants may be emitted to the atmosphere, but may also serve as the carbon source for growth of plant-associated microbes in the aerial parts of plants leaves, the phyllosphere [see section 1.7 further below]. In the latter environment, bacteria capable of using compounds containing a single carbon atom are of particular relevance, and are presented in the next section [Section 1.4].

Methylotrophy

Methylotrophy is defined as the ability of organisms to grow with organic compounds containing a single carbon atom (C-1) or lacking C-C bonds such as methanol. Other methylotrophic C-1 substrates include methane, formaldehyde, methylamine, methylated sulfur compounds and halogenated methanes. Methylotrophic microorganisms are ubiquitous and can be found in soils and sediments [START_REF] Kolb | Aerobic methanol-oxidizing bacteria in soil[END_REF], fresh and marine waters [START_REF] Neufeld | Marine methylotrophs revealed by stable-isotope probing, multiple displacement amplification and metagenomics[END_REF], roots and leaves of plants (Delmotte et al., 2009;[START_REF] Knief | Metaproteogenomic analysis of microbial communities in the phyllosphere and rhizosphere of rice[END_REF], and air [START_REF] Deleon-Rodriguez | Microbiome of the upper troposphere: Species composition and prevalence, effects of tropical storms, and atmospheric implications[END_REF]. Methylotrophic microbes can be found in all three domains of life, i.e. bacteria, Archaea (e.g. methylotrophic methanogens) and Eukarya (e.g. yeasts growing with methanol), but methylotrophic metabolism has mainly been investigated in bacteria (Chistoserdova et al., 2009;[START_REF] Kolb | Aerobic methanol-oxidizing bacteria in soil[END_REF]Kawaguchi et al., 2011;[START_REF] Peyraud | Coconsumption of methanol and succinate by Methylobacterium extorquens AM1[END_REF]. More than 150 methylotrophic bacterial species belonging to the Alpha-, Beta-, Gamma-Proteobacteria, Verrucomicrobia, Cytophagales, Bacteroidetes, Firmicutes, and Actinobacteria, have been described so far [START_REF] Kolb | Aerobic methanol-oxidizing bacteria in soil[END_REF]. These bacteria can be aerobic or anaerobic, either strictly or facultatively [START_REF] Lidstrom | Aerobic methylotrophic prokaryotes[END_REF]. In terms of carbon usage, methylotrophic bacteria are categorized into two main groups: those which can utilize methane, called methanotrophs, and those which cannot, which are often called nonmethanotrophic methylotrophs [START_REF] Lidstrom | Aerobic methylotrophic prokaryotes[END_REF]. Bacteria capable of utilizing methane gas as the only source of carbon and energy possess an enzyme, methane monooxygenase (MMO), involved in the oxidation of methane to methanol [START_REF] Hakemian | The biochemistry of methane oxidation[END_REF]. This enzyme exists in two different forms: soluble methane monooxygenase and membrane bounded (or particulate) methane monooxygenase [START_REF] Lidstrom | Aerobic methylotrophic prokaryotes[END_REF][START_REF] Semrau | Facultative methanotrophy: false leads, true results, and suggestions for future research[END_REF].

Methanotrophs are principally obligate methylotrophs; and only a few genera so far (e.g.

Methylocella, Methylocapsa, and Methylocystis) can also utilize multicarbon compounds

Chapter 1

16 [START_REF] Dedysh | Methylocella species are facultatively methanotrophic[END_REF][START_REF] Kolb | Aerobic methanol-oxidizing bacteria in soil[END_REF][START_REF] Semrau | Facultative methanotrophy: false leads, true results, and suggestions for future research[END_REF]. Basing on their remarkable internal membranes, carbon assimilation pathways and 16S rRNA gene sequences, methanotrophs have been classified into two groups [START_REF] Hanson | Methanotrophic bacteria[END_REF][START_REF] Lidstrom | Aerobic methylotrophic prokaryotes[END_REF] pathways for carbon assimilation. However, some methylotrophic bacteria use another pathway, the ribulose bis-phosphate pathway, instead (Chistoserdova et al., 2009).

Methylotrophic bacteria are quite often facultative methylotrophs, and may use different multi-carbon compounds for their growth (Chistoserdova et al., 2009).

Chloromethane-degrading bacteria

While cometabolic oxidation of chloromethane was observed for nitrifying bacteria [START_REF] Rasche | Biodegradation of halogenated hydrocarbon fumigants by nitrifying bacteria[END_REF] and methanotrophs [START_REF] Han | Chloromethane stimulates growth of Methylomicrobium album BG8 on methanol[END_REF] and is attributed to the activity of ammonium-or methane monooxygenase, respectively, different types of methylotrophic bacteria capable of utilizing chloromethane as the only source of carbon and energy were isolated over the years (Table 1.4) (Schäfer et al., 2007). The first aerobic bacterium for which growth with chloromethane was described was Hyphomicrobium sp. strain MC1, isolated from a sewage treatment plant in Switzerland in 1986 (Hartmans et al., 1986;Vuilleumier et al., 2011) and growing under aerobic conditions. In subsequent years, Acetobacterium dehalogenans (formerly named strain MC) [START_REF] Messmer | Methyl chloride metabolism of the strictly anaerobic, methyl chloride-utilizing homoacetogen strain MC[END_REF] and

Pseudomonas aeruginosa NB1 (Freedman et al., 2004) were described to utilize chloromethane anaerobically. (Schäfer et al., 2005) and Leisingeria methylohalidivorans MB2 [START_REF] Schaefer | Leisingera methylohatidivorans gen. nov., sp nov., a marine methylotroph that grows on methyl bromide[END_REF] were obtained from marine environments (Table 1.4). Strikingly, despite the fact that terrestrial plants represent the largest source of chloromethane, chloromethane-degrading bacteria had not been isolated from plant environments until the initiation of this project.

Methylotrophic pathways in Methylobacterium

Pink-pigmented, strictly aerobic and facultative methylotrophs of the genus Methylobacterium are perhaps the most studied methylotrophic bacteria, and have been investigated by a wide variety of classical microbiological, genetic, proteomic, metabolomic, and genomic approaches (see e.g. (Vuilleumier et al., 2009, and references cited therein;Muller et al., 2011a;[START_REF] Schneider | Oxalyl-coenzyme a reduction to glyoxylate is the preferred route of oxalate assimilation in Methylobacterium extorquens AM1[END_REF][START_REF] Roselli | The 380 kb pCMU01 plasmid encodes cloromethane utilization genes and redundant genes for vitamin B12-and tetrahydrofolate-dependent chloromethane metabolism in Methylobacterium extorquens CM4: A proteomic and bioinformatics study[END_REF]. Three major aspects of methylotrophic metabolism can be differentiated: (i) initial oxidation of C1 compounds, (ii) assimilation of carbon into biomass and (iii) complete oxidation of carbon to CO 2 for energy production (Fig. 1.4) (Chistoserdova et al., 2009). Formaldehyde (HCHO) usually constitutes the central intermediate compound in methylotrophic metabolism in Methylobacterium as well as in other methylotrophic microorganisms, and results from initial oxidation reactions of diverse C-1 compounds (Fig. 1.4) (Vuilleumier et al., 2009, and references cited therein).

In the case of methanol, pyrrolo-quinoline quinone (PQQ) dependent methanol dehydrogenase is involved (Fig. 1.4) [START_REF] Lidstrom | Aerobic methylotrophic prokaryotes[END_REF], although the importance of an alternative XoxF-dependent reaction has recently also been demonstrated [START_REF] Schmidt | Functional investigation of methanol dehydrogenase-like protein XoxF in Methylobacterium extorquens AM1[END_REF][START_REF] Skovran | XoxF Is required for expression of methanol dehydrogenase in Methylobacterium extorquens AM1[END_REF].

Fig. 1.4. Metabolism of C-1 compounds in Methylobacterium extorquens

Methylobacterium lacks the ability to transform methane or to utilize it for growth. Different C-1 substrates methanol (CH 3 OH), methylamine (CH 3 NH 2 ), dichloromethane (CH 2 Cl 2 ) and chloromethane (CH 3 Cl) are metabolized to synthesize biomass through the serine cycle (reactions in light blue) and oxidized to CO 2 to produce energy (reactions in dark blue). The specific pathway for chloromethane utilization (bold arrows) in Methylobacterium extorquens CM4 is different from the canonical one, which involves a tetrahydromethanopterindependent pathway [START_REF] Vorholt | Cofactor-dependent pathways of formaldehyde oxidation in methylotrophic bacteria[END_REF]Marx et al., 2012) Similarly, the oxidation of methylamine was shown to be mainly performed by TQQdependent methylamine dehydrogenase in some Methylobacterium extorquens [START_REF] Lidstrom | Aerobic methylotrophic prokaryotes[END_REF], whereas other strains appear to use an alternative enzymatic system involving glutamyl-methylamide as the intermediate [START_REF] Latypova | Genetics of the glutamate-mediated methylamine utilization pathway in the facultative methylotrophic beta-proteobacterium Methyloversatilis universalis FAM5[END_REF] and unpublished data from the team). For dichloromethane, dichloromethane dehydrogenase / glutathione S-transferase affords the hydrolytic transformation of dichloromethane to formaldehyde with release of hydrochloric acid (Fig. 1.4) (Muller et al., 2011a).

Formaldehyde produced as a result of initial oxidations of C-1 compounds must be metabolized rapidly and efficiently because of its toxicity [START_REF] Vorholt | Cofactor-dependent pathways of formaldehyde oxidation in methylotrophic bacteria[END_REF][START_REF] Marx | Formaldehyde-detoxifying role of the tetrahydromethanopterin-linked pathway in Methylobacterium extorquens AM1[END_REF].

Part of this formaldehyde is assimilated into biomass through the serine cycle (Fig. 1.4), and the remainder is oxidized to CO 2 for energy production [START_REF] Lidstrom | Aerobic methylotrophic prokaryotes[END_REF]. Two pathways have been characterized in Methylobacterium for oxidation of formaldehyde. One pathway involves tetrahydrofolate (H 4 F, Fig. 1.5) while the other depends on tetrahydromethanopterin (H 4 MPT,Fig. 1.5) and formaldehyde is oxidized to CO 2 through formic acid (HCOOH) for energy production (Fig. 1.4) [START_REF] Vorholt | Cofactor-dependent pathways of formaldehyde oxidation in methylotrophic bacteria[END_REF]. The pathway involving H 4 F was shown to be mainly funneling carbon into the serine cycle for biomass production (Fig. 1.4) with methylene-H 4 F (CH 2 =H 4 F) as the key intermediate [START_REF] Marx | Flux analysis uncovers key role of functional redundancy in formaldehyde metabolism[END_REF]. Rather, it is the H 4 MPT-dependent pathway (Fig. 1.4) which is the preferred pathway for the oxidation of formaldehyde into CO 2 and energy production in Methylobacterium [START_REF] Vorholt | Novel formaldehyde-activating enzyme in Methylobacterium extorquens AM1 required for growth on methanol[END_REF][START_REF] Vorholt | Cofactor-dependent pathways of formaldehyde oxidation in methylotrophic bacteria[END_REF]. In the case of chloromethane, however, the situation was shown to be quite different, in that chloromethane is directly transformed to CH 2 =H 4 F without intermediate formaldehyde production (Fig. 1.4) (Schäfer et al., 2007, and references cited therein).as described in the following section. At the end of the 1990s, biochemical and genetic studies were carried out to elucidate the utilization of chloromethane in Methylobacterium extorquens CM4 and suggested the existence of a novel and metabolic pathway (Fig. 1.6) (Vannelli et al., 1998;Vannelli et al., 1999). Vannelli et al. (1998) first observed that Methylobacterium extorquens CM4 metabolized chloromethane as the only source of carbon and energy, releasing stoichiometric amounts of chloride in the medium during growth. It was observed that 1 mole chloromethane was oxidized to 1 mole carbon dioxide, producing 1 mole of hydrochloric acid and consuming 1.5 moles of oxygen. Proteomic analysis by one-dimensional SDS-PAGE showed that two proteins of 67 kDa and 36 kDa were induced in cells of CM4 grown

O OH O N H N N H N H N H 2 N H O OH OH OH O H OH C-1 Tetrahydromethanopterin (H 4 MPT) N H O N H COOH N N H N H N H 2 N H O COOH COOH C-1

Tetrahydrofolate (H 4 F)

Fig. 1.5. Structures of cofactors involved in the metabolism of C-1 compounds.

The asterisk with C-1 shows the attachment position of methyl, methylene, methenyl and formyl groups.

Chapter 1 22 with chloromethane, and were not detected in methanol-grown cells (Vannelli et al., 1998).

Random mutagenesis of strain CM4 with a miniTn5 transposon was then performed and led to identify mutants unable to grow with chloromethane (Fig. 1.6) (Vannelli et al., 1998).

Mutants unable to grow with chloromethane were able to utilize other C-1 substrates such as methanol, methylamine and formate, while mutants defective in the utilization of these compounds could still grow normally with chloromethane (Vannelli et al., 1998). This suggested that chloromethane was metabolized in strain CM4 strain by reactions different from those involved in the metabolism of other C-1 compounds (Fig. 1.4). Further analysis of mutants unable to grow with chloromethane allowed the identification of chloromethaneutilization (cmu) genes which mapped to two different approx. 10 kb DNA fragments (Fig. 1.6), and to define a specific pathway for chloromethane utilization (Fig. 1.4) (Vannelli et al., 1999). Sequence analysis showed that two of the genes essential for chloromethane utilization corresponded to the previously identified 67 kDa and 36 kDa proteins induced by chloromethane (Vannelli et al., 1998). Thes genes were predicted to encode two different types of methyltransferases and were named cmuA and cmuB respectively (Vannelli et al., 1999). The corresponding protein CmuA is a two-domain protein; with an N-terminal Genes responsible for dehalogenation of chloromethane (green), for growth with chloromethane (blue), and genes involved in the metabolism of cobalamine (orange) required for the dehalogenation, are localized in two clusters, subsequently found to be closely associated on megaplasmid pCMU01 (380 kb) of strain CM4 (cf. Chapter 2 below and Marx et al., 2012). Triangles indicate the insertion sites of miniTn5 transposon in characterized mutants and red flashes indicate the chloromethane-induced promoter regions that were subsequently identified (Studer et al 2002). (Fig. adapted fromVannelli et al., 1999) Chapter 1 23 methyltransferase domain at and a C-terminal corrinoid-binding domain, similar to methyl transfer systems of methylotrophic archaeal methanogens [START_REF] Burke | Clustered genes encoding the methyltransferases of methanogenesis from monomethylamine[END_REF]Vannelli et al., 1999). Protein CmuB showed methyltransferase activity with tetrahydrofolate as suggested from sequence similarity searches (Studer et al., 1999). Both proteins CmuB (Studer et al., 1999) and CmuA (Studer et al., 2001) were purified to homogeneity and allowed to demonstrate that chloromethane dehalogenase of strain CM4 required two subsequent reactions involving these two proteins (Studer et al., 2001) et al., 2001). The function of cmuC, a gene essential for growth with chloromethane (Fig. 1.6), and of its close homolog cmuC2 located upstream of cmuA, are still unknown.

cmuB metF cobQ cobD cmuC cbiD cobC 1kb cmuA cmuC2 purU folD folC cobU
Chapter 1

24

The resulting CH 3 -H 4 F is then transferred to formate by a tetrahydrofolate-dependent pathway that appears distinct from those found in Methylobacterium strains (Fig. 1.4) [Section 1.4.2]. This was inferred from the phenotype of minitransposon mutants of strain CM4 in metF, folD and purU genes (Fig. 1.6) coding for homologs of methylene-H 4 F reductase, methylene-H 4 F dehydrogenase/methenyl-H 4 F cyclohydrolase and formyl-H 4 F hydrolase, respectively (Vannelli et al., 1999). These genes were found nearby cmuA or cmuB genes (Fig. 1.6 ) and the mutants of these genes were also unable to grow with chloromethane, but exhibited wild-type levels of chloromethane dehalogenase activity (Vannelli et al., 1999). As shown in Fig. 1.4, the chloromethane-utilization pathway is similar to H 4 F dependent oxidation of formaldehyde to CO 2 defined by other enzymes methylene-H 4 F dehydrogenase (MtdA) [START_REF] Vorholt | The NADPdependent methylene tetrahydromethanopterin dehydrogenase in Methylobacterium extorquens AM1[END_REF] and methenyl-H 4 F cyclohydrolase (FchA) [START_REF] Pomper | A methenyl tetrahydromethanopterin cyclohydrolase and a methenyl tetrahydrofolate cyclohydrolase in Methylobacterium extorquens AM1[END_REF]. In addition, gene fusion studies showed chloromethane-dependent expression of metF, folD, purU genes (Studer et al., 2002).

Following these studies elucidating the detailed metabolic pathway of chloromethane, Methylobacterium extorquens CM4 became the model organism for the study for bacterial degradation of chloromethane. Its genome sequence was subsequently determined (Marx et al., 2012) and analyzed, allowing global proteomic [START_REF] Roselli | The 380 kb pCMU01 plasmid encodes cloromethane utilization genes and redundant genes for vitamin B12-and tetrahydrofolate-dependent chloromethane metabolism in Methylobacterium extorquens CM4: A proteomic and bioinformatics study[END_REF] and transcriptomic (Bringel et al., work in progress), investigations of chloromethane utilization in Methylobacterium, as well as application of strain CM4 to address this topic in the context of the most relevant niche with regard to the chloromethane budget, the phyllosphere

The phyllosphere as a microbial niche

The parts of the plants in immediate contact with air (mainly leaves) are defined as the phyllosphere (Fig. 1.8) [START_REF] Vorholt | Microbial life in the phyllosphere[END_REF]. Due to the presence of various carbon compounds including methanol but also sugars leaching from the interior of the plant [START_REF] Mercier | Role of leaf surface sugars in colonization of plants by bacterial epiphytes[END_REF][START_REF] Lindow | Microbiology of the phyllosphere[END_REF], the phyllosphere is a hospitable habitat for microorganisms. Estimated at 1 x 10 9 km² globally, leaf surfaces represent a very important surface on earth, i.e. approximately twice the land surface area [START_REF] Vorholt | Microbial life in the phyllosphere[END_REF]. Global bacterial population in the phyllosphere habitat is estimated to be 10 26 [START_REF] Vorholt | Microbial life in the phyllosphere[END_REF] references cited therein). Bacteria are the most abundant microbes in the phyllosphere at 10 6

Chapter 1 25 and yeasts are also observed [START_REF] Whipps | Phyllosphere microbiology with special reference to diversity and plant genotype[END_REF]. Microbes can be found on the plant surface as epiphytes and also within plant tissues as endophytes [START_REF] Whipps | Phyllosphere microbiology with special reference to diversity and plant genotype[END_REF][START_REF] Vorholt | Microbial life in the phyllosphere[END_REF].

1.6.1. Bacterial diversity of the phyllosphere Phyllosphere bacteria have been explored in a number of culture-dependent [START_REF] Enya | Culturable leaf-associated bacteria on tomato plants and their potential as biological control agents[END_REF][START_REF] Ceballos | Cultivable bacteria populations associated with leaves of banana and plantain plants and their antagonistic activity against Mycosphaerella fijiensis[END_REF] and culture-independent (Delmotte et al., 2009;[START_REF] Knief | Metaproteogenomic analysis of microbial communities in the phyllosphere and rhizosphere of rice[END_REF] studies, revealing the abundance of Proteobacteria in the phyllosphere. Studies on culturable epiphytes and endophytes of Oryza sativa (rice) showed that lphaproteobacteria made up more than 70 % of the microbial compartment [START_REF] Elbeltagy | Isolation and characterization of endophytic bacteria from wild and traditionally cultivated rice varieties[END_REF][START_REF] Mano | Culturable endophytic bacterial flora of the maturing leaves and roots of rice plants (Oryza sativa) cultivated in a paddy field[END_REF]. It was reported that culturable epiphytic and endophytic isolates from the rice phyllosphere mainly belonged to Sphingomonas (28% of endophytes, 27% of epiphytes), Methylobacterium (19% of endophytes, 12% of epiphytes), and Bacillus (13% of endophytes, 12% of epiphytes) [START_REF] Mano | Culturable endophytic bacterial flora of the maturing leaves and roots of rice plants (Oryza sativa) cultivated in a paddy field[END_REF]. [START_REF] Yang | Microbial phyllosphere populations are more complex than previously realized[END_REF] were the first to use culture- thaliana and rice plants. They also observed the dominance of Proteobacteria phylum.

Alphaproteobacteria was the most dominant class accounting for more than 70% abundance of the bacterial community in all samples investigated (Delmotte et al., 2009;[START_REF] Knief | Metaproteogenomic analysis of microbial communities in the phyllosphere and rhizosphere of rice[END_REF][START_REF] Vorholt | Microbial life in the phyllosphere[END_REF]. Bacteroidetes was the second most abundant phylum in the soybean, clover and A. thaliana communities (Delmotte et al., 2009) while in the phyllosphere of rice plant, the class Actinobacteria was the most represented class [START_REF] Knief | Metaproteogenomic analysis of microbial communities in the phyllosphere and rhizosphere of rice[END_REF].

Agrobacterium, Burkholderia, Methylobacterium, Mycobacterium, Pseudomonas, Sphingobium, Sphingomonas and Xanthomonas were of the most abundantly colonizing genera in all phyllosphere samples (Delmotte et al., 2009;[START_REF] Knief | Metaproteogenomic analysis of microbial communities in the phyllosphere and rhizosphere of rice[END_REF][START_REF] Vorholt | Microbial life in the phyllosphere[END_REF]. Knief et al. (2010b) evaluated the effects of geographical site, host plant species, time and the presence of other phyllosphere bacteria on Methylobacterium community composition.

They observed different Methylobacterium communities in the phyllosphere of the same plants from different locations, and also observed a similar Methylobacterium community on different plants growing at the same site, suggesting that site-specific factors had a stronger impact on the Methylobacterium community composition (Knief et al., 2010b). In contrast, [START_REF] Redford | The ecology of the phyllosphere: geographic and phylogenetic variability in the distribution of bacteria on tree leaves[END_REF]) observed more differences in phyllosphere communities on different tree species from the same site, than those of the same tree species from different locations.

Although the species/site dependent bacterial diversity in the phyllosphere is substantial, it appears to be lower as compared to the bacterial diversity in the rhizosphere or bulk soil [START_REF] Vorholt | Microbial life in the phyllosphere[END_REF], and references cited therein).

1.6.2. Plant-bacterial relationships in the phyllosphere Bacteria inhabiting the phyllosphere can have beneficial, neutral or pathogenic effects on host plants [START_REF] Kutschera | Plant-associated methylobacteria as co-evolved phytosymbionts: a hypothesis[END_REF]. During phyllosphere colonization, Methylobacterium bacteria benefit from the abundance of methanol (as carbon and energy source) released from plant leaves, while Sphingomonas (due to the presence of TonB-dependent receptors) can utilize various carbohydrates and sugars leached from plant leaves (Delmotte et al., 2009). It is believed that plant-bacteria interactions in the phyllosphere are often mutually advantageous.

Plants provide the carbon and other nutrients to bacteria, while bacteria impart beneficial effects on plant growth through one or more mechanisms directly or indirectly. For example, experiments showed that the growth of axenically cultured protonemal filaments of a moss Chapter 1 27 plant was promoted by methylobacteria [START_REF] Hornschuh | Epiphytic bacteria associated with the bryophyte Funaria hygrometrica: Effects of Methylobacterium strains on protonema development[END_REF]; This effect was attributed to the production of cytokinins excreted by the bacteria into the culture medium and taken up by the plant cells. An auxin (indole-3-acetic acid) based plant-growth promoting effect of Methylobacterium was also observed [START_REF] Hornschuh | Moss-associated methylobacteria as phytosymbionts: an experimental study[END_REF]. Other beneficial aspects of phyllosphere bacteria to plants include nitrogen fixation [START_REF] Furnkranz | Nitrogen fixation by phyllosphere bacteria associated with higher plants and their colonizing epiphytes of a tropical lowland rainforest of Costa Rica[END_REF], regulation of ethylene through 1-aminocyclopropane-1-carboxylate deaminase activity in higher plants [START_REF] Chinnadurai | Characterization of 1aminocyclopropane-1-carboxylate deaminase producing methylobacteria from phyllosphere of rice and their role in ethylene regulation[END_REF], production of siderophores [START_REF] Idris | Bacterial communities associated with flowering plants of the Ni hyperaccumulator Thlaspi goesingense[END_REF][START_REF] Idris | Characterization of Ni-tolerant methylobacteria associated with the hyperaccumulating plant Thlaspi goesingense and description of Methylobacterium goesingense sp[END_REF], and reduction of phytotoxicity of heavy metals [START_REF] Baldi | Mercury, arsenic and boron resistant bacteria isolated from the phyllosphere as positive bioindicators of airborne pollution near geothermal plants[END_REF]. On the other hand, citrus canker, bacterial blight, bacterial spot on pepper and tomato, bacterial blight of rice, black rot of crucifers, brown spot of beans and crown-gall tumor are some of the diseases caused by phyllosphere-colonizing strains of Xanthomonas [START_REF] Xu | AvrAC(Xcc8004), a type III effector with a leucine-rich repeat domain from Xanthomonas campestris pathovar campestris confers avirulence in vascular tissues of Arabidopsis thaliana ecotype Col-0[END_REF], Pseudomonas [START_REF] Hirano | Bacteria in the leaf ecosystem with emphasis on Pseudomonas syringae -a pathogen, ice nucleus, and epiphyte[END_REF] and Agrobacterium [START_REF] Hu | Agrobacterium tumefaciens recognizes its host environment using ChvE to bind diverse plant sugars as virulence signals[END_REF]. Several isolates of Sphingomonas from phyllosphere have been reported to have a remarkable plant-protective effect by suppressing disease symptoms and reducing plant pathogens growth in A. thaliana [START_REF] Innerebner | Protection of Arabidopsis thaliana against leaf-pathogenic Pseudomonas syringae by Sphingomonas strains in a controlled model system[END_REF].A model system comprising of Sphingomonas spp., Pseudomonas syringae and Arabidopsis thaliana, was proposed to study plant protection affect of phyllosphere bacteria against palnt pathogens [START_REF] Innerebner | Protection of Arabidopsis thaliana against leaf-pathogenic Pseudomonas syringae by Sphingomonas strains in a controlled model system[END_REF]. Recently, mini-Tn5 transposon insertional mutants of Sphingomonas sp. Fr1 screened on the basis of their plant protective and plant colonizing traits suggested the possibility of nutrient competition along with other mechanisms to be further investigated, in the biocontrol activity of phyllosphere bacteria [START_REF] Vogel | forward genetic in planta screen for identification of plant-protective traits of Sphingomonas sp strain Fr1 against Pseudomonas syringae DC3000[END_REF].

Adaptation of bacteria to the phyllospheric environment

Bacteria on the surface of leaves are exposed to highly variable micro-and macroenvironments, resulting in irregular distribution and uneven growth rates [START_REF] Vorholt | Microbial life in the phyllosphere[END_REF].

Various factors such as climatic conditions, ultraviolet radiation from sunlight, the waxy characteristics of the leaf cuticle layer and other leaf features (e.g. veins, trichomes and hydathodes) which affect water and nutrient availability, and plant metabolites and antimicrobial compounds will influence the structure and the growth dynamics of bacterial communities in the phyllosphere [START_REF] Lindow | Microbiology of the phyllosphere[END_REF][START_REF] Vorholt | Microbial life in the phyllosphere[END_REF]. Plant genotypes may also affect bacterial colonization of the phyllosphere [START_REF] Whipps | Phyllosphere microbiology with special reference to diversity and plant genotype[END_REF]. It thus seems Chapter 1 28 clear that phyllospheric bacteria will have adaptive mechanisms to counteract the various stresses associated with life on aerial plant surfaces. For instance, formation of aggregates on leaf surfaces may provide bacteria with the ability to modify their local environment [START_REF] Whipps | Phyllosphere microbiology with special reference to diversity and plant genotype[END_REF]. Production of extracellular polysaccharides may also increase nutrient availability, protection against desiccation of epiphytic bacteria, and shield cells from antimicrobial compounds through the formation of biofilms [START_REF] Morris | The ecological significance of biofilm formation by plant-associated bacteria[END_REF][START_REF] Rigano | Biofilm formation, epiphytic fitness, and canker development in Xanthomonas axonopodis pv citri[END_REF]. Release of plant growth regulators (e.g. indole-3-acetic acid) associated with enhanced nutrient leakage from plants is another widespread adaptive mechanism in phyllosphere colonization [START_REF] Brandl | Contribution of indole-3-acetic acid production to the epiphytic fitness of Erwinia herbicola[END_REF]. Biosurfactants released by phyllosphere bacteria may also enhance nutrient availability by increasing wettability and substrate leaching [START_REF] Mousivand | Characterization and antagonistic potential of some native biofilm-forming and surfactant-producing Bacillus subtilis strains against six pathotypes of Rhizoctonia solani[END_REF], and references cited therein), and production of pigments afford protection from ultraviolet radiations (Jacobs et al., 2005). Further, the existence of bacteria capable of harvesting sunlight through bacteriorhodopsins (Atamna- et al., 2012b) and anoxygenic photosynthesis in the phyllosphere has been reported recently, suggesting yet another mechanism of adaptation to the phyllosphere under nutrient limited conditions (Atamna-Ismaeel et al., 2012a). In recent years, proteomic and transcriptomic studies have helped to better understand adaptive mechanisms of phyllosphere bacteria. For example, [START_REF] Gourion | A proteomic study of Methylobacterium extorquens reveals a response regulator essential for epiphytic growth[END_REF] identified 40 proteins which were up-regulated in M. extorquens specifically during phyllosphere colonization. These proteins include enzymes involved in C-1 metabolism, general stress proteins e.g.; heat-shock proteins, enzymes involved in DNA repair, and catalases and superoxide dimutases. In addition, a novel two-component response regulator named PhyR (phyllosphere induced regulator) was also discovered. PhyR was demonstrated to be essential for phyllosphere colonization and is responsible for the induction of several proteins specifically expressed during epiphytic growth [START_REF] Gourion | A proteomic study of Methylobacterium extorquens reveals a response regulator essential for epiphytic growth[END_REF][START_REF] Gourion | PhyR is involved in the general stress response of Methylobacterium extorquens AM1[END_REF].

Ismaeel
Chapter 1 29

Objectives of the PhD project

The degree to which the biosphere contributes to the atmospheric burden of certain climaterelevant trace gases such as chloromethane is poorly understood. On the one hand, plant phyllospheric bacteria may therefore also constitute a significant sink for chloromethane. In The general question at the basis of the project (Fig. 1.9) was whether epiphytic chloromethane-degrading bacteria in the phyllosphere may act as a filter for emissions of chloromethane by plants.

To address this question, the following three major experimental objectives were defined in this project:

1) Develop molecular tools for the detection of chloromethane-related processes in the phyllosphere of A. thaliana

2)

Determine if bacteria can be isolated from the phyllosphere that degrade thaliana, chosen for emitting different amounts of chloromethane as verified using the bioreporter developed in Chapter 3, is presented in Chapter 5.

An exploratory preliminary study of the bacterial diversity of the A. thaliana phyllosphere in different HOL1 gene variants of A. thaliana evaluated by 16S pyrosequencing is reported in Chapter 6.

The conclusions of the work performed are summarised in a final Chapter 7, in which the perspectives arising from this work are also presented.

Chapter 1

Chapter 2. Comparative genomics of chloromethanedegrading strains

This Chapter also includes two published articles: This chapter presents a summary of the main results of comparative genomic analyses performed during the thesis period that focused on genomes of strains capable of degrading chloromethane. The main objectives of these analyses were to: i) determine and analyse the genome sequences of two reference chloromethanedegrading methylotrophs investigated by our group;

ii) check for the occurrence of the chloromethane dehalogenase cmuA gene in complete bacterial genomes found in sequence databases;

iii) identify genes common and/or unique to methylotrophs able to utilize chloromethane as the sole carbon and energy source; iv) assess the diversity of chloromethane utilization pathways within genomes of chloromethane-degrading bacteria beyond the Proteobacteria phylum.

This analysis was also expected to yield valuable insights for future experimental investigations of bacterial chloromethane utilization.

Sequencing and genome analysis of chloromethane-degrading strains

The genome sequence of the chloromethane-degrading strain mainly investigated in our laboratory, Methylobacterium extorquens CM4, was determined as part of a collaborative project initiated in 2005 by Prof. Chris J. Marx (Harvard University) leading an international consortium of labs from Switzerland, USA and France, and aiming at Chapter 2 obtaining the compete genome sequence of of six Methylobacterium strains at the Joint Genomic Institute (JGI) (USA). Subsequently, the genome of another chloromethanedegrading strain, Hyphomicrobium sp. MC1, the first chloromethane-degrading strain to be reported (Hartmans et al., 1986) The complete and assembled genome sequences were determined for six strains of the alphaproteobacterial genus Methylobacterium, chosen for their key adaptations to different plant-associated niches and environmental constraints.

G enomic and metagenomic investigations have highlighted the prevalent role of methylotrophic microorganisms in a variety of marine, freshwater, and terrestrial environments (3)(4)6). These data have propelled new understanding of the molecular intricacies of microbial methylotrophic metabolism (1) and have sparked continued interest in their potential for biotechnological applications (15). In this work, the assembled complete genome sequences of six strains of the alphaproteobacterial genus Methylobacterium were determined. The selected strains were chosen for key characteristics, in terms of ecology, physiology, and metabolism (Table 1), in order to investigate how such adaptive features are reflected at the level of genome composition and architecture.

Genomes were sequenced at the Joint Genome Institute (JGI) using combinations of small to medium DNA libraries (3, 6, and 8 kb), as well as fosmid libraries (35 and 40 kb), with Sanger sequencing (7.3 to 9.6ϫ coverage) completed with 454 pyrosequencing (20ϫ coverage). All general aspects of library construction and sequencing can be found at http://www.jgi.doe.gov /sequencing/protocols/prots_production.html. Draft assemblies and quality assessment were obtained using the Phred/Phrap/ Consed software package. Possible misassemblies were corrected with Dupfinisher (8), PCR amplification, and transposon bombing of bridging clones (Epicentre Biotechnologies, Madison, WI). Gaps between contigs were closed by editing in Consed, custom primer walking, and PCR amplification. A final assembly (7.5 to 10.5ϫ coverage) was obtained for all 6 genomes (Table 1), and automatic annotation was performed using the JGI-Oak Ridge National Laboratory annotation pipeline (12). Additional automatic and manual sequence annotations, as well as comparative genome analysis, were performed using the MicroScope platform at Genoscope (16).

The six Methylobacterium strains show significant variation in chromosome size and plasmid content (Table 1), and each possesses several conserved gene clusters known to be involved in methylotrophy in Methylobacterium (2,[START_REF] Vuilleumier | Methylobacterium genome sequences: a reference blueprint to investigate microbial metabolism of C1 compounds from natural and industrial sources[END_REF]. Five of the strains possess conserved clusters of genes associated with photosynthesis, including genes encoding the light-harvesting complex and the reaction center, and genes involved in biosynthesis of bacteriochlorophyll and carotenoids. Further analyses of these six genomes will include comparisons to the two Methylobacterium genomes already reported [START_REF] Vuilleumier | Methylobacterium genome sequences: a reference blueprint to investigate microbial metabolism of C1 compounds from natural and industrial sources[END_REF], i.e., M. extorquens AM1, a major model strain in studies of methylotrophy (2) and genome evolution (5), and the dichloromethanedegrading strain M. extorquens DM4 ( 14). This will define both core-and strain-specific features of Methylobacterium strains and provide new insights into the metabolic flexibility of these facultative methylotrophs and into the modes of bacterial adaptation to specific ecological niches.

Nucleotide sequence accession numbers. GenBank accession numbers for all the chromosomes and plasmids sequenced in this study are shown in Table 1. a Number of annotated protein-coding sequences in MicroScope (16).

b This strain, originally reported as M. populi strain BJ001 [START_REF] Van Aken | Methylobacterium populi sp. nov., a novel aerobic, pink-pigmented, facultatively methylotrophic, methane-utilizing bacterium isolated from poplar trees (Populus deltoides x nigra DN34)[END_REF], was assigned to the species M. extorquens based on 16S rRNA gene identity (99.3%) and overall genome similarity with the four other sequenced M. extorquens strains (~80% identity over 75% of its genome sequence) (also see reference 18).

Strains of the genus Hyphomicrobium originally attracted interest for their distinctive prosthecae and atypical complex growth cycle (9). Strains of this genus are ubiquitous but were detected in wastewater treatment plants in particular, and often under denitrifying conditions (3,8). Following the description of strain MC1 ( 6), several chloromethane-degrading Hyphomicrobium strains were isolated from various aquatic and soil environments, and most recently also from the surfaces of plant leaves (11). Strain MC1 features the consecutive cmuBCA gene arrangement; these genes encode chloromethane dehalogenase, which has been found in all aerobic chloromethane-degrading bacteria characterized so far with the exception of Methylobacterium extorquens CM4, from which chloromethane dehalogenase was purified (13) and in which regulation of chloromethane dehalogenase expression was investigated (14).

The assembled genome sequence of Hyphomicrobium sp. strain MC1 was obtained using a mix of sequencing technologies. A mate-paired 454 library with an 8-kb insert size was constructed (version Titanium), and 559,691 reads (173,407,941 bp; approximately 36ϫ coverage) were assembled using Newbler (version 2.3, release 091027_1459). Assembly validation was made via Consed (www.phrap.org), and 129 PCRs between contigs were performed and sequenced for gap closure. For quality assessment, a total of 25,299,825 Illumina 36-bp reads were mapped onto the whole genome sequence using SOAP (http://soap.genomics.org.cn) (2), allowing us to correct potential base errors and confirming the final closed circular 4,757,528-bp assembly. Sequence annotation and comparative genome analysis are under way using the MicroScope platform at Genoscope (15).

Based on the 16S rRNA sequence of its single rRNA operon, strain MC1 appears to be most closely related to Hyphomicrobium facile subspecies type strains. Of its 4,679 predicted open reading frames (ORFs), 947 (20%) have close homologs (Ͼ80% amino acid identity over Ͼ80% of protein length, almost all of them in synteny) in Hyphomicrobium denitrificans ATCC 51888, whose genome sequence has been determined (NC_014313) (4).

Genes encoding enzymes and proteins for oxidation of methanol (mxa) and methylamine (mgs and mgd [5,7] but not mau genes) were identified, together with proteins and enzymes involved in pyrroloquinoline quinone synthesis and tetrahydrofolate-and tetrahydromethanopterin-linked pathways. Genes for complete serine and ethylmalonyl coenzyme A pathways (1) for carbon assimilation were identified. The genome of strain MC1 also encodes a complete glycolysis pathway and a closed tricarboxylic acid cycle, but no genes for the glyoxylate shunt (isocitrate lyase and malate synthase) were detected. Six terminal oxidases of different types were identified. Unlike in H. denitrificans ATCC 51888, genes for N 2 fixation, a complete uptake hydrogenase gene cluster, and gene systems for both assimilative (nas) and dissimilative (nar) reduction of nitrate, ammonia and nitrate/nitrite transport, a putative nitrate-inducible formate dehydrogenase, an alkane sulfonate oxidation and transport system, and an acetone carboxylase (acxRABC) cluster (12) were detected. In contrast, the dichloromethane dehalogenation genes (dcmRABC) (10) allowing H. denitrificans ATCC 51888 to grow on DCM were not found in strain MC1.

Hyphomicrobium sp. strain MC1 thus emerges as a promising model for investigating the degradation of halogenated pollutants in the context of methylotrophic metabolism using genomic information (16) and for studies of morphological and metabolic features supporting bacterial growth under nutrientscarce conditions.

Nucleic acid sequence accession number. The Hyphomicrobium sp. strain MC1 genome sequence was deposited in GenBank/EMBL under accession number FQ859181.

This work was supported by a GIS IbiSA grant to S.V. (2009 campaign).

Comparative genomics of chloromethane-degrading strains

Comparative genomics using the Microscope platform of Genoscope

MicroScope (http://www.genoscope.cns.fr/agc/microscope) is a platform developed by Genoscope (Evry, France) which provides easy-to-use tools to study, annotate and perform comparative genomic analysis of bacterial genomes (Fig. 2.1) [START_REF] Vallenet | MicroScope, an integrated microbial resource for the curation and comparative analysis of genomic and metabolic data[END_REF]. This platform comprises three major components: (i) a resource providing a relational database for completed and ongoing genome projects named PkGDB (prokaryotes genome database), which stores information on organisms, sequences and genomic objects; (ii) a panel of bioinformatics tools linked to the PkGDB database to provide results of syntactic and functional annotation pipelines as well as metabolic pathway tools (e.g. KEGG, MicroCyc) analysis specific to each genome; and (iii) an interface, MaGe (Magnifying genomes), allowing manual annotations and comparative genomic analyses [START_REF] Vallenet | MaGe: a microbial genome annotation system supported by synteny results[END_REF][START_REF] Vallenet | MicroScope: a platform for microbial genome annotation and comparative genomics[END_REF]. The MaGe interface combines graphical interfaces and analysis of synteny (i.e., conservation of organization of genes among different genomes), together with genome data sources and metabolic pathway tools to assist in data evaluation in order to manually assign the best possible annotation to a given gene product (Fig. 2.1) [START_REF] Vallenet | MaGe: a microbial genome annotation system supported by synteny results[END_REF][START_REF] Vallenet | MicroScope: a platform for microbial genome annotation and comparative genomics[END_REF].

It also offers useful tools and functionalities to explore genome data according to the specifications and requests of the annotator. MicroScope can be used as an open access resource (for publicly available genomes) or as a restricted access resource (where access can be restricted to a limited group of annotators defined by the project leader).

MicroScope resource currently contains data for more than 1600 microbial genomes from 129 different projects [START_REF] Vallenet | MicroScope, an integrated microbial resource for the curation and comparative analysis of genomic and metabolic data[END_REF]. According to the latest report of MicroScope, there are several projects related to the study of genomes of bacteria of environmental relevance and capable of utilizing a large variety of compounds of high industrial and environmental interest [START_REF] Vallenet | MicroScope, an integrated microbial resource for the curation and comparative analysis of genomic and metabolic data[END_REF]. Our laboratory coordinates several Microscope annotation projects, notably Methyloscope, Methanoscope, and Hyphoscope, which include both public and newly determined private genome sequences in the field of methylotrophic and methanotrophic bacterial metabolism. These projects have already led to the publication of 10 genome announcements so far, including the two presented in the previous section (Vuilleumier et al., 2011;Marx et al., 2012), and have also provided the basis for more detailed bioinformatics and experimental studies (Muller et al., 2011a;Muller et al., 2011b;Nadalig et al., 2011;[START_REF] Roselli | The 380 kb pCMU01 plasmid encodes cloromethane utilization genes and redundant genes for vitamin B12-and tetrahydrofolate-dependent chloromethane metabolism in Methylobacterium extorquens CM4: A proteomic and bioinformatics study[END_REF]. To date, 7 complete genome sequences are directly relevant to the analysis of bacterial chloromethane utilization. They include those of strains known to grow with chloromethane, and those containing close homologs of cmu genes known to be involved in chloromethane dehalogenation [Chapter 1]. They are briefly described in the following, and their key properties are described in Table 2.1.  M. extorquens CM4 is the strain for which the only known metabolic pathway of aerobic chloromethane utilization so far, the cmu pathway, has been studied in detail (Fig. 2.2) (Vannelli et al., 1999;Studer et al., 2002). The genome of this strain has been published (Marx et al., 2012), and a detailed study of its megaplasmid harboring cmu has been recently reported by [START_REF] Roselli | The 380 kb pCMU01 plasmid encodes cloromethane utilization genes and redundant genes for vitamin B12-and tetrahydrofolate-dependent chloromethane metabolism in Methylobacterium extorquens CM4: A proteomic and bioinformatics study[END_REF].

 Hyphomicrobium sp. MC1 is the first strain for which growth with chloromethane was described (Hartmans et al., 1986). Its genome sequence was determined at Genoscope, and published by our group (Vuilleumier et al., 2011).

 Roseovarius sp. 217 is a chloromethane-degrading strain isolated from marine waters (Schäfer et al., 2005). Its sequence, determined relatively early on in the sequencing age, was not completely assembled, but its is publically available as a set of 37 contigs (http://img.jgi.doe.gov/cgi-bin/w/main.cgi).

 Leisingera methylohalidivorans DSM 14336 is another aerobic chloromethanedegrading strain isolated from marine waters [START_REF] Schaefer | Leisingera methylohatidivorans gen. nov., sp nov., a marine methylotroph that grows on methyl bromide[END_REF]. Its genome sequence, fully determined but not yet published, is publically available on the IMG platform of the Joint Genomic Institite (http://img.jgi.doe.gov/cgibin/w/main.cgi).

 Desulfomonile tiedjei DSM 6799 is an anaerobic bacterium involved in the reductive dehalogenation of chlorophenol and chlorobenzoate [START_REF] Villemur | The Desulfitobacterium genus[END_REF] (http://img.jgi.doe.gov/cgi-bin/w/main.cgi).

 Thermosediminibacter oceani DSM 16646, isolated from a core sample of deep sea sediment, uses thiosulfate, elemental sulfur and MnO 2 as electron acceptors, and grows at 68°C [START_REF] Pitluck | Complete genome sequence of Thermosediminibacter oceani type strain (JW/IW-1228P T )[END_REF]..  Thermincola potens JR, an electricity-producing bacterium isolated from a thermophilic microbial fuel cell, is capable of using carbon monoxide as its only Chapter 2 source of carbon and energy and thus also is a methylotrophic bacterium [START_REF] Byrne-Bailey | Complete genome sequence of the electricity-producing "Thermincola potens" strain JR[END_REF].

The first five of these strains belong to the phylum Proteobacteria, while the last two belong to the Firmicutes. The last three strains contain homologs of cmuA and cmuB chlormethane dehalogenase. Unlike other chloromethane-degrading strains with the cmu pathway, however, they have an anaerobic lifestyle and utilization of chloromethane has not been reported for these strains. (Table 2.1).

In addition, available complete genome sequences of other methylotrophic bacteria unable to degrade chloromethane for growth and belonging to the genera Methylobacterium and Hyphomicrobium were used in comparative genomic analysis.

These included the sequences of Methylobacterium extorquens AM1 (Vuilleumier et al., 2009), M. extorquens DM4 (Vuilleumier et al., 2009;Muller et al., 2011a), M. extorquens PA1 (Marx et al., 2012), M. extorquens BJ001 (Marx et al., 2012), M. nodulans ORS 2060 (Marx et al., 2012), Methylobacterium sp. 4-46 (Marx et al., 2012), M. radiotolerans JCM 2831 (Marx et al., 2012), Hyphomicrobium denitrificans ATCC 51888 (Brown et al., 2011), Hyphomicrobium sp. GJ21 (unpublished genome sequence of the DCM-degrading strain (see e.g. [START_REF] Vuilleumier | Sequence variation in dichloromethane dehalogenases/glutathione S-transferases[END_REF]) and Hyphomicrobium sp. VS [START_REF] Pol | Isolation of a dimethylsulfide-utilizing Hyphomicrobium species and its application in biofiltration of polluted air[END_REF].

Comparative genomics analysis of sequenced strains featuring cmu genes

The main objective of the analysis was to investigate if the genes involved in the cmu pathway (Fig. 2.2) (Studer et al., 2002) [see Section 1.5, Chapter 1 for details], are conserved among the strains capable of utilizing chloromethane. The cmu pathway involves three main aspects: (i) dehalogenation; (ii) assimilation of carbon for biomass production; (iii) generation of energy through carbon mineralization to CO 2 (Fig. 2.

2).

Genes conserved in chloromethane-degrading strains include genes essential for dehalogenation of chloromethane (cmuA, cmuB, and cmuC), genes essential for growth with chloromethane as the sole carbon (metF2) and energy source (folD, purU), and genes found in the vicinity of cmuA, cmuB and cmuC in methylotrophic chloromethanedegrading strains (fmdB, hutI) (Table 2.1) [START_REF] Roselli | The 380 kb pCMU01 plasmid encodes cloromethane utilization genes and redundant genes for vitamin B12-and tetrahydrofolate-dependent chloromethane metabolism in Methylobacterium extorquens CM4: A proteomic and bioinformatics study[END_REF].

Key genes of chloromethane utilization in the context of the cmu pathway were compared in all sequenced strains reported to utilize chloromethane for growth or shown to harbor homologs of the chloromethane dehalogenase gene cmuA (Table 2.1).

Chapter 2 The cmuA gene encodes the CmuA protein, a two-domain protein with an N-terminal methyltransferase domain and a C-terminal corrinoid-binding domain (Fig. 2.3) (Vannelli et al., 1999). The cmuA gene has been found in all the aerobic chloromethane-degrading strains isolated from different environments (Nadalig et al., 2011). A majority of the chloromethane-degrading strains compared here (Table 2 reported to contain cmuABC genes and to utilize chloromethane through the cmu pathway (Schäfer et al., 2005).

Surprisingly, homologs of cmu genes for chloromethane dehalogenase CmuAB with over 60% identity at the protein level were also detected in the recently reported genome sequences of Desulfomonile tiedjei DSM 6799, Thermosediminibacter oceani DSM 16646 [START_REF] Pitluck | Complete genome sequence of Thermosediminibacter oceani type strain (JW/IW-1228P T )[END_REF] and Thermincola potens JR (Byrne-Bailey et al., 2010) (Table 2.1) , which suggest that they may be capable of chloromethane dehalogenation.

Interestingly, Desulfomonile tiedjei DSM 6799 was isolated for its capacity to dehalogenate multicarbon halogenated compounds (e.g. chlorobenzoate) through reductive dehalogenation in an energy yielding process [START_REF] Villemur | The Desulfitobacterium genus[END_REF][START_REF] Ding | Molecular techniques in the biotechnological fight against halogenated compounds in anoxic environments[END_REF]. However, none of these three strains were described to perform methylotrophic metabolism, and all feature an anaerobic lifestyle, unlike all chloromethane-degrading strains containing the cmu pathway so far.

Genes associated to carbon assimilation and energy production

The gene metF2 encoding methylene tetrahydrofolate reductase in strain CM4 and responsible for funnelling the methyl group of chloromethane into the serine cycle for carbon assimilation (Fig. 2.2) features homologs in all chloromethane-utilizing strains compared here (Table 2.1). The corresponding protein is thought to convert CH 3 -H 4 F produced after dehalogenation of chloromethane to CH 2 =H 4 F, which then enters the serine cycle (Fig. 2.2). In Thermosediminibacter oceani DSM 16646 and Thermincola potens JR, the detected homologs of methylene tetrahydrofolate reductase were closer to the chromosomal paralog of metF in M. extorquens CM4 which, unlike its megaplasmid homolog metF2, has no proven role in growth with chloromethane (Vannelli et al., 1999;Studer et al., 2002;[START_REF] Roselli | The 380 kb pCMU01 plasmid encodes cloromethane utilization genes and redundant genes for vitamin B12-and tetrahydrofolate-dependent chloromethane metabolism in Methylobacterium extorquens CM4: A proteomic and bioinformatics study[END_REF]. It is actually more closely homologous to metF of E. coli [START_REF] Sheppard | Purification and properties of NADH-dependent 5,10-methylenetetrahydrofolate reductase (MetF) from Escherichia coli[END_REF] and Thermus thermophilus HB8 [START_REF] Igari | Properties and crystal structure of methylenetetrahydrofolate reductase from Thermus thermophilus HB8[END_REF], which are actually involved in the reverse reaction from CH 2 =H 4 F to CH 3 -H 4 F during methionine synthesis [START_REF] Sheppard | Purification and properties of NADH-dependent 5,10-methylenetetrahydrofolate reductase (MetF) from Escherichia coli[END_REF].

Similarly, comparative analysis of folD and purU genes essential for growth with chloromethane in strain CM4 (Studer et al., 2002) (Fig. 2.2) were also conserved in the different strains considered in this analysis, but to a lesser extent than genes cmuA and cmuB (Table 2.1). At the protein level, levels of identity of all considered homologs of FolD exceed 40%, whereas homologs of PurU, highly identical betwen M. extorquens the PurU protein length in chloromethane-degrading strains Roseovarius sp. 217 and Leisingera methylohalidivorans (Table 2.1).

Organization of cmu genes

The organization of genes in gene clusters and operons may be used to infer their role in a metabolic pathway. In M. extorquens CM4, cmu genes are located in two clusters on the megaplasmid pCMU01, which are separated by a 27 kb DNA region containing genes involved in cobalamin (vitamin B12) metabolism, and vitamin B12 is indeed required for chloromethane utilization (Fig. 2.4) [START_REF] Roselli | The 380 kb pCMU01 plasmid encodes cloromethane utilization genes and redundant genes for vitamin B12-and tetrahydrofolate-dependent chloromethane metabolism in Methylobacterium extorquens CM4: A proteomic and bioinformatics study[END_REF]. In Hyphomicrobium sp. MC1, potens JR, the detected homologs of chloromethane dehalogenation genes cmuABC, along with another gene coding for the putative regulatory protein FmdB, were found in the same putatively operonic organisation as in strain MC1. Interestingly, in Desulfomonile tiedjei DSM 6799, no cmuC homolog was detected between cmuA and cmuB or elsewhere in the genome (Table 2.1) (Fig. 2.4). This is actually in fair agreement with the fact that the cmuC gene is the least conserved among chloromethane-degrading strains with the cmu pathway (Nadalig et al., 2011), and confirms the elusive role of cmuC in chloromethane utilisation despite its essential character in strain CM4 [START_REF] Roselli | Génomique fonctionnelle de la dégradation des méthanes chlorés[END_REF][START_REF] Roselli | Génomique fonctionnelle de la dégradation des méthanes chlorés[END_REF] The capacity for chloromethane dehalogenation of these three strains will need to be addressed. Investigations along these lines are all the more interesting given the fact that these strains have an anaerobic lifestyle which was not yet encountered in a chloromethane-degrading bacterium possessing the cmu pathway. Arrows represents cmu genes associated with chloromethane dehalogenation (green), genes essential for assimilation of carbon into biomass (blue), genes involved in the oxidation of chloromethane to CO 2 (red), and genes of unknown function (white). These genes are located in distinct but close gene clusters on the megaplasmid of M. extorquens CM4 (A) [START_REF] Roselli | The 380 kb pCMU01 plasmid encodes cloromethane utilization genes and redundant genes for vitamin B12-and tetrahydrofolate-dependent chloromethane metabolism in Methylobacterium extorquens CM4: A proteomic and bioinformatics study[END_REF] and on a putative unique operon in the chromosome of Hyphomicrobium MC1 and in other chloromethane-degrading strains with the cmu pathway (B) (Nadalig et al., 2011), Thermosediminibacter oceani DSM 16646 and Thermincola potens JR (C), and Desulfomonile tiedjei DSM 6799 (D). In Roseovarius sp. 217 and Leisingera methylohalidivorans DSM 14336, cmu genes for dehalogenation were absent, and identified homologs of accessory genes (Table 2 HYPMCv2_3968 (42%) a Analysis performed using the MicroScope platform at Génoscope [START_REF] Vallenet | MicroScope: a platform for microbial genome annotation and comparative genomics[END_REF]Muller et al., 2011b;[START_REF] Vallenet | MicroScope, an integrated microbial resource for the curation and comparative analysis of genomic and metabolic data[END_REF]. b Proteins common to M. extorquens CM4 and Hyphomicrobium sp. MC1 without close homologs in any of the other completely sequenced Methylobacterium and Hyphomicrobium strains, i.e. Methylobacterium extorquens strains AM1 (6531 proteins, accession number NC_012808), M. extorquens DM4 (5773 proteins, accession number NC_012988), M. extorquens PA1 (5357 proteins, accession number NC_01017), M. extorquens BJ001 (6027 proteins, accession number NC_010725), M. nodulans ORS 2060 (10161 proteins, accession number NC_011894), Methylobacterium sp. 4-46 (8356 proteins, accession number NC_010511), M. radiotolerans JCM 2831 (7293 proteins, accession number NC_010505), Hyphomicrobium denitrificans ATCC 51888 (3948 proteins, accession number NC_014313), Hyphomicrobium sp. GJ21(4055 proteins, accession number: not public) and Hyphomicrobium sp. VS (3598 proteins, accession number: not public) Threshold for common proteins: over 35% identity on over 75% of the protein sequences compared. Threshold for excluded proteins: any homolog with over 35% identity over 75% of the protein sequence in any of the considered genomes. c Located on the chromosome of M. extorquens CM4 Chapter 2 55 2.4.4. The chloromethane-specific gene set in Alphaproteobacteria M. extorquens CM4 and Hyphomicrobium sp. MC1 are the only sequenced methylotrophic strains using the cmu pathway (Table 2.1) for utilization of chloromethane. The genomes of these two strains were compared to ten other publically available genome sequences of Methylobacterium and Hyphomicrobium strains that do not grow with chloromethane (Table 2.2), in order to try to define a set of proteins that is common and unique to chloromethanedegrading strains with the cmu pathway.

cmu
For this analysis, the comparative genomic tool 'Gene Phyloprofile' of the MicroScope platform of Génoscope was used. This yielded a short list (Table 2.2) of 12 proteins common to M. extorquens CM4 and Hyphomicrobium sp. MC1, but lacking close homologs in any of the other completely sequenced Methylobacterium and Hyphomicrobium strains (excluded proteins with over 35% identity over 75% of the protein sequence in any of the considered genomes). All of these proteins except one (Mchl_4782 of unknown function) are encoded by genes located on the megaplasmid of M. extorquens CM4, on which cmu genes are located [START_REF] Roselli | The 380 kb pCMU01 plasmid encodes cloromethane utilization genes and redundant genes for vitamin B12-and tetrahydrofolate-dependent chloromethane metabolism in Methylobacterium extorquens CM4: A proteomic and bioinformatics study[END_REF]. Along with the key genes involved in chloromethane metabolism already described (i.e. cmuABC and purU, Table 2.1), cobA and a gene coding for putative regulatory protein (FmdB) were found (Table 2.2). Protein CobA is involved in the synthesis of cobalamin (also known as vitamin B12), an essential cofactor for the function of the CmuAB dehalogenase (Fig. 2.2) (Studer et al., 1999;[START_REF] Roselli | The 380 kb pCMU01 plasmid encodes cloromethane utilization genes and redundant genes for vitamin B12-and tetrahydrofolate-dependent chloromethane metabolism in Methylobacterium extorquens CM4: A proteomic and bioinformatics study[END_REF].

The gene encoding for a putative regulatory protein FmdB adjacent to cmuA in both MC1 and CM4 strains is located close to cmu genes, and may represent the still unknown regulator of chloromethane dehalogenase. Further experiments involving mutation of this gene in CM4 or MC1 may be performed to confirm its involvement in chloromethane utilization.

A few other genes highly conserved and unique in both CM4 and MC1 strains were detected in addition to cmu genes involved in chloromethane dehalogenation. Interestingly, these genes define a complete acetone-utilizing gene cluster encoding the acetone carboxylase subunits (acxABC genes) and its transcriptional activator (acxR gene) (Table 2.2), and thereby also involve transformation of another key C1 compound, carbon dioxide. Acetone carboxylase is the key enzyme of bacterial acetone metabolism, catalyzing the ATPdependent carboxylation of acetone to form acetoacetate [START_REF] Sluis | Biochemical, molecular, and genetic analyses of the acetone carboxylases from Xanthobacter autotrophicus strain Py2 and Rhodobacter capsulatus strain B10[END_REF]. In preliminary experiments designed to address the question of the presence of acx genes together with cmu Chapter 2 56 genes on the pCMU01 megaplasmid, [START_REF] Roselli | The 380 kb pCMU01 plasmid encodes cloromethane utilization genes and redundant genes for vitamin B12-and tetrahydrofolate-dependent chloromethane metabolism in Methylobacterium extorquens CM4: A proteomic and bioinformatics study[END_REF] recently showed that M. extorquens CM4, a facultative methylotroph growing with several multicarbon compounds including acetate, ethanol and succinate (Anthony, 2011;[START_REF] Peyraud | Coconsumption of methanol and succinate by Methylobacterium extorquens AM1[END_REF], is indeed also able to grow and utilize acetone as the sole source of carbon of energy. The comparative genome analysis presented here (Table 2.2) suggests that Hyphomicrobium sp. MC1 may also degrade acetone and grow with this compound.

Conclusions and perspectives

Summarizing, comparative genomic analysis of chloromethane-degrading strains performed during this PhD project showed that i) The cmu pathway is conserved and consists of closely colocalized genes and gene clusters in the genomes of chloromethane-degrading Methylobacterium and Hyphomicrobium strains, and other Proteobacteria for which the entire genome sequence is unknown (Nadalig et al., 2011); ii) Genes cmuABC are absent in marine chloromethane-degrading strains Roseovarius sp. 217 and Leisingera methylohalidivorans DSM 14336, suggesting the existence of at least one alternative pathway for bacterial growth with chloromethane. In future experiments, Roseovarius sp. 217 and Leisingera methylohalidivorans DSM 14336 could be grown with C 13 -labelled chloromethane to discover new genes and enzymes expressed during growth with chloromethane and involved in chloromethane utilization.

iii) Some strains of known genome sequence contain cmu genes but have not been reported to utilize chloromethane, e.g. Desulfomonile tiedjei DSM 6799, Thermosediminibacter oceani DSM 16646 and Thermincola potens JR. These strains also harbor homologs of genes involved in the serine cycle for assimilation of C-1 compounds (notably glyA and eno) (Kalyuzhnaya and Lidstrom, 2005;Vuilleumier et al., 2009), suggesting that they may also be capable of growing with C1 compounds iii) Strains CM4 and MC1 not only showed conservation of cmu genes but also of acetone utilization genes. Growth of Hyphomicrobium sp. MC1 with acetone could be tested.

Acetone is the second most abundant carbonyl carbon in the atmosphere after formaldehyde [START_REF] Fischer | The role of the ocean in the global atmospheric budget of acetone[END_REF]. Bacterial utilization of chloromethane and acetone may be correlated considering that sinks and sources of these compounds are quite Chapter 2 57 similar, with largest emissions from plants and oceans for both compounds [START_REF] Montzka | Scientific assessment of ozone depletion: 2010. Global ozone research and monitoring project[END_REF][START_REF] Fischer | The role of the ocean in the global atmospheric budget of acetone[END_REF].

iv) The cmuA gene is the most conserved of all cmu genes in chloromethane-degrading strains (Nadalig et al., 2011), including in the three anaerobic strains discussed here for the first time in the context of chloromethane utilization. This strengthens its status as a biomolecular tool of choice in experimental investigations of chloromethane utilization, including those presented in the following Chapters of this manuscript. [START_REF] Roselli | The 380 kb pCMU01 plasmid encodes cloromethane utilization genes and redundant genes for vitamin B12-and tetrahydrofolate-dependent chloromethane metabolism in Methylobacterium extorquens CM4: A proteomic and bioinformatics study[END_REF], with an initial unique 10 mM portion chloromethane as the sole carbon source Kit: Wizard Genomic DNA Purification Kit 1. Centrifuge 500 mL of grown culture (OD 600 approx. 0.3) using Sorvall Evolution/F10 rotor (8000 rpm, 15 min). Discard supernatant.

2. Resuspend and wash the cell pellet in 15 mL fresh M3 medium; transfer to a single sterile SS34 rotor tube. Centrifuge (Sorvall Evolution/SS34 rotor) for 5 minutes at 12000 rpm (17000 g) and discard supernatant. 7. Centrifuge at 15000 rpm (Sorvall Evolution/SS34 rotor) for 15 min at 4°C. 8. Mark the position of the pellet on the outside of the tube with a permanent marker.

Carefully remove the supernatant using a 5 mL pipetman (taking care not to dislodge the DNA pellet), carefully invert the tube and dry on absorbing paper in a laminar flow hood. Les gènes cmu de ces isolats ont été analysés à l'aide de nouvelles amorces PCR conçues au laboratoire, et leurs séquences ont été comparées avec celles des souches aérobies dégradant le chlorométhane précédemment décrites. Les trois isolats présentaient un arrangement colinéaire des gènes cmuBCA, similaire à ceux de toutes les autres souches précédemment caractérisées, à l'exception de Methylobacterium extorquens CM4. Cette étude est la première à décrire des souches bactériennes dégradant le chlorométhane et isolées de la phyllosphère.

Article Chapter 4

Introduction Chloromethane, the most abundant volatile halocarbon in the atmosphere (600 p.p.t.), may be responsible for about 15% of chlorine-catalysed ozone destruction in the stratosphere (Clerbaux et al., 2007). The global chloromethane budget is uncertain, with estimated known sources not fully accounting for identified sinks. The major sink for chloromethane likely involves reactions with hydroxyl radicals in the troposphere (Yoshida et al., 2004). Industrial emissions of the compound estimates range from o 10% (Yoshida et al., 2004) to 60% [START_REF] Trudinger | Atmospheric histories of halocarbons from analysis of Antarctic firn air: methyl bromide, methyl chloride, chloroform, and dichloromethane[END_REF] of total known emissions. Natural sources of chloromethane emissions include higher plants, grasslands, salt marshes, peatlands, wood-rotting fungi, senescent leaves, biomass burning and oceans, with vegetation possibly representing the major biotic source (Saito & Yokouchi, 2008). A methyltransferase responsible for chloromethane production was purified from leaves of Brassica oleracea [START_REF] Attieh | Purification and characterization of a novel methyltransferase responsible for biosynthesis of halomethanes and methanethiol in Brassica oleracea[END_REF](Attieh et al., , 2000)). In Arabidopsis thaliana, a protein encoded by the gene HOL (harmless to ozone layer) catalyses S-adenosyl-L-methionine-dependent methylation of chloride (Rhew et al., 2003;Nagatoshi & Nakamura, 2009). This enzyme is involved in the transformation of thiocyanate produced upon the degradation of glucosinolate, and its product methylisothiocyanate appears to play a role in the resistance of A. thaliana to bacterial infection (Nagatoshi & Nakamura, 2009). A physiological role for enzyme-produced chloromethane remains to be demonstrated.

MICROBIOLOGY ECOLOGY

Certain methylotrophic bacteria are able to use chloromethane as the sole carbon and energy source for growth (McDonald et al., 2002;[START_REF] Trotsenko | The biology of methylobacteria capable of degrading halomethanes[END_REF]reviewed in Schäfer et al., 2007; Table 1). Chloromethanedegrading bacteria have been isolated from diverse environments such as soils (Doronina et al., 1996;[START_REF] Miller | Bacterial oxidation of methyl bromide in fumigated agricultural soils[END_REF]Coulter et al., 1999;McAnulla et al., 2001a), activated sludge (Hartmans et al., 1986;Traunecker et al., 1991;Freedman et al., 2004), freshwater (McAnulla et al., 2001a), and seawater (Schäfer et al., 2005), and include representatives affiliated to a wide range of genera including Aminobacter, Hyphomicrobium, Leisingera, Methylobacterium, Roseovarius (Alphaproteobacteria), Pseudomonas (Gammaproteobacteria) and Acetobacterium (Actinobacteria). However, the association of such bacteria with plants has not yet been investigated.

The only pathway for chloromethane catabolism by aerobic bacteria characterized so far was investigated in detail for strain Methylobacterium sp. CM4 (Doronina et al., 1996). This strain may now be affiliated to the species Methylobacterium extorquens on the basis of its complete genome sequence, including revised 16S rRNA gene sequences (GenBank NC_011757). A set of genes essential for growth on chloromethane (Vannelli et al., 1998(Vannelli et al., , 1999)), termed cmu genes for chloromethane utilization, was identified by minitransposon mutagenesis of strain CM4. These genes and the corresponding enzymes defined a specific pathway for chloromethane utilization in methylotrophic metabolism that depends on corrinoid and folate cofactors (Vannelli et al., 1999). The chloromethane dehalogenase enzyme, consisting of a corrinoid methyltransferase protein encoded by cmuA and of a tetrahydrofolate-dependent methyltransferase encoded by cmuB, was purified and characterized (Studer et al., 1999(Studer et al., , 2001)). The role in chloromethane utilization of cmuC, encoding another puta-tive methyltransferase essential for chloromethane utilization (Vannelli et al., 1999), remains to be elucidated. Chloromethane-dependent expression of cmu genes was also demonstrated (Studer et al., 2002).

Several other chloromethane-degrading strains were subsequently reported [START_REF] Woodall | Identification of methyl halide-utilizing genes in the methyl bromide-utilizing bacterial strain IMB-1 suggests a high degree of conservation of methyl halide-specific genes in Gram-negative bacteria[END_REF]McAnulla et al., 2001b;Schäfer et al., 2005;[START_REF] Warner | Analysis of genes involved in methyl halide degradation in Aminobacter lissarensis CC495[END_REF] and also contained cmu genes. The cmuA gene has been used as a molecular biomarker of bacterial methyl halide metabolism in a large variety of environments (McAnulla et al., 2001a;Miller et al., 2004;[START_REF] Borodina | Use of DNA-stable isotope probing and functional gene probes to investigate the diversity of methyl chloride-utilizing bacteria in soil[END_REF]Schäfer et al., 2005). The plant environment, however, has not yet been explored in the context of bacterial chloromethane metabolism, despite the fact that some of the genera featuring representatives of chloromethane-degrading bacteria, notably Hyphomicrobium, Methylobacterium and Pseudomonas, are known to be efficient colonizers of the phyllosphere [START_REF] Kinkel | Microbial population dynamics on leaves[END_REF][START_REF] Andrews | The ecology and biogeography of microorganisms on plant surfaces[END_REF][START_REF] Trotsenko | Aerobic methylotrophic bacteria as phytosymbionts[END_REF]Knief et al., 2008Knief et al., , 2010;;[START_REF] Raja | Genetic diversity and phylogeny of pink-pigmented facultative methylotrophic bacteria isolated from the phyllosphere of tropical crop plants[END_REF]Delmotte et al., 2009).

To investigate the occurrence of chloromethane-degrading bacteria in the phyllosphere, bacterial enrichments from leaves of A. thaliana, which is the best-characterized chloromethane-emitting plant at the molecular level (Rhew et al., 2003;Nagatoshi & Nakamura, 2009), were performed with the aim of isolating new bacterial strains able to grow with chloromethane as the sole carbon source, and to develop new molecular tools to efficiently detect and characterize gene signatures for chloromethane utilization in isolated strains and environmental DNA, in particular in the phyllosphere environment.

Materials and methods

DNA and bacterial strains

Strains M. extorquens CM4 and Hyphomicrobium chloromethanicum CM2, isolated in Russia from petrochemical factory soil (Doronina et al., 1996), and Hyphomicrobium sp. strain MC1, isolated from industrial sewage sludge (Hartmans et al., 1986), were laboratory stocks. Genomic DNA extraction of bacterial strains was performed using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI). Total DNA of Aminobacter lissarensis CC495, Aminobacter ciceronei IMB-1, Rhodobacteraceae 179 and Rhodobacteraceae 198 was kindly provided by H. Schäfer (University of Warwick, UK).

Growth media

Liquid cultures for enrichment and growth were performed in a mineral medium for methylotrophic bacteria (M3) containing (L -1 of distilled water) KH 2 PO 4 (6.8 g), (NH 4 ) 2 SO 4 (0.2 g), NaOH (5 M) (5.85 mL), yielding a final pH of 7.2. After autoclaving, 1 mL L À1 medium each of (14.4), NiSO 4 Á 6H 2 O ( 10), H 3 BO 3 (10) and 0.5 mL L À1 of H 2 SO 4 (95%) were added.

Bacterial strains were cultivated and isolated on a solid medium containing (L -1 of distilled water) K 2 HPO 4 (1.04 g), (NH 4 ) 2 SO 4 (0.2 g), NaH 2 PO 4 Á H 2 O (0.65 g), MgSO 4 Á 7H 2 O (0.1 g), bromothymol blue (0.1 g) and agar (15 g) at a pH of 7.2. Calcium nitrate and trace elements were added after autoclaving as described for liquid M3 medium.

Enrichment culture and strain isolation from plant leaves

Enrichment cultures were set up by adding one leaf of greenhouse-grown A. thaliana Columbia (Col-0, $40 mg) to a 300-mL vial fitted with a sealed mininert valve cap (Sigma) and containing 50 mL of M3 medium. Following the addition of 12 mL (approximately 10 mM) chloromethane gas (Fluka, approximately 5 atm) to the headspace, enrichment cultures were incubated under shaking (100 r.p.m.) at 30 1C. After 24 h, leaf material was removed and 12 mL of chloromethane gas was again added. Enrichment cultures obtained after three successive subcultures were spread onto mineral agar plates and incubated in sealed, gas-tight jars containing 1.3% chloromethane (v/v). Chloromethane dehalogenation was indicated by the development of yellow colour on a green background around dehalogenating colonies, which were selected and purified on fresh solid M3 medium.

Dehalogenation activity

Resting cell suspensions were prepared from exponentialphase cultures (50 mL, OD 600 nm o 0.3), obtained with chloromethane as the sole carbon source. After centrifugation at 14 700 g for 10 min, cells were washed twice in 50 mM chloride-free phosphate buffer pH 7.0, and the cell pellet was resuspended in the same buffer (6 mL final volume). Protein determination was performed with 1 mL of cell suspension using the bicinchoninic acid assay and a commercial kit (Pierce). For activity measurements, 5 mL of cell suspensions of chloromethane-degrading strains were added to 17-mL Hungate vial capped with a gas-tight mininert valve (Sigma). Chloromethane gas (10 mL) was added in excess and the vial was incubated at 30 1C. At different times, aliquots (0.5 mL) were sampled through the valve with a 1-mL syringe, transferred to Eppendorf tubes on ice, centrifuged immediately, and the resulting supernatants were transferred to fresh Eppendorf tubes and kept frozen until further use. Control experiments with phosphate buffer and with cell suspensions of the nondechlorinating strain M. extorquens AM1 (Vuilleumier et al., 2009) were performed to evaluate the nonenzymatic degradation of chloromethane.

Chloride concentration was determined spectrophotometrically as [FeCl] 21 (l max = 340 nm) formed in a highly acidic medium using the method of [START_REF] Jörg | Thiol-tolerant assay for quantitative colorimetric determination of chloride released from wholecell biodehalogenations[END_REF]. Chloride concentration was determined by comparison with a calibration curve (0-5 mM) obtained with a sodium chloride solution in the same buffer, and dehalogenase activity was expressed as nmol min À1 mg À1 protein.

DNA extraction from bacterial cultures and environmental samples

Genomic DNA extraction from enrichment cultures (10 mL at OD 600 nm = 0.6) was performed using the Wizard Genomic DNA Purification Kit (Promega) according to the manufacturer's recommendations. DNA was extracted from epiphytic bacteria on the surface of A. thaliana leaves. Briefly, 10-15 leaves of A. thaliana plants (360-860 mg fresh weight) were washed as described previously (Delmotte et al., 2009), with 30 mL TE buffer, pH 7.5, containing 0.1% Silwet L-77 (GE Bayer Silicones). After centrifugation, total DNA was prepared using the FastDNA spin kit (MP Biomedicals, Santa Ana, CA), as described by Knief et al. (2008). Cell lysis was performed using a Mikro-Dismembrator S (Sartorius Stedim Biotech, France) by three consecutive 1-min treatments at 3000 min -1 .

Quantitative PCR (qPCR)

CmuA and 16S rRNA gene copy numbers were evaluated through qPCR using an ABI PRISM 5700 sequence detection system (Applied Biosystem, Foster City, CA). qPCR analysis was carried out in triplicate using phyllospheric DNA (5-10 ng) in 96-well plates, using the primer pairs cmuA802f and MF2 (5 0 -CCRCCRTTRTAVCCVACYTC) for the cmuA gene and BACT1369F and PROK1492R (Suzuki et al., 2000) for the 16S rRNA gene, respectively. The PCR reaction mix contained 1 Â qPCR Mastermix Plus for SYBR Green I (Eurogentec S.A., Belgium), 0.3 and 12.8 mM of cmuA802f and MF2 primers, respectively, for cmuA amplification, or 0.4 mM of each primer BACT1369F and PROK1492R for 16S rRNA gene amplification. Reaction conditions were 10 min at 95 1C, followed by 45 cycles of denaturation at 95 1C for 15 s, followed by annealing and elongation at 60 1C for 60 s. Calibration curves were obtained using genomic DNA from M. extorquens CM4 for both cmuA and 16S rRNA gene analysis.

PCR and RFLP analysis

The primers used in this study are listed in (Mastercycler Personal, Eppendorf, Germany). Each PCR reaction mixture consisted of 2.5 mL of PCR buffer (New England Biolabs), 0.25 mL of dNTPs (200 mM), 1 mL of each forward and reverse primers (20 mM), 17.9 mL of distilled water, 0.3 mL of Taq polymerase (5 U mL À1 , New England Biolabs), 0.05 mL of Pfu polymerase (3 U, Promega) and 2 mL of template DNA solution (25 ng). After initial denaturation (94 1C, 3 min), DNA amplification was performed by 30 cycles of 45-s denaturation at 94 1C, annealing for 1 min (at 52 1C for the 16S rRNA gene and between 61 and 67 1C for cmu genes; see Table 2 for details), extension for 1-4.5 min (depending of fragment length) at 72 1C and a final extension step of 7 min at 72 1C.

A two-step semi-degenerate PCR strategy [START_REF] Jacobs | Comprehensive transposon mutant library of Pseudomonas aeruginosa[END_REF] was used to access the 5 0 -upstream region of the cmuB gene fragments obtained. In the first PCR, primer cmuBrev was used with a mix of the three semi-degenerate primers cekg2A, cekg2B and cekg2C [START_REF] Jacobs | Comprehensive transposon mutant library of Pseudomonas aeruginosa[END_REF]. The second PCR involved using the reverse primer cmuBrev2 and the primer cekg4 targeting the tail of the semi-degenerate primers used in the first PCR [START_REF] Jacobs | Comprehensive transposon mutant library of Pseudomonas aeruginosa[END_REF].

Amplified cmuA fragments from each strain and from enrichment cultures were purified using the 'GENECLEAN Turbo' kit (MP Biomedicals Europe, France). Purified products (2 mg) were digested with the enzyme DdeI (30 U; Fermentas, France) and the appropriate buffer at 37 1C for 20 h. Digested products were size fractionated on 2% agarose/Nusieve (3/1) gels.

Sequencing

PCR products were purified with ExoSAP-IT reagent (USB Corporation) according to the manufacturer's recommendations; DNA sequences were obtained from PCR products with appropriate primers (Table 2) on an ABI Prism 3130 XL Genetic Analyzer (Applied Biosystems, UK). The assembled sequences for the cmuBCA clusters of Hyphomicrobium strains MC1, AT2, AT3 and AT4 obtained were deposited in the EMBL database under numbers FN667867, FN667868, FN667869 and FN667870 respectively, together with the corresponding partial rRNA gene sequences (FN667863, FN667864, FN667865 and FN667866, respectively).

Phylogenetic analysis

DNA sequences were compared with databases by online BLAST searches (http://www.ebi.ac.uk/tools/blast). DNA and protein sequences of interest were analysed using the Mobyle platform (http://mobyle.pasteur.fr). Multiple alignments were obtained using CLUSTALW, and manually adjusted using JALVIEW, as implemented on the Myhits online portal (http://myhits.isb-sib.ch). Multiple alignments were analysed using the PHYLIP suite of programs, and in particular the DNADIST, NEIGHBOR, SEQBOOT and CONSENSE, as implemented on the Mobyle platform. Bootstrap analysis was performed with 100 replicates.

Results

Quantification of 16S rRNA and cmuA genes on the leaf surfaces of A. thaliana Total DNA was extracted from the leaf surface of leaves A. thaliana, yielding approximately 0.4-1.2 ng DNA mg À1 fresh weight of leaves (range 30-1000 ng DNA per leaf). The cmuA gene was detected in all samples, with a copy number of 8.0 AE 0.8 copies ng À1 DNA. The 16S rRNA gene was detected in the same samples at a copy number of 10 100 AE 2000 copies ng À1 DNA. Assuming a single copy of 16S rRNA gene per bacterial genome, this suggested that on average, o 1 in 1000 bacterial cells on leaf surfaces carried the cmuA gene and may be capable of dehalogenating chloromethane.

Isolation, characterization and identification of chloromethane-utilizing bacteria

Enrichment cultures were set up with A. thaliana leaves as the inoculum in a chloride-free mineral medium with chloromethane as the sole carbon and energy source (1.3%, v/v). Chloromethane-dependent growth with concomitant chloride production was observed in enrichment cultures.

Three chloromethane-degrading bacterial strains termed AT2, AT3 and AT4 (AT in reference to A. thaliana) were obtained from such enrichment cultures, as single colonies on a solid mineral medium with chloromethane as the sole carbon and energy source. These isolates grew aerobically on both liquid and solid mineral medium with chloromethane, methanol or succinate as the sole carbon source, indicating that they were facultative methylotrophs. All three strains displayed characteristic hyphae, indicative of the genus Hyphomicrobium (Moore, 1981). Taxonomical affiliation to cluster II [START_REF] Rainey | Phylogenetic analysis and intrageneric structure of the genus Hyphomicrobium and the related genus Filomicrobium[END_REF] was confirmed using 16S rRNA gene sequence analysis (Fig. 1). The ability of strains AT2, AT3 and AT4 to transform chloromethane and to use it as the sole carbon and energy source for growth was compared with reference strains M. extorquens CM4 and H. chloromethanicum CM2 (Table 1). Hyphomicrobium strains AT2 grew with chloromethane with similar doubling times as the previously described strains CM2 and MC1 (t d $5 h). The growth of M. extorquens CM4 was slightly slower (t d 7.3 h), whereas newly isolated Hyphomicrobium strains AT3 and AT4 were the slowest growing (t d over than 18 h). In contrast, specific chloromethane dehalogenation activities inferred from measurements of chloride concentration in the supernatants of cell suspensions were similar for all strains (Table 1).

Organization and diversity of cmu genes in chloromethane-degrading strains

Genes cmuA, cmuB and cmuC could be amplified and sequenced from total DNA of chloromethane-degrading phyllosphere isolates using both previously described primers (McAnulla et al., 2001b;Miller et al., 2004) and primers newly designed from the better conserved sequence regions in previously reported cmu gene clusters (Table 2). A two-step PCR strategy [START_REF] Jacobs | Comprehensive transposon mutant library of Pseudomonas aeruginosa[END_REF] afforded access to the unknown sequence region upstream of amplified cmuB gene fragments yielding complete sequences of the cmuB gene for the phyllosphere isolates. The sequences and organization of cmu genes in strains isolated from A. thaliana were compared with those of previously described strains (Figs 2 and 3, Supporting Information, Fig. S1).

Phylogenetic analysis of partial cmuA gene sequences from phyllosphere isolates obtained using the primer pair cmuA802f-cmuA1609R (Miller et al., 2004) was compared with those of previously reported chloromethane-degrading strains, and from selected cmuA gene fragments available in sequence databases and obtained from environmental DNA of different origins (Miller et al., 2004;[START_REF] Borodina | Use of DNA-stable isotope probing and functional gene probes to investigate the diversity of methyl chloride-utilizing bacteria in soil[END_REF]Schäfer et al., 2005) (Fig. 3). This analysis yielded a picture congruent with that obtained for the analysis of the 16S rRNA gene (Fig. 1). It also suggested that phyllosphere isolates, together with strain MC1, belong to a clade that includes sequences from woodland soil covered with leaflitter and garden soils [START_REF] Borodina | Use of DNA-stable isotope probing and functional gene probes to investigate the diversity of methyl chloride-utilizing bacteria in soil[END_REF]. The levels of sequence identity between cmuA gene fragments were 75-80% between Hyphomicrobium strains and either M. extorquens CM4 or Aminobacter strains. cmuA amplicons of strains AT3 and AT4 showed identical sequences, differing from those of strains AT2, CM2 and MC1 which, with over 99% pairwise identity, clustered tightly together (Fig. 3).

In contrast with cmuA, only a few partial or full cmuB and cmuC gene sequences are available so far, all obtained from cultivated and isolated strains (Fig. 2). The new degenerate primer pairs developed in this work allowed the detection and retrieval of cmuB and cmuC gene sequences from the newly isolated chloromethane-degrading strains reported here, and comparison with previously described cmu genes (Table 2). Overall, the sequences for cmuB gene fragments (Fig. S2a) showed high levels of identity, but cmuB sequences of Hyphomicrobium strains were only about 60% identical to that of M. extorquens CM4. Regarding cmuC (Fig. S2b), sequence analysis of amplicons again showed that sequences from AT3 and AT4 were most closely related Fig. 2. Comparisons of cmu gene organization in chloromethane-degrading bacteria. Arrows represent protein-coding genes, and homologous genes are given with identical shading. Annotations above arrows indicate the name of the gene. Chloromethane degradation genes are part of two different clusters in Methylobacterium extorquens CM4. Amplified PCR products used to characterize the new isolates (highlighted in bold), as well as the primers used, are indicated. Dotted lines refer to products of the 3 0 end of the cmu gene cluster obtained by two-stage semi-degenerate PCR [START_REF] Jacobs | Comprehensive transposon mutant library of Pseudomonas aeruginosa[END_REF]. Gene clusters are drawn to scale.

(97.9% identity), and that sequences from strains MC1, AT2 and CM2 formed a closely related cluster ( 4 91% identity). Notably, cmuC sequences of Hyphomicrobium strains including the new isolates were equally distant ($47% identity) to cmuC and to cmuC2 of unknown function found immediately upstream of cmuA, of strain CM4. This emphasizes the lesser degree of conservation of cmuC despite it being essential for growth with chloromethane in strain CM4 (Vannelli et al., 1999).

The cmuBCA cluster organization of cmu genes for Hyphomicrobium strains isolated from A. thaliana leaves and for strain MC1 was the same as that found previously for H. chloromethanicum CM2 and all other previously isolated strains, with the exception of M. extorquens CM4 (Fig. 2). In all cases where it was characterized, this singlecluster cmu gene arrangement also featured genes paaE and hutI, encoding a putative oxidoreductase and a putative imidazolone hydrolase, respectively. In this work, PCR reactions with cmuA802f-paaErev1 and cmuA802f-hutIrev2 primer pairs (Table 2, and data not shown) provided evidence that paaE and hutI genes were also present in the three new strains reported here and in the same arrangement as in strain H. chloromethanicum CM2 (McAnulla et al., 2001b).

New PCR primers for cmuA analysis

The published reverse primer cmuA1609R used for the detection of cmuA sequences in environmental samples (Miller et al., 2004) allowed to accommodate the shorter cmuA sequence of A. ciceronei. In this work, a frameshift in the cmuA sequence originally reported for A. ciceronei IMB1 (AF307143) sequence was detected and corrected, extending its predicted cmuA gene from 1704 to 1851 nt. Amplification using the primer pair cmuA802f together with cmuA1802r newly designed in this work yields a larger cmuA gene PCR fragment of approximately 1 kb (Table 2). Similar sensitivity was achieved with the newly designed cmuA primers and with the previously described primers (see Fig. S1). However, the new primer cmuA1802r may allow the detection of a wider diversity of cmuA sequences, because it could be successfully used to amplify cmuA from marine strain Rhodobacteraceae 198, unlike primer cmuA1609R (Schäfer et al., 2005) (Fig. 4). Also, the 193-bp 3 0 -end cmuA sequence also amplified with this newly defined primer pair is slightly less conserved than the sequence between primers cmuA802f and cmuA1609R (see Table S1), thus potentially allowing better discrimination of cmuA sequences retrieved from environmental DNA.

Restriction fragment profiling of PCR-amplified cmuA in chloromethane-degrading enrichment cultures obtained from plant leaves A protocol for monitoring chloromethane-degrading enrichment cultures obtained from leaves of A. thaliana as inocula was developed using restriction digestion of PCRamplified cmuA gene fragments (Fig. 4). At the timepoint chosen to isolate chloromethane-degrading strains by plating out of the liquid enrichment culture on a solid selective mineral medium (OD 600 nm = 0.6, 8 days), the detection limits using primer pair cmuA802f-cmuA1802r developed here were typically 0.5 and 10 pg of the DNA template for the reference strain M. extorquens CM4 and for the enrichment culture, respectively (Fig. S1). This suggested that the chloromethane-degrading bacterial subpopulation in enrichment cultures represented about 5% of the total bacteria present in the cultures at that stage.

The amplicons obtained were digested with the restriction enzyme DdeI, which cuts one to three times and at variable positions in the cmuA sequence of previously characterized chloromethane-degrading strains (Schäfer et al., 2005) (Fig. 4). The digestion patterns of amplicons in enrichment cultures were distinct from all reference strains, except for H. chloromethanicum CM2 and Hyphomicrobium sp. MC1. Strain AT2 showed the same pattern as the enrichment culture from which it was isolated (Fig. 4), and the same situation was found for strains AT3 and AT4 and the corresponding enrichment culture from which these strains originated (data not shown). The restriction profiling method applied here to amplicons of 1000 nucleotides thus holds promise for a focused, time-saving exploration of chloromethane utilization genes aiming at discovering new, more divergent cmu gene sequences in environmental samples, and at characterizing the corresponding bacteria.

Discussion

Bacteria growing aerobically with chloromethane as the sole source of carbon and energy had previously been isolated from a variety of environments, and so far, all feature the cmu pathway for chloromethane utilization (Studer et al., 2002;Schäfer et al., 2007). However, the phyllosphere compartment of vegetation, possibly the quantitatively most important source of chloromethane (Clerbaux et al., 2007), had not yet been investigated in this respect. In this work, the key gene cmuA involved in dehalogenation of chloromethane was detected and quantified in DNA from the leaf surface of the model plant A. thaliana, and three chloromethane-degrading Hyphomicrobium strains were isolated from enrichment cultures originating from leaves of A. thaliana grown with chloromethane as the sole carbon source. In addition, several degenerate primer pairs and an associated genotyping approach were developed for the detection and characterization of cmu genes in enrichment cultures and isolated strains.

The Hyphomicrobium chloromethane-degrading strains isolated in this work possessed cmu genes in the same arrangement as in most previously isolated strains from other ecosystems, confirming the dominant status of the cmu pathway in the bacterial degradation of chloromethane. However, the isolation of strains belonging to the Hyphomicrobium genus was unexpected, inasmuch as Methylobacterium strains were recently shown to be efficient leaf colonizers and predominant in the A. thaliana phyllosphere, with Hyphomicrobium likely representing only a minor contribution (Delmotte et al., 2009;Knief et al., 2010). Indeed, enrichment cultures obtained from plant leaves in the same medium, but with methanol as the sole carbon and energy source led to the enrichment of strains belonging to the genus Methylobacterium (data not shown). However, chloromethane-degrading Hyphomicrobium isolates also grew well with methanol as the carbon source, suggesting that Hyphomicrobium strains may be better adapted to growth with chloromethane than Methylobacterium strains, albeit in an as yet unknown way. Other aspects of Hyphomicrobium metabolism require further investigation, in particular, the fact that similar chloromethane dehalogenase activity was detected in cell-free extracts of all chloromethane-degrading strains despite differences in the growth rates of the strains with chloromethane (Table 1).

The demonstration of chloromethane-degrading bacteria at the surface of A. thaliana leaves is of relevance for the overall budget of chloromethane in the environment in the light of current estimates for chloromethane emissions above plant areas ($1.8 Tg Cl year À1 ) [START_REF] Yoshida | Inverse modeling of the global methyl chloride sources[END_REF]. If indeed, as suggested from this work, some phyllosphere bacteria function as a filter for emissions of chloromethane from plants, then measurements and estimates of chloromethane emissions above plant areas will actually tend to reflect the difference between total chloromethane emissions from vegetation and bacterial degradation of chloromethane in the phyllosphere, rather than the total chloromethane potential from plants. Whether this may contribute towards explaining the deficit in identified sources of chloromethane (Clerbaux et al., 2007) is a topic for further investigation. Clearly, assessing the importance of chloromethane degradation by specialized methylotrophic bacteria in the phyllosphere will require further work, especially considering that plant emissions of methanol, itself a growth substrate for most methylotrophic bacteria, exceed those of chloromethane by over three orders of magnitude [START_REF] Nemecek-Marshall | Methanol emission from leaves[END_REF]Rhew et al., 2003). Chapter 7

153

The degree to which the biosphere contributes to the atmospheric burden of climate-relevant trace gases is still not well understood. Novel observations and measurements have confirmed not only the important role of the plant biosphere in emissions of one-carbon volatile organic compounds, which play a role in global warming and ozone depletion [START_REF] Harren | Online, real-time detection of volatile emissions from plant tissue[END_REF] , but also of the microbial compartment associated with plants in mitigating such emissions. Plants and bacteria are thought to play a key role in the global cycling of chloromethane (Clerbaux et al., 2007), but have not yet been studied together in the context of chloromethane metabolism.

This context provided the incentive to propose a PhD thesis project aimed at exploring the topic of bacterial chloromethane degradation in the phyllosphere. A laboratory model system comprising Arabidopsis thaliana, a well-characterized chloromethane-emitting plant (Nagatoshi and Nakamura 2009), and the most studied chloromethane degrading bacterial strain, Methylobacterium extorquens CM4 (Studer et al., 2002), using bacterial chloromethane dehalogenase gene cmuA and the gene HOL1 involved in the production of chloromethane in A. thaliana as molecular biomarkers.

Taken together, the results obtained in the PhD work using biomolecular approaches indirectly but conclusively confirmed that different HOL1 variants of A. thaliana emit chloromethane to different extents, and that these emissions, although quite minor in comparison to emissions of methanol, affect the phyllosphere microflora, and methyl halide degrading bacteria in particular. A synthetic summary of the main conclusions of this work is given in the following, together with a brief presentation of work in progress, and an outline of proposed future experiments. First, chloromethane-degrading bacteria were detected in the phyllosphere of A. thaliana using both culture-dependent and culture-independent methods (Nadalig et al., 2011) [Chapter 4]. Chloromethane-degrading strains were isolated from enrichment cultures of leaves of A. thaliana. They possessed cmu genes in the same arrangement as most previously described chloromethane-degrading strains isolated from other environments (Schäfer et al., 2007), confirming the dominant status of the cmu pathway in the bacterial degradation of chloromethane.

In parallel, the abundance and expression of cmuA was investigated, and showed that the proportion of chloromethane-degrading bacteria on the surface of leaves of A. thaliana, although relatively low (<1‰), was sufficient to significantly affect net A. thaliana emissions of chloromethane on the basis of previously published values of chloromethane dehalogenase activity (Vannelli et al., 1998) and A. thaliana emissions of chloromethane (Rhew et al., 2003) [Chapter 5]. These observations also suggested that available values for emissions of chloromethane may actually reflect the difference between total chloromethane emissions from vegetation and bacterial degradation of chloromethane in the phyllosphere, rather than the total potential of plants for emissions of chloromethane In order to identify further functional genes of interest in the context of chloromethane metabolism, the team has recently embarked on applying the RNA-seq approach for global transcriptomic analysis of chloromethane utilization by strain M. extorquens CM4 (project led by Françoise Bringel and funded by IBiSA Génoscope, France). In the RNASeq approach, purified total mRNAs are transcribed into cDNA, and subjected to highthroughput deep sequencing. The obtained sequence reads are mapped onto a reference genome sequence to infer expressions of transcripts in the investigated samples [START_REF] Haas | How deep is deep enough for RNA-Seq profiling of bacterial transcriptomes?[END_REF]Wang et al., 2012a;[START_REF] Westermann | Dual RNA-seq of pathogen and host[END_REF]. This project is expected to define the global adaptive response of bacteria to chloromethane through the discovery of transcriptional units specifically expressed in chloromethane-grown versus methanol-grown cultures.

Knowledge of cmu genes identified in this RNA-seq project will be useful in metagenomics studies of the A. thaliana phyllosphere. However, chloromethane-degrading strains exist that do not have cmu genes [Chapter 2], and which metabolize chloromethane by yet unknown pathways. To detect such bacteria in the environment, and characterize the pathways of corresponding strains which have been isolated, stable isotope probing DNA-SIP may be used, a method in which the growth substrate is labelled by a stable isotope and leads to incorporation of the label in the biomass (including DNA and proteins) of metabolically active bacteria. Today, this approach is applied in combination with metagenomics in particular [START_REF] Chen | Towards "focused genomics"a case study combining DNA Stable isotope probing, multiple displacement amplification and metagenomics[END_REF][START_REF] Uhlik | Stable isotope probing in the metagenomics era: A bridge towards improved bioremediation[END_REF]. [START_REF] Chen | Towards "focused genomics"a case study combining DNA Stable isotope probing, multiple displacement amplification and metagenomics[END_REF][START_REF] Henneberger | Field-scale labelling and activity quantification of methane-oxidizing bacteria in a landfill-cover soil[END_REF]. In the context of the present project, enrichment of epiphytic communities capable of using 13 C-labelled chloromethane may help uncover the phyllosphere chloromethane degradome, defined as the metagenome of chloromethane-utilizing epiphytic communities, and thereby investigate the diversity of chloromethane-utilization pathways associated with the phyllosphere beyond the cmu pathway. Funding for this work has also been applied for in the context of the ANR BIOADAPT program.

Chapter 7 162

The different approaches outlined in Fig. 7.1 have the potential to achieve the long-term objective of the team of characterizing the functional adaptation of bacteria and microbial communities to chloromethane. Clearly, the work reported in this manuscript only represented a first attempt to unravel chloromethane-related metabolism at the low ambient concentrations encountered in the environment. The tools developed in this project will now also be used to follow chloromethane emissions and its degradation in different environments. For example, a PhD project ("thèse en cotutelle" of Pauline Chaignaud, in collaboration with University of Bayreuth, codirected by Françoise Bringel and Steffen Kolb and cofunded by the German DFG and Région Alsace), was recently initiated in the lab to explore bacterial chloromethane metabolism in forest soils, basing on studies of global gene expression in reference dehalogenating strains. This project is expected to contribute to the team's overall objectives of developing effective bacterial bioindicators and bioreporters to answer ecological questions of adaptation and response to environmental stress specifically addressing bacterial dehalogenative metabolism of chlorinated methanes in particular, but also of chlorinated pollutants in general, in natural environments.

  2000); b WHO (2003); c WHO (2004a),; d WHO (2004b); e Chen et al. (2012); f Montzka et al.

Fig. 1 . 1 .

 11 Fig. 1.1. Formation and destruction of ozone layer in the stratosphere. Natural reactions of ozone are disturbed by halomethanes (CHxCly) in presence of ultraviolet A (UVA, wavelength 315 -400 nm) and ultraviolet B (UVB, wavelength 280 -315) radiations. (Figure adapted from http://www.cec.org)

Fig. 1 . 2 . 1 12ferns

 121 Fig. 1.2. Function of HOL1 in A. thaliana.HOL1 catalyzes the S-adenosylmethionine (SAM) dependent methylation of thiocyanate (SCN -) produced during glucosinolate hydrolysis, yielding methyl thiocyanate (CH 3 SCN) (A), or methylation of chloride (Cl -) to produce chloromethane

Fig. 1 . 3 .

 13 Fig. 1.3. Biosynthesis of methanol in plants.(A) Polygalacturonic acid is demethylated by pectin methylesterase (PME) to produce methanol and galacturonic acid residues during elongation of the primary cell wall. (B) General structure of the backbone of pectin consisting of polygalacturonic acid (a homopolymer of α-D-[1][2][3][4]-galactosyluronic acid units) with different degrees of methyl esterification (adapted from[START_REF] Galbally | The production of methanol by flowering plants and the global cycle of methanol[END_REF].

Chapter 1 21 1 . 5 .

 115 Biochemistry and genetics of chloromethane degradation in Methylobacterium extorquens CM4

Fig. 1 . 6 .

 16 Fig. 1.6. Chloromethane-utilization (cmu) genes in Methylobacterium extorquens CM4.Genes responsible for dehalogenation of chloromethane (green), for growth with chloromethane (blue), and genes involved in the metabolism of cobalamine (orange) required for the dehalogenation, are localized in two clusters, subsequently found to be closely associated on megaplasmid pCMU01 (380 kb) of strain CM4 (cf. Chapter 2 below andMarx et al., 2012). Triangles indicate the insertion sites of miniTn5 transposon in characterized mutants and red flashes indicate the chloromethane-induced promoter regions that were subsequently identified(Studer et al 2002). (Fig. adapted fromVannelli et al., 1999)

Fig. 1 . 7 .

 17 Fig. 1.7. Dehalogenation of chloromethane by CmuA and CmuB proteins. (A & B) CmuA acting as a bifunctional protein (methyltransferase I and corrinoid-binding domains) and serves as an intermediate methyl carrier. In a second reaction, CmuB methyltransferase II transfers the methyl group (-CH 3 ) from CmuA to tetrahydrofolate (H 4 F). (C) In combination; CmuA and CmuB proteins catalyze the in vitro transfer of the methyl group of chloromethane to tetrahydrofolate to form methyltetrahydrofolate (CH 3 -H 4 F). (Fig. adapted fromStuder et al., 2001). The function of cmuC, a gene essential for growth with chloromethane (Fig.1.6), and of its close homolog cmuC2 located upstream of cmuA, are still unknown.

Fig. 1 . 8 .

 18 Fig. 1.8. The phyllosphere of Arabidopsis thaliana. The left panel drawing shows a rosette of an A. thaliana plant. The right panel shows a schematic representation of a magnified cross-section of an A. thaliana leaf. Bacteria are found on both leaf surfaces (epiphytes) as well as within leaf tissues (endophytes). Fig. adapted from Vorholt (2012).

  leaves possibly represent the quantitatively most important source of chloromethane produced in the biosphere [discussed in Sections 1.2 and 1.3 above], but this has not yet been investigated comprehensively. On the other hand, the importance of bacteria capable of utilizing chloromethane as the only source of biomass and energy [see Sections 1.4 -1.6 above] has not been addressed in this context. Since several carbon compounds including methanol are released from plants on the surface of leaves, making the phyllosphere an important habitat for bacteria and especially methylotrophic bacteria [see Section 1.6 above],

Fig. 1 . 9 .

 19 Fig. 1.9. Objectives and questions of research project. The objectives of the research project were to develop tools to detect chloromethane emissions [Chapter 3] based on the sequencing data of genomes of chloromethanedegrading strains [Chapter 2], especially to detect chloromethane emissions from the model plant A. thaliana and its mutants with varying expression of HOL1, the gene responsible for chloromethane emissions [Chapter 5], to detect and isolate chloromethane-degrading strains from the phyllosphere of A. thaliana [Chapter 4], to investigate if chloromethane emissions from plants provide selective advantage to bacteria for phyllosphere colonization [Chapter 5] and to evaluate the possible influence of chloromethane emissions on the phyllosphere bacterial diversity [Chapter 6], in order to address the question whether phyllosphere bacteria can act as filters for plant emissions of chloromethane

chloromethane 3 ) 1 31A

 31 Explore the diversity of bacteria residing in the phyllosphere in relation to chloromethane emissions from plants, and investigate in particular if chloromethane emissions from plants provide a selective advantage to chloromethane-degrading bacteria for phyllosphere colonization The results of the work performed in this PhD project to address these objectives are presented in the following Chapters of the thesis manuscript. In the next chapter [Chapter 2], an overview of comparative genomic analysis of chloromethane-degrading strains, which has resulted in a clearer picture of the cmu pathway for chloromethane utilisation at the basis of the development of robust molecular tools to detect chloromethane-degrading bacteria in the phyllosphere, is first presented. In Chapter 3, the development of a bacterial bioreporter based on M. extorquens CM4 for the detection of chloromethane is presented. The work resulting in the first isolation of chloromethane-degrading bacteria from the phyllosphere of A. thaliana is presented in Chapter 4.Chapter detailed investigation on the abundance and expression of cmuA in different variants of A.

Chapter 2 Fig. 2 . 1 .

 221 Fig. 2.1. A screen capture of the "MicroScope" web platform.The screenshot was taken during analysis of the genome of Methylobacterium extorquens CM4 in the "Genome Browser" window. Several tools (A) can be used to explore the genome of the selected (pivot) organism (B). Genomic objects (predicted genes in the 6 frames) (C) are displayed as rectangles, along with (D) genes in synteny in other organisms in the MicroScope database (PkGBD, http://www.genoscope.cns.fr/agc/microscope) and publically available genome sequences (Refseq).

2 .

 2 Complete genome sequences of chloromethane-degrading strains and cmu gene containing strains

Fig. 2 . 1 .

 21 Fig. 2.1. Comparison of cmu gene organization in different genomes.Arrows represents cmu genes associated with chloromethane dehalogenation (green), genes essential for assimilation of carbon into biomass (blue), genes involved in the oxidation of chloromethane to CO 2 (red), and genes of unknown function (white). These genes are located in distinct but close gene clusters on the megaplasmid of M. extorquens CM4 (A)[START_REF] Roselli | The 380 kb pCMU01 plasmid encodes cloromethane utilization genes and redundant genes for vitamin B12-and tetrahydrofolate-dependent chloromethane metabolism in Methylobacterium extorquens CM4: A proteomic and bioinformatics study[END_REF] and on a putative unique operon in the chromosome of Hyphomicrobium MC1 and in other chloromethane-degrading strains with the cmu pathway (B)(Nadalig et al., 2011), Thermosediminibacter oceani DSM 16646 and Thermincola potens JR (C), and Desulfomonile tiedjei DSM 6799 (D). In Roseovarius sp. 217 and Leisingera methylohalidivorans DSM 14336, cmu genes for dehalogenation were absent, and identified homologs of accessory genes (Table2.1) were not colocalized.

  Protocol : Total DNA extraction from large volume bacterial cultures for genome sequencing Bacterial strain: Hyphomicrobium MC1 grown in M3 medium

3 . 5 .

 35 Resuspend in 13 mL of Nuclei Lysis Solution from the kit. Add 250 µl of DNase-free Proteinase K (20 mg/mL) and 0.5 mL of 0.5M EDTA solution. Incubate for 5 min at 80°C to lyse cells and cool to room temperature. 4. Add 50 µl of DNase-free RNase (from the kit). Invert the tube 2-5 times for homogeneisation. Incubate at 37°C for 30 min, at 80°C for 5 min and cool to room temperature. Add 13 mL of protein precipitation solution (from the kit). Mix thoroughly by repeated inversion, but *without* vortexing. Centrifuge at 15000 rpm (Sorvall Evolution/SS34 rotor) for 15 min at 4°C. 6. Transfer the supernatant (16 mL) very carefully (*without any fines*) to a fresh sterile SS34 tube containing 12 mL of -20°C isopropanol. Homogeneize the mixture by inverting the tube very gently until appearance of turbidity.

Chapter 2 59 9 . 2 Résumé 3 Résumé

 2923 Carefully add 10 mL cold 70% ethanol (kept at -20°C) and incubate at room temperature for 15 minutes.10. Centrifuge again at 15000 rpm (Sorvall Evolution/SS34 rotor) for 15 min at 4°C; take care to centrifuge with the tube in exactly the same orientation as in the first centrifugation (i.e. with the pellet facing outwards). 11. Carefully remove the supernatant with a 1 mL pipetman. Dry the pellet in a laminar flowhood. 12. Add 1 mL of DNA Rehydration Solution (from the kit). Incubate at 65°C for 1 hour and/or at 4°C overnight. Measure the DNA concentration by NanoDrop, agarose gel electrophoresis (and PicoGreen dsDNA quantification kit Molecular Probes if -2.1; 260/230 ratio 1.75-1.85). 13. Store at -20°C until further use. Chapter Les monohalométhanes (halogénures de méthyle) sont des composés organiques volatils qui jouent un rôle important dans l'environnement, tant en raison de leur toxicité pour les organismes vivants que pour leur implication dans le fonctionnement de l'atmosphère. Ainsi, le chlorométhane et le bromométhane sont responsables de la destruction d'ozone stratosphérique, alors que l'iodométhane influe sur la formation d'aérosols à l'interface océan-atmosphère. Le bilan atmosphérique global de ces halométhanes, en particulier le chlorométhane, est encore mal évalué en raison d'un grand nombre d'incertitudes dans les estimations et la non prise en compte possible de certaines sources et puits en raison des difficultés de détection et de quantification du chlorométhane. L'objectif principal de cette étude était de développer un bio-rapporteur pour la détection spécifique du chlorométhane et des autres mono-halométhanes. Methylobacterium extorquens CM4, une souche qui peut utiliser le chlorométhane comme seule source de carbone et d'énergie, a été sélectionnée pour cette étude. Le gène cmuA a été choisi en tant que gène marqueur et l'induction de son expression spécifique par le chlorométhane a été vérifiée par qRT-PCR. Un plasmide, comportant une cassette d'expression du gène codant la protéine fluorescente jaune (YFP) placée sous le contrôle du promoteur du gène cmuA, a été construit. En présence de chlorométhane, une émission de fluorescence, proportionnelle à la concentration de chlorométhane sur une large gamme de valeurs (2 pM -20 mM), a été quantifiée chez M. extorquens CM4. Le temps nécessaire pour détecter la production de fluorescence est de 20 minutes environ, et une concentration seuil de chlorométhane correspondant à seulement 60 molécules de chlorométhane par cellule de bio-rapporteur a été déterminée. Ce rapporteur biologique pourrait être utilisé comme méthode de détection sensible des émissions de chlorométhane provenant de différents milieux (par exemple les sols et les plantes) au laboratoire ainsi que in situ. Chapter Le chlorométhane est un composé volatil produit naturellement dans la phyllosphère, le compartiment défini comme les parties aériennes des plantes et qui abrite une flore bactérienne riche. Le chlorométhane peut servir comme substrat pour la croissance des bactéries méthylotrophes aérobies qui possèdent les gènes cmu. Ainsi, des bactéries ont été isolées à partir de sols et d'eaux douce ou marine mais aucune n'a été décrite provenant de la phyllosphère. Dans notre étude, nous avons mis en évidence la présence de bactéries dégradant le chlorométhane sur la surface des feuilles d'Arabidopsis thaliana, par PCR quantitative spécifique du gène cmuA codant la protéine chlorométhane déhalogénase. A partir de cultures d'enrichissement contenant du chlorométhane comme seule source de carbone et inoculés avec des feuilles d'Arabidopsis thaliana, des souches bactériennes ont été isolées. Le génotypage basé sur l'analyse des produits PCR du gène cmuA après digestion par une enzyme de restriction (DdeI) a été utilisé pour évaluer la diversité des bactéries dégradant le chlorométhane pendant l'enrichissement et après l'isolement des souches. Sur la base des séquences des gènes de l'ARNr 16S, les isolats obtenus ont été affiliés au genre Hyphomicrobium dont la présence caractéristique d'hyphe a pu être observée au microscope.

FEMS

  European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

w

  IUPAC-code (M = A/C, R = A/G, W = A/T, Y = C/T, S = C/G, K = G/T, H = A/C/T, V = A/C/G, D = A/G/T, B = C/G/T, N = A/C/G/T).z Positions correspond to the chloromethane utilization cluster of Hyphomicrobium chloromethanicum strain CM2 GenEMBL accession no. AF281259. ‰ Used for sequencing only. FEMS Microbiol Ecol 77 (2011) 438-448 c 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

Fig. 1 .

 1 Fig. 1. Taxonomic affiliation of alphaproteobacterial chloromethane-degrading bacteria based on 16S rRNA gene sequences. Strains isolated in this study are shown in bold. The gammaproteobacterium Pseudomonas aeruginosa, which is unable to degrade chloromethane, was used as the outgroup. Multiple sequence alignments (1403 bp) were obtained with CLUSTALW and analysed with PHYLIP (see Materials and methods). Bootstrap analysis was performed on 100 replicate trees, and the values of nodes recovered in more than 75% of cases are shown. Scale bar = 1% sequence divergence.

Fig. 3 .

 3 Fig. 3. Phylogenetic analysis of characterized cmuA genes from chloromethane-degrading strains. Strains characterized in this study are shown in bold. Bootstrap analysis of the multiple alignments of cmuA gene fragments (765 nt) obtained with CLUSTALW was performed on 100 replicate trees, and the values of nodes recovered in more than 75% of the cases are shown. Scale bar = 2% sequence divergence.

Fig. 4 .

 4 Fig. 4. DdeI restriction digestion patterns of PCR-amplified cmuA fragments obtained with the primers cmuA802f and cmuA1802r. M, lanes with molecular mass marker 1 kb ladder (Fermentas); lane 1, Aminobacter sp. IMB1; lane 2, Aminobacter sp. CC495; lane 3, Rhodobacteraceae 179; lane 4, Rhodobacteraceae 198; lane 5, Methylobacterium extorquens CM4; lane 6, Hyphomicrobium chloromethanicum CM2; lane 7, Hyphomicrobium strain MC1; lane 8, Hyphomicrobium strain AT2; lane 9, Hyphomicrobium strain AT3; lane 10, Hyphomicrobium strain AT4; lane 11, representative chloromethane-degrading enrichment culture with chloromethane as the sole additional carbon source (OD 600 nm = 0.6, 8 days) obtained after inoculation with a single leaf of Arabidopsis thaliana; lane 12, undigested PCR-amplified cmuA fragment of the same enrichment culture.

Figure S1 .

 S1 Figure S1. Analysis of PCR-amplified fragments of cmuA obtained with primers cmuA802f and cmuA1802r. PCR products separated by agarose gel electrophoresis were obtained from Methylobacterium extorquens CM4 (lanes 1 to 8) and from an enrichment culture obtained after incubation with a single A. thaliana leaf (lanes 9 to 16). M, lanes with molecular mass marker 1 kb ladder (Fermentas); lanes 1 and 9, 500 pg of DNA template; lanes 2 and 10, 100 pg of DNA template; lanes 3 and 11, 50 pg of DNA template; lanes 4 and 12, 10 pg of DNA template; lanes 5 and 13, 1 pg of DNA template; lanes 6 and 14, 0.5 pg of DNA template; lanes 7 and 15, 0.1 pg of DNA template; lanes 8 and 16, no DNA (water). 172x129mm (300 x 300 DPI)

Chapter 7 154 7 . 1 . 7 155 7 . 2 .

 771772 Biomolecular tools to study chloromethane-associated processes in the phyllosphere New tools were developed to efficiently detect and estimate chloromethane emissions in the phyllosphere environment, and to follow their effect on the phyllosphere microflora, with particular emphasis on bacteria involved in chloromethane degradation. Relative expression of the HOL1 gene responsible for chloromethane emissions in A. thaliana was quantified using qRT-PCR in different variants of the plant [Chapter 5]. On the other hand, basing on knowledge acquired during this work on genome sequences of both dechlorinating and nondechlorinating Alphaproteobacteria [Chapter 2], degenerate oligonucleotide primers were developed for quantitative PCR analysis at both DNA and RNA levels that targeted the chloromethane dehalogenase gene cmuA of chloromethane-degrading bacteria (Nadalig et al., 2011) [Chapter 4 & 5]. In addition, a highly sensitive bacterial bioreporter was developed to detect and quantify methyl halides and chloromethane in particular [Chapter 3]. A plasmid-based transcriptional reporter construct featuring a promotorless YFP gene cassette fused downstream of the cmuA gene promoter region afforded halomethane-dependent production of fluorescence in Methylobacterium extorquens CM4. The fluorescence signal was proportional to the presence of halomethane over a wide range of concentrations (pM -mM). The time required to detect onset of YFP fluorescence production was as low as 20 minutes, and the threshold concentration was approximately 2 pM (about 60 molecules of chloromethane per bioreporter cell). The characteristics of this bioreporter for detection of methyl halides are attractive, considering the high sensitivity to evaluate low level natural emissions of chloromethane from plants and other natural sources using gas chromatography. Such analytical sensitivity is out of reach for most laboratories today, and thus represents a major bottleneck on the way to establish a reliable global budget of chloromethane and other methyl halides.Chapter Chloromethane-degrading bacteria as filters of chloromethane emissions to the atmosphere New insights obtained in this work supported our initial working hypothesis, i.e. that chloromethane-degrading bacteria are present in the phyllosphere, and that they may act to filter plant emissions of chloromethane to the atmosphere.

7. 3 .Fig. 7 . 1 .

 371 Fig. 7.1. Current perspectives for the A. thaliana-based model developed in the project. Ongoing rapid developments in sequencing technology allow to envisage further work on the developed model under methyl halide concentration regimes typical of laboratory conditions (left) or environmental conditions (right), and at the DNA level (top) as well as at the RNA level (bottom).An inmmediate and already ongoing approach (top right, see Chapter 6) will consist in highthroughput sequencing of PCR amplicons of cmuA obtained from total DNA from leaf surfaces of different HOL variants using 454 pyrosequencing technology, as well as random shotgun sequencing of this DNA using Illumina technology. Other genes of interest, identified by ongoing RNAseq studies of model strains designed to characterise the adaptations involved in metabolism of chloromethane (bottom left), may also be investigated by this approach. Further, stable isotope probing approaches involving exposure of A. thaliana to artificially high concentrations of 13C-labelled chloromethane (top left), by affording the isolation by ultracentrifugation of "heavy" 13C-labelled DNA enriched in genetic material from metabolically active chloromethane-degrading populations, may uncover novel pathways for utilisation of chloromethane beyond the cmu pathway characterised in model chloromethanedegrading strains such as M. extorquens CM4 and Hyphomicrobium sp. strain MC1. The outcome of these different approaches will feed in the ultimate and most demanding long-term objective of this work (bottom right), i.e. the characterisation of the functional adaptation of bacterial communities in the phyllosphere to exposure to ambient concentrations of chloromethane.

  approach will be to develop the pyrosequencing experiments initiated on the structural diversity of the phyllosphere based on the 16S rRNA gene [Chapter 6],by focussing on amplification of the chloromethane dehalogenase cmuA gene from nucleic acids extracted from the phyllosphere of the three HOL1 variants of A. thaliana.

  ., Woodall, C.A., McDonald, I.R., Studer, A., Vuilleumier, S., Leisinger, T., and Murrell, J.C. (2001a) Chloromethane utilization gene cluster from Hyphomicrobium chloromethanicum strain CM2 T and development of functional gene probes to detect halomethane-degrading bacteria. Appl. Environ. Microbiol. 67: 307-316. McClenny, W.A., Oliver, K.D., Jacumin, H.H., and Daughtrey, E.H. (2002) Ambient level volatile organic compound (VOC) monitoring using solid adsorbents -Recent US EPA studies. J Environ. Monit. 4: 695-705. ., chloromethane-utilizing bacteria isolated from a polluted environment. Int. J. Syst. Evol. Microbiol. 51: 119-122.

Table 1 .1. Important properties of chlorinated methanes

 1

	Properties	Chloromethane (CH 3 Cl) a	Dichloromethane (CH 2 Cl 2 ) b	Trichloromethane (CHCl 3 ) c	Tetrachloromethane (CCl 4 ) d
	Physico-chemical properties
	Molecular weight	50.5	84.9	119.5	153.8
	Density (101.3 kPa)	0.0023 g/cm 3 (at 0°C)	1.3255 (at 20°C)	1.48 g/cm 3 (at 25°C)	1.594 g/cm 3 (at 25°C)
	Water solubility (at 25°C)	4.8 g/L	13 g/L	7.2 -9.3 g/L	0.8 g/L
	Henry's law constant (atm cm 3 per mol)	0.0102 e	0.0023 e	0.0035 e	0.0275 e
	Carbon oxidation state	-2	0	2	4
	Vapor pressure	575 kPa (at 25°C)	46.53 kPa (at 20°c)	21.3 kPa (at 20°c)	12.2 kPa (at 20°c)
	Environmental data
	Toxic effects to humans	neurotoxicity, renal tumor and infertility	possible carcinogen, affects nervous system	possible carcinogen, liver and kidney tumors	possible carcinogen, neurological & renal damage
	Genotoxicity	weak	none	very weak	none
	Tolerable daily intake	-	6 µg/kg of body wt	0.015 mg/kg of body wt 1.45 g/kg of body wt
	Tolerable daily inhalation	0.018 mg/m 3 of air	20 µg/m 3 of air	0.14 mg/m 3 of air	6.1 mg/m 3 of air
	Atmospheric conc. (ppt)	550 ± 30 f	22.21 ± 6.7 f	7 ± 1.5 f	95 ± 5 f
	Half life (years)	1.30 f	0.38 f	0.40 f

Table 1 .2. Estimated source and sink strengths for atmospheric chloromethane from observations and three-dimensional model studies Source/sink type Best estimates a (Gg y -1 ) Range b (Gg y -1 ) Sources

 1

	Tropical plants

Table 1 .3. Major volatile organic compounds released from plants

 1

	Volatile organic compound	Tg C y -1	Reference
	Monoterpenes	530 -560	Guenther et al. (2012)
	Isoprene	157 -177	Guenther et al. (2012)
	Methanol	100 -105	Galbally and Kirstine (2002)
	Methane	62 -236	Keppler et al. (2006)
	Chloromethane	0.8 -8	Clerbaux et al. (2007)

Chapter 1 17 Table 1.4 Chloromethane degrading strains isolated from different environments. Bacterial strain Origin Gram type Reference

			Chapter 1
	A collection of aerobic chloromethane-degrading bacterial strains was isolated from a
	petrochemical factory soil belonging to genra Methylobacterium and Hyphomicrobium. Two
	of these, strains CM2 and CM4, were investigated in more detail and named
	Hyphomicrobium sp. strain MC1 Hyphomicrobium chloromethanicum and Methylobacterium chloromethanicum respectively industrial sewage plant negative	Hartmans et al. (1986)
	(Switzerland) (McDonald et al., 2001). In particular, strain CM4, subsequently assigned to the species
	Acetobacterium dehalogenans MC Methylobacterium extorquens [see below, Chapter 2, and Marx et al., 2012] became the activated sludge (Germany) positive	Traunecker et al. (1991)
	Hyphomicrobium sp. strain CM2 workhorse organism to unravel the biochemistry and genetics of chloromethane utilisation soil from a petrochemical negative	Doronina et al. (1996)
	factory (Russia) [see below, section 1.6]. While two other strains, Aminobacter ciceronei CC495 and
	Methylobacterium extorquens sp. strain CM4 a Aminobacter lissarensis IMB-1, were isolated from soils (McDonald et al., 2005), strains soil from a petrochemical negative	Doronina et al. (1996)
	factory (Russia) fumigated strawberry field Roseovarius sp. 179, Roseovarius sp. 217, Ruegeria sp. 198 Aminobacter ciceroni IMB1	negative	Hancock et al. (1998)
		soil (USA)
	Aminobacter lissarensis CC495	beech woodland soil	negative	Coulter et al. (1999)
		(Ireland)
	Pseudomonas aeruginosa NB1	activated sludge	negative	Freedman et al. (2004)
		(USA)
	Leisingera methylohalidivorans MB2	marine tide pool	negative	Schaefer et al. (2002)
		(USA)
	Roseovarius sp. strain 179	coastal seawater	negative	Schäfer et al. (2005)
		(Scotland)
	Roseovarius sp. strain 198	coastal seawater	negative	Schäfer et al. (2005)
		(Scotland)
	Roseovarius sp. strain 217	English channel seawater	negative	Schäfer et al. (2005)
		(England)
	a Methylobacterium extorquens sp. CM4 was previously known as Methylobacterium chloromethanicum CM4 (Kato et al., 2005)
	(McDonald et al., 2001).
			18

  (Fig. 1.7).

	A	CH 3 Cl		Cl -	B
				CmuA	CH 3 Cl	CH	3 -H	4 F
				(MTI)		+ HCl
	cmuC?
	cmuC2?	Co I	CH	3 -Co III
						Co	H	4 F (H + )
				CmuB
				(MTII)
						CmuA	CmuB
			H	4 F	CH	3 ̶ H	4 F + H +
				C
					0.4
						CmuA + CmuB
				(mM)	0.3
				CH 3 -H 4 F formed	0.1 0.2	CmuA
						CmuB
					0.0
						0	60	120	180
						Reaction time (min)

  Ce chapitre présente les résultats de génomique comparative obtenus par l'analyse de génomes séquencés des souches utilisant le chlorométhane. Le but principal de cette étude a été de définir les gènes spécifiques des souches dégradant le chlorométhane, ou qui leur sont communs, et d'évaluer si la seule voie d'utilisation du chlorométhane décrite à ce jour est conservée parmi les différentes souches capables d'utiliser le chlorométhane.The comparison of structural and functional characteristics of different organisms is a primordial theme in the field of biological research. Since the sequencing of the first complete genome of Haemophilus influenzae in 1995(Fleischmann et al., 1995) rapidly

	Chapter 2 Chapter 2
	Résumé 2.1. Introduction
	developing sequencing techniques have been used to determine the genome sequence of
	numerous organisms, and especially bacteria with their relatively small genomes. Indeed,
	Ce travail a bénéficié des programmes de séquençage et d'assemblage des génomes de as of March 2013, 2,175 finished and 14,643 ongoing projects for sequencing bacterial
	deux souches étudiées au laboratoire, Methylobacterium extorquens CM4 et genomes are now publically inventoried (http://www.genomesonline.org). These
	Hyphomicrobium sp. MC1, ainsi que d'autres souches des mêmes genres advances have led to the emergence of a new discipline, comparative genomics (Koonin
	Methylobacterium et Hyphomicrobium menés au Génoscope (France) et au Joint and Aravind, 2009), whereby the genomes of different organisms of interest are compared
	Genomic Institute (USA) durant la période de thèse.
	clés impliqués dans l'utilisation du chlorométhane, d'autres gènes, notamment ceux
	impliqués dans l'utilisation de l'acétone, ont été mis en évidence.

1) Complete genome sequence of the chloromethane-degrading strain Hyphomicrobium sp. strain MC1. Vuilleumier S., Nadalig T. , Farhan Ul Haque M., Magdelenat G., Lajus A, Roselli S., Muller E.E.L., Gruffaz C., Barbe V., Médigue C., and Bringel F. (2011). J. Bacteriol. 193, 5035-5036. 2) Complete genome sequences of six strains of the genus Methylobacterium. J. Bacteriol. 194, 4746-4748. Marx C.J., Bringel F., Chistoserdova L., Moulin L., Farhan Ul Haque M., Fleischman D.E., Gruffaz C., Jourand P., Knief C., Lee M.-C., Muller E.E.L., Nadalig T., Peyraud R., Roselli S., Russ L., Goodwin L.A., Ivanova N., Kyrpides N., Lajus A., Land M.L., Médigue C., Mikhailova N., Nolan M., Woyke T., Stolyar S., Vorholt J.A., Vuilleumier S. (2012). L'analyse par génomique comparative a confirmé que les gènes cmuABC codant les protéines impliqués dans la déshalogénation et les gènes encodant les protéines nécessaires à l'assimilation du carbone et pour l'oxidation complète du chlorométhane en CO 2 pour produire de l'énergie (metF, folD et purU) sont fortement conservés chez les souches CM4 et MC1. L'exploration comparative des génomes bactériens disponibles à ce jour a aussi permis de retrouver les gènes cmu dans la séquence génomique complète de trois bactéries anaérobies et dont l'utilisation du chlorométhane n'a pas encore été décrite, à savoir Desulfomonile tiedjei DSM 6799, Thermosediminacter oceani DSM 16646 et Thermincola potens JR. En revanche, les gènes cmu ne sont pas conservés chez les autres souches chlorométhane-dégradantes Leisingera methylohalidivorans DSM 14336 et Roseovarius sp. 217 dont la séquence génomique a aussi été déterminée, confirmant ainsi que d'autres voies d'utilisation bactériennes du chlorométhane que la voie cmu existent, et qu'elles restent encore à découvrir. Par ailleurs, les outils de la plateforme MicroScope du Génoscope ont permis de définir l'ensemble des gènes communs et uniques aux souches chloromethane-dégradantes M. extorquens CM4 et Hyphomicrobium sp. MC1 par comparaison aux génomes d'autres souches de Methylobacterium et Hyphomicrobium déjà séquencées. En plus des gènes-to identify the genes or proteins that are conserved among organisms, and which confer to each organism its features and functions. Comparative genomics also provides a powerful tool to obtain information about the structure and plasticity of genomes, the evolution of different species, and the adaptation mechanisms of organisms to diverse physicochemical constraints in the environment.

  , was sequenced at Génoscope (France) following a find of original laboratory stocks of this strain in Strasbourg. I became co-author of the two published genome announcement articles describing the genomes of these two strains, on the basis of my contributions to the preparation of cultures and DNA extraction for sequencing purposes [see Appendix ofChapter 2], and to subsequent manual gene annotation and genome analysis of these strains using the Microscope platform at Genoscope [see following section].The two genome announcement publications are reproduced in the following.

2.2.1. Complete genome sequences of six strains of the genus Methylobacterium Journal of Bacteriology, volume 193, number 18, pages 5035-5036 2.2.2. Complete genome sequence of the chloromethane-degrading Hyphomicrobium sp.

strain MC1

Journal of

Bacteriology, volume 194, number 17,

TABLE 1

 1 Characteristics of the six complete Methylobacterium genomes sequenced in this study

			Genome analysis
				No. of			GenBank
			Size	rrn	No. of	No. of	accession
	Organism	Key characteristic(s)	(Mb) % GC	operons	tRNAs	CDS a	no.	Reference
	M. extorquens strain PA1	Arabidopsis thaliana epiphyte	5.471 68.2	5	58	5,410	NC_010172 11
	M. extorquens strain CM4	Chloromethane degrader	5.778 68.2	5	61	3,112	NC_011757 13
			0.380 66.3			388	NC_011758
			0.023 63.9			44	NC_011760
	M. extorquens strain BJ001 b	Populus deltoides x nigra DN34 endophyte	5.800 69.4	5	58	6,017	NC_010725 17
			0.025 64.9			30	NC_010727
			0.023 66.8			31	NC_010721
	M. radiotolerans strain JCM 2831 Radioresistant strain	6.078 71.5	4	56	6,325	NC_010505 9
			0.586 69.6	2	1	650	NC_010510
			0.047 62.5			66	NC_010509
			0.043 63.2		1	75	NC_010514
			0.038 63.7		1	60	NC_010517
			0.036 62.0			64	NC_010518
			0.028 61.0		1	45	NC_010502
			0.022 61.1			38	NC_010504
			0.021 65.1			33	NC_010507
	Methylobacterium sp. strain 4-46 Lotononis bainesi nodulating, photosynthetic 7.659 71.6	6	63	8,337	NC_010511 7
			0.058 65.1			108	NC_010373
			0.020 59.2			34	NC_010374
	M. nodulans strain ORS 2060	Nonpigmented, nitrogen fixing, Crotalaria	7.772 68.9	7	71	8,879	NC_011894 10
		nodulating	0.488 65.9		2	630	NC_011892
			0.458 65.7			609	NC_011887
			0.040 64.2			84	NC_011893
			0.038 61.6			66	NC_011895
			0.020 61.4			30	NC_011888
			0.013 60.5			16	NC_011889
			0.010 67.2			14	NC_011890

Table 2 .1. Characteristics of bacterial genomes harboring homologs of the cmuA gene a Strains Methylobacterium extorquens CM4 Hyphomicrobium sp. MC1 Roseovarius sp. 217 Leisingera methylo- halidivorans DSM 14336 Desulfomonile tiedjei DSM 6799 Thermosediminibacter oceani DSM 16646

 2

	Thermincola
	potens JR

.3. Comparison of CmuA of Methylobacterium extorquens CM4 and its homologs in Roseovarius sp. 217 and Leisingera methylohalidivorans

  .1) have predicted cmuA homologs with over 64% protein identity to CmuA of demonstrated function of strain CM4, with the exception of the two marine strains of the Rhodobacteraceae family, i.e.

	Roseovarius sp. 217 and Leisingera methylohalidivorans DSM 14336, which only
	contain a predicted protein with 34% protein identity to the C-terminal domain of CmuA
	of strain CM4 (Table 2.1) (Fig. 2.3).
		CmuA protein
	M. extorquens CM4			617 aa
	(cmuA, gene # MchI_5697)	Methyltransferase I domain	Corrinoid-binding domain
		(N-terminal)	(C-terminal)
			34% identity
	Roseovarius sp. 217			227 aa
	(gene # ROS217MchI_5797)
	Leisingera methylohalidivorans DSM 14336 (gene # Leime_2531)	34% identity	227 aa
	Fig. 2
	strains Roseovarius sp. 217 and Leisingera methylohalidivorans DSM 14336 (Fig. 2.3,
	Table 2.1), suggest that an alternative pathway for dehalogenation of chloromethane is
	used in these strains. However, it should be noted that other closely related

Similarly, two other methyltransferase proteins involved in chloromethanedehalogenation (i.e. CmuB and CmuC encoded by cmuB and cmuC genes) conserved in chloromethane-degrading strains with the cmu pathway, lack a corresponding homolog in Roseovarius sp. 217 and Leisingera methylohalidivorans DSM 14336 (Table 2.1). Taken together, the lack of homologs of cmuB and cmuC, and the only distant cmuA homolog in chloromethane-utilizing strains from the same family (Rhodobacteraceae), i.e. Roseovarius sp. 179 and Ruegeria sp. 198, whose sequences are not yet available, were ROS217_19562 Chapter 2

  genes are located in a single cluster, presumably as an operon, and on a putative

genomic island. The organization of cmu in Hyphomicrobium sp. MC1 is conserved in other chloromethane-degrading strains isolated from different environments

[START_REF] Nadalig | Detection and isolation of chloromethane-degrading bacteria from the Arabidopsis thaliana phyllosphere, and characterization of chloromethane utilization genes[END_REF]

[see Chapter 4]. In Roseovarius sp. 217 and Leisingera methylohalidivorans DSM 14336, in contrast, homologs of cmu genes are not clustered as in strains CM4 and MC1, confirming the suggestion that such distant gene homologs to bona fide cmu gene counterparts in strains CM4 and MC1 may actually not be involved in chloromethane utilization. The reverse situation applies to strains Thermosediminibacter oceani DSM 16646, Desulfomonile tiedjei DSM 6799, and Thermincola potens JR, in which cmu gene homologs were found, but which are not known to dehalogenate chloromethane or to use it as a growth substrate. In Thermosediminibacter oceani DSM 16646 and Thermincola

Table 2 .2. Common and unique genes found in the genomes of Methylobacterium extorquens CM4 and Hyphomicrobium sp. MC1 a, b

 2 .1) were not colocalized.

	Chapter 2

Table 1 .

 1 Growth of chloromethane-degrading strains and dehalogenase activity of resting cells with chloromethane Control experiments in buffer without cells yielded an abiotic degradation rate under the same conditions of 0.054 AE 0.008 nmol min À1 corresponding to o 0.1% the observed biotic rates. Similarly, the observed rate for cell suspensions of the nondechlorinating strain Methylobacterium extorquens AM1 (Vuilleumier et al., 2009) was 0.0093 AE 0.0018 nmol min À1 mg À1 protein. All rights reserved calcium nitrate solution (25 g L À1 ) and of trace elements solution containing (mg L À1 ) FeSO 4 Á 7H 2 O (100), MnSO 4 Á H 2 O (100), ZnSO 4 (29.5), Co(NO 3 ) 2 Á 6H 2 O (25), CuCl 2 Á 2H 2 O (25), Na 2 MoO 4 Á 2H 2 O (25), NH 4 VO 3

			Specific activity
		Doubling	(nmol min À1 mg À1
	Strains	time (h)	protein) Ã
	Methylobacterium extorquens	7.3 AE 1.1	26.1 AE 5.8
	CM4
	Hyphomicrobium	4.9 AE 0.3	24.8 AE 2.2
	chloromethanicum CM2
	Hyphomicrobium sp. strain MC1	5.1 AE 0.3	29.5 AE 4.0
	Hyphomicrobium sp. strain AT2	5.9 AE 0.7	28.0 AE 0.3
	Hyphomicrobium sp. strain AT3	18.1 AE 0.5	22.8 AE 2.0
	Hyphomicrobium sp. strain AT4	19.8 AE 0.5	21.6 AE 0.2

à FEMS Microbiol Ecol 77 (2011) 438-448 c 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd.

Table 2 .

 2 Reactions were performed in 0.2-mL microcentrifuge tube

	FEMS Microbiol Ecol 77 (2011) 438-448

Table 2 .

 2 Primers used in this study

	Target gene	Primer Ã	Sequence (5 0 -3 0 ) w	Position z	References
	cmuA	cmuA802f	TTCAACGGCGAYATGTATCCYGG	7404-7426	Miller et al. (2004)
		cmuAfor2 ‰	CAAGAACGTAAAGCCTGAGCA	8180-8200	This study
		cmuA1609R	TCTCGATGAACTGCTCRGGCT	8212-8190	Miller et al. (2004)
		cmuArev ‰	GGCATGTCGGTGATGACMAAYTC	8263-8241	This study
		cmuArev2 ‰	ATGCACGGATGGACGACGGA	7519-7500	This study
		cmuA1802r	TTVGCRTCRAGVCCGTA	8404-8388	This study
	cmuB	cmuBfor	GGCRTGCARATGGCGTTCGACG	4630-4651	This study
		cmuBfor3 ‰	TTCCCCAAGTGGACGG	5094-5109	This study
		cmuBfor4 ‰	GCGAATGGGTCA	5128-5139	This study
		cmuBrev	CTACGCTTCGCTGCGCAGGAACT	5273-5252	This study
		cmuBrev2 ‰	ATGATCAACGCATCAGAGGC	4742-4723	This study
	cmuC	cmuCfor ‰	GGCGACGACCTTGGCTTTCAG	5954-5974	This study
		cmuCfor2 ‰	TCCGAAATTGATTTTC	5980-5995	This study
		cmuCfor3 ‰	GCATGTTCGTGTCCGAAAT	5969-5987	This study
		cmuCfor4 ‰	GCGCCGATGGACAATATTTC	6313-6332	This study
		cmuCrev2 ‰	ACGCCGGACGATGT	6401-6388	This study
	paaE	paaErev1	TSTCGTCGAARTCGAT	9865-9850	This study
	hutI	hutIrev2	TCVTCRCARHAVRCYTCDAC	10 655-10 635	This study
	Ã Annealing temperatures for PCR: 67 1C, cmuA802f-cmuArev; 55 1C, cmuA802f-cmuA1609R and cmuA802f-cmuA1802r; 61 1C, cmuA802f-
	paaErev1; 65 1C, cmuA802f-hutIrev2; 62 1C, cmuBfor-cmuBrev; 67 1C, cmuBfor-cmuArev.

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Arabidopsis thalina plant (picture taken by Myhammad FARHAN UL HAQUE)Methylobacterium extorquens CM4 cells (1000x) (picture taken by Sandro ROSELLI)

Scientific communications

Acknowledgements

With the humble praise and gratitude to almighty ALLAH, the compassionate and the merciful, for bestowing me His endless blessings throughout my life and my affectionate to the Holy Prophet Muhammad (peace be upon him), whose moral and spiritual teachings enlightened our hearts and minds towards achieving high ideas of life.

The work of the thesis was performed at the laboratory "Génétique Moléculaire, Génomique, Microbiologie" UMR7156-CNRS-Université de Strasbourg. Thanks to all the members of the unit, it was a pleasure to work here. I would like to express my sincere thanks to Dr Steffen Kolb, Dr. Lionel Moulin, Prof. Julia Vorholt and Prof. Anne-Catherine Schmit for accepting to be the members of jury of my thesis defense. I would like to express my deep and sincere gratitude to my supervisor Prof. Stéphane Vuilleumier for having confidence in me and providing me the opputunity to be the part of this project and his lab. Thanks Stéphane for all the guidance and teaching throughout this ACKNOWLEDGMENTS This research resulted from an approved proposal (Marx_0165_051130) evaluated during the DOE-CSP-05 program supported by the Office of Biological and Environmental Research in the DOE Office of Science. The team of C.J.M. was also supported by an NSF grant (IOB-0612591), and the team of S.V. was also supported by a mobility grant from CNRS (USA mobility program). The work conducted by the U.S. Department of Energy Joint Genome Institute was supported by the Office of Science of the U.S. Department of Energy under contract DE-AC02-05CH11231.

We are grateful to the JGI personnel who participated in the sequencing, assembly, and automated annotation processes.

Acknowledgements

Support for this project by REALISE, the Alsace Network for Engineering and Environmental Sciences (http://realise. u-strasbg.fr) and from the EC2CO program of CNRS-INSU is gratefully acknowledged. We also thank SFERE, the Government of Pakistan and the French Ministry of Foreign Affairs for a PhD grant to M.F.U.H., and the French Ministry of Research and Higher Education for a PhD grant to S.R.

annotation and analysis platform of Génoscope (https://www.genoscope.cns.fr/agc/microscope/mage/) , Integrated Microbial Genomes platform of the US American Joint Genomic Institute (http://img.jgi

Methyl halides are volatile one-carbon compounds which play an important role in the functioning of the atmosphere. Global budgets of methyl halides are still poorly understood due to uncertainties in estimations of their natural sources and sinks. Among them, chloromethane (CH 3 Cl) is the most abundant halogenated hydrocarbon in the atmosphere, and responsible for substantial destruction of stratospheric ozone. A bacterial bioreporter for the detection of methyl halides was developed on the basis of detailed knowledge of the physiology and genetics of the Alphaproteobacterium Methylobacterium extorquens CM4, which utilizes chloromethane as the sole source of carbon and energy for growth. A plasmid construct with the promoter region of the chloromethane dehalogenase gene cmuA fused to a promotorless yellow fluorescent protein gene cassette resulted in methyl halide dependent fluorescence when introduced into M. extorquens CM4. Fluorescence production of the bacterial bioreporter to methyl halides was shown to be rapid, specific and highly sensitive.

This bioreporter may provide an attractive alternative to analytical chemical methods to screen for methyl halide emissions from different environments, including plants

Supporting Information

Additional Supporting Information may be found in the online version of this article: Fig. S1. Analysis of PCR-amplified fragments of cmuA obtained with primers cmuA802f and cmuA1802r. Fig. S2. Phylogenetic analysis of cmuB and cmuC genes in chloromethane-degrading strains. Table S1. Sequence identity of amplified cmuA gene fragments.

Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

Conclusions and perspectives

Chapter 7 156 be involved in plant defense against pathogens [START_REF] Innerebner | Protection of Arabidopsis thaliana against leaf-pathogenic Pseudomonas syringae by Sphingomonas strains in a controlled model system[END_REF][START_REF] Vogel | forward genetic in planta screen for identification of plant-protective traits of Sphingomonas sp strain Fr1 against Pseudomonas syringae DC3000[END_REF], varied significantly among the three HOL1 gene variants investigated.

However, the observed slight differences in bacterial community structure [Chapter 6] of the HOL1 variants might not only be due to differences in chloromethane emissions, but could follow from production of other volatile compounds (such as methyl thiocyanate) or the metabolism of glucosinolate metabolic products previously reported to affect the bacterial population and community composition in the phyllosphere [START_REF] Ruppel | Composition of the phyllospheric microbial populations on vegetable plants with different glucosinolate and carotenoid compositions[END_REF][START_REF] Schreiner | Interaction between plants and bacteria: Glucosinolates and phyllospheric colonization of cruciferous vegetables by Enterobacter radicincitans DSM 16656[END_REF], and also dependent on the activity of the HOL1 enzyme these data strongly suggest that plant emissions of chloromethane, however minor compared to those of methanol for example (Clerbaux et al., 2007;[START_REF] Bracho-Nunez | Plant-specific volatile organic compound emission rates from young and mature leaves of Mediterranean vegetation[END_REF]) (Table 1.3), may provide a selective advantage for growth of chloromethane-degrading bacteria in the phyllosphere, for reasons which need to be understood in more detail in future work.

Perspectives for future work

The insights obtained in the different aspects of this project suggest many avenues for further research in order to gain a better understanding of chloromethane degradation by bacteria in the phyllosphere. may in some cases be below the detection or quantification limits of analytical instruments, at least when no pre-concentration step is used before analysis. For example the sensitive limits for chloromethane detection based on GC/MS methods described previously are 1 -100 ppt (µM range) or even higher (McClenny et al., 2002;Rhew et al., 2003;[START_REF] Blei | Methyl bromide and methyl chloride fluxes from temperate forest litter[END_REF][START_REF] Derendorp | Methyl chloride and C2-C5 hydrocarbon emissions from dry leaf litter and their dependence on temperature[END_REF]. While the bioreporter developed in this work may help in this context, the use of stable isotope analysis will also be considered. Stable isotope analysis is a powerful tool to constrain fluxes of chloromethane between various environmental compartments [START_REF] Greule | Stable hydrogen-isotope analysis of methyl chloride emitted from heated halophytic plants[END_REF]. In collaboration with the team of Frank Keppler of the Max Planck Institute of Chemistry in Mainz, Germany, our group has measured hydrogen and carbon isotope fractionation of the remaining untransformed chloromethane following its degradation by Methylobacterium extorquens CM4 (Nadalig et al., submitted) Use of this technique may help to further demonstrate the existence of both chloromethane emissions by A. thaliana and its consumption in the phyllosphere.

In addition, the emissions from the phyllosphere with respect to differential HOL1 expression in A. thaliana variants also need to be investigated further. Plant metabolites emitted by A.

thaliana may be detected by gas chromatography coupled to mass spectrometry (GC-MS) [START_REF] Brodersen | Isoprenoid biosynthesis is required for miRNA function and affects membrane association of Argonaute 1 in Arabidopsis[END_REF]. For these experiments, the phyllosphere of HOL1 gene variant plants grown from sterile seeds, under axenic conditions on MS medium in closed jars using well-established laboratory procedures, will be analyzed. Targeted volatile compounds to be monitored will include chloromethane and other volatile chemicals such as C-1 compounds (CO 2 , methanol, and formaldehyde), plant leaf reference metabolites such as phytohormones (indole-3-acetic acid, cytokinin), monoterpenes and ethylene. Other metabolites associated with the HOL1 gene, e.g. thiocyanate and methylthiocyanate (Nagatoshi and Nakamura, 2009) will also be followed. Gases will be captured and extracted using a gas flux column for given periods of time to help concentrate and circumvent temporal fluctuations of plant-Chapter 7

158 emitted volatile compounds. Headspace volatile organic compounds can be collected using glass jars or using Teflon bags and measured by thermodesorption-GC-MS (TD-GC-MS) [START_REF] Houshyani | Three-step pathway engineering results in more incidence rate and higher emission of nerolidol and improved attraction of Diadegma semiclausum[END_REF][START_REF] Pearse | Headspace volatiles from 52 oak species advertise induction, species identity, and evolution, but not defense[END_REF]. This work, planned in collaboration with IBMP, CNRS Strasbourg, will require substantial further funding, which has been applied for in the context of the ANR BIOADAPT program. This strategy may also allow to address the question of a potential selective advantage provided by the chloromethane degrading ability of bacteria in colonization of the phyllosphere. Alternatively, this question may also be investigated by competition experiments such as those developed by the Vorhjolt group (Knief et al., 2010a;[START_REF] Innerebner | Protection of Arabidopsis thaliana against leaf-pathogenic Pseudomonas syringae by Sphingomonas strains in a controlled model system[END_REF]. For such experiments, we envisage to inoculate A. thaliana plants with varying

Chapter 7

Chapter 7