Larry J Millet
email: lmillet1@utk.edu

Jayde Aufrecht

Jessy Labbé

Jessie Uehling

Rytas Vilgalys

Myka L Estes

Cora Miquel Guennoc

Aurélie Deveau

Stefan Olsson

Gregory Bonito

Mitchel J Doktycz

Scott T Retterer

Increasing access to microfluidics for studying fungi and other branched biological structures

Keywords: Fungi, Microfluidics, Cell culture, Bacterial-fungal interactions, Plant root, Arabidopsis, Neuron, Branching biology

Background: Microfluidic systems are well-suited for studying mixed biological communities for improving industrial processes of fermentation, biofuel production, and pharmaceutical production. The results of which have the potential to resolve the underlying mechanisms of growth and transport in these complex branched living systems. Microfluidics provide controlled environments and improved optical access for real-time and high-resolution imaging studies that allow high-content and quantitative analyses. Studying growing branched structures and the dynamics of cellular interactions with both biotic and abiotic cues provides context for molecule production and genetic manipulations. To make progress in this arena, technical and logistical barriers must be overcome to more effectively deploy microfluidics in biological disciplines. A principle technical barrier is the process of assembling, sterilizing, and hydrating the microfluidic system; the lack of the necessary equipment for the preparatory process is a contributing factor to this barrier. To improve access to microfluidic systems, we present the development, characterization, and implementation of a microfluidics assembly and packaging process that builds on self-priming point-of-care principles to achieve "ready-to-use microfluidics. "

Results:

We present results from domestic and international collaborations using novel microfluidic architectures prepared with a unique packaging protocol. We implement this approach by focusing primarily on filamentous fungi; we also demonstrate the utility of this approach for collaborations on plants and neurons. In this work we (1) determine the shelf-life of ready-to-use microfluidics, (2) demonstrate biofilm-like colonization on fungi, (3) describe bacterial motility on fungal hyphae (fungal highway), (4) report material-dependent bacterial-fungal colonization, (5) demonstrate germination of vacuum-sealed Arabidopsis seeds in microfluidics stored for up to 2 weeks, and (6) observe bidirectional cytoplasmic streaming in fungi.

Background

Morphological branching is a common and fundamental mode of biological propagation and growth [START_REF] Devauchelle | Ramification of stream networks[END_REF][START_REF] Perron | The root of branching river networks[END_REF][START_REF] Ebert | Streamers, sprites, leaders, lightning: from micro-to macroscales[END_REF][START_REF] Lu | Patterning mechanisms of branched organs[END_REF]. Branched biological structures are evident across all taxonomic kingdoms and size scales. Aggregates of unicellular bacteria form branching communities, which gives rise to colonial phenotypes that are often distinct (e.g. morphology and color) [START_REF] Golding | Adaptive branching during colonial development of lubricating bacteria[END_REF][START_REF] Be'er | Lethal protein produced in response to competition between sibling bacterial colonies[END_REF][START_REF] Be'er | Paenibacillus dendritiformis bacterial colony growth depends on surfactant but not on bacterial motion[END_REF][START_REF] Richards | Mechanistic basis of branch-site selection in filamentous bacteria[END_REF][START_REF] Flärdh | Regulation of apical growth and hyphal branching in Streptomyces[END_REF][START_REF] Wardell | Decreasing the hyphal branching rate of Saccharopolyspora erythraea NRRL 2338 leads to increased resistance to breakage and increased antibiotic production[END_REF]. From roots to branches, and stems to leaf structures; plants arborize to develop high surface area tissues; a plants branching structure is vital for nutrient absorption in photosynthesis, energy transport and storage, reproduction, and waste release [START_REF] Hallé | Branching in plants[END_REF]. Hyphal branching in fungi is involved in beneficial and detrimental interactions among plants and microbes, it is through these high surface-area structures that nutrient uptake, environmental signaling, and communication are achieved [START_REF] Martinuz | The application of Arabidopsis thaliana in studying tripartite interactions among plants, beneficial fungal endophytes and biotrophic plantparasitic nematodes[END_REF][START_REF] Van Overbeek | Impact of bacterial-fungal interactions on the colonization of the endosphere[END_REF][START_REF] Deveau | Bacterial-fungal interactions: ecology, mechanisms and challenges[END_REF]. Neuronal cells and tissues branch to connect with targets cells and organs for efficient computation of information and the coordination of physiological processes [START_REF] Chklovskii | Synaptic connectivity and neuronal morphology[END_REF][START_REF] Tsai | Fibrillar amyloid deposition leads to local synaptic abnormalities and breakage of neuronal branches[END_REF]. Understanding the physical and molecular cues that initiate the formation and function of branching structures and resolve the underlying mechanisms of growth and transport in branched tissues will benefit relevant industries including those involved in fermentation, biofuel production, and health care. To capture the dynamics of this process at such a fine spatial scale requires a culturing platform that enables real-time and high-resolution imaging. While microtechnological methods are well established for culturing neurons and mammalian cells, advancements for increasing the precision and sophistication for measuring plant, fungal, and microbial structures and dynamics (e.g. growth, forces, secretions) are in demand [START_REF] Reinmets | Microfluidic quantification of yeast surface adhesion[END_REF][START_REF] Sun | A microfluidic platform with integrated sensing pillars for protrusive force measurements on neurospora crassa[END_REF][START_REF] Jones S Jr | A microfluidic device for massively parallel, whole-lifespan imaging of single fission yeast cells[END_REF][START_REF] Li | Microfluidic system for rapid detection of airborne pathogenic fungal spores[END_REF][START_REF] Beneyton | Droplet-based microfluidic high-throughput screening of heterologous enzymes secreted by the yeast Yarrowia lipolytica[END_REF].

Microfluidic platforms are particularly well-suited for guiding, restraining, and imaging branched structural growth and specification; through a modular on-chip approach, branched biological systems can be interrogated in ways not possible via conventional culture. For example, customized microfluidic designs with interconnected compartments may be used to facilitate and isolate desired branching features. Highly-controlled microfluidic environments also improve the ability to study innate mechanisms and environmental influences guiding process outgrowth, cellular physiology, and biological interactions [START_REF] Millet | New perspectives on neuronal development via microfluidic environments[END_REF][START_REF] Held | Microfluidics structures for probing the dynamic behaviour of filamentous fungi[END_REF][START_REF] Hanson | Fungi use efficient algorithms for the exploration of microfluidic networks[END_REF][START_REF] Hu | Microfluidic analysis of pressure drop and flow behavior in hypertensive micro vessels[END_REF][START_REF] Stanley | Probing bacterial-fungal interactions at the single cell level[END_REF]. Despite these benefits, the intersection of microfluidics and biology is highly interdisciplinary and often requires strong cross-departmental or multi-institutional collaborations in order to apply the precision technology toward answering deeplyrooted biological questions. Microfluidic applications for rhizosphere-on-a-chip are providing high-resolution access for studying the dynamics of root-bacterial interactions [START_REF] Massalha | Live imaging of root-bacteria interactions in a microfluidics setup[END_REF][START_REF] Morrell-Falvey | Microscope slide-in chamber[END_REF][START_REF] Aufrecht | Quantifying the spatiotemporal dynamics of plant root colonization by beneficial bacteria in a microfluidic habitat[END_REF]. Recent studies on fungal polarity and growth, and plant development and metabolism demonstrate the ability to increase throughput and semi-automate work to resolve molecular regulations of mesoscale biological interactions using microfluidics [START_REF] Geng | Compartmentalized microchannel array for high-throughput analysis of single cell polarized growth and dynamics[END_REF][START_REF] Grossmann | The RootChip: an integrated microfluidic chip for plant science[END_REF].

Despite the numerous papers that have sought to simplify microfluidics use for biological inquiry [START_REF] Mu | Microfluidics for manipulating cells[END_REF][START_REF] Kovarik | Micro total analysis systems for cell biology and biochemical assays[END_REF], technical and logistical barriers still exist that counteract the effective deployment of microfluidics in biological disciplines. Technical barriers to bio-microfluidic collaborations include equipment, such as vacuum pumps, house vacuum systems, syringe pumps, fabrication facilities. Physical barriers include institutional proximity. Unfortunately, the lack of familiarity with microfluidic systems (creation and implementation) and personal contacts are also primary impediments to initiating collaborations. Overcoming these limitations will promote the broad use of a microfluidics tool-set for biological inquiry. For example, microfluidic platforms can readily be designed to provide an environment that allows for resolving the biological response to local physical, chemical, and biological cues integrated within the platform design [START_REF] Pagano | Optimizing design and fabrication of microfluidic devices for cell cultures: an effective approach to control cell microenvironment in three dimensions[END_REF][START_REF] Kim | Biological applications of microfluidic gradient devices[END_REF][START_REF] Zervantonakis | Three-dimensional microfluidic model for tumor cell intravasation and endothelial barrier function[END_REF][START_REF] Hv | Directional memory arises from long-lived cytoskeletal asymmetries in polarized chemotactic cells[END_REF]. Complex barriers, topographical cues, compartmentalization of chemically and physically interacting biological systems, and complex chemical gradients can be controlled and manipulated within microfluidic systems to replicate natural interactions and conditions in a fundamental form [START_REF] Millet | New perspectives on neuronal development via microfluidic environments[END_REF][START_REF] Chiu | Small but perfectly formed? Successes, challenges, and opportunities At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research ? Choose BMC and benefit from: for microfluidics in the chemical and biological sciences[END_REF][START_REF] Jain | A shear gradient-activated microfluidic device for automated monitoring of whole blood haemostasis and platelet function[END_REF][START_REF] Möller | Steep pH gradients and directed colloid transport in a microfluidic alkaline hydrothermal pore[END_REF][START_REF] Schwarz | A microfluidic device for measuring cell migration towards substratebound and soluble chemokine gradients[END_REF][START_REF] Millet | Nanofluidic interfaces in microfluidic networks[END_REF].

Assembling, sterilizing, and hydrating the microsystem is one of the principle technical barriers that limits the use and implementation of microfluidics in biological investigation. The lack of both simple and precision equipment is a key contributing factor that further complicates the use of microfluidics [START_REF] Boyd-Moss | Self-contained microfluidic systems: a review[END_REF]. In the effort to help collaborators overcome barriers to implementing microfluidics, we have resolved a preparation strategy that greatly minimizes the hurdles associated with using microfluidics in biology. Here, we present the development, characterization, and implementation of a microfluidics assembly process that builds on principles employed in self-priming and point-of-care technologies [START_REF] Dimov | Stand-alone self-powered integrated microfluidic blood analysis system (SIMBAS)[END_REF][START_REF] Choi | Powering point-of-care diagnostic devices[END_REF][START_REF] Xu | Vacuum-driven power-free microfluidics utilizing the gas solubility or permeability of polydimethylsiloxane (PDMS)[END_REF][START_REF] Li | A selfpowered one-touch blood extraction system: a novel polymer-capped hollow microneedle integrated with a pre-vacuum actuator[END_REF][START_REF] Li | A "place n play" modular pump for portable microfluidic applications[END_REF][START_REF] Liang | Systematic characterization of degas-driven flow for poly(dimethylsiloxane) microfluidic devices[END_REF]. We designed and implemented novel microfluidic chips, and also tested previously implemented plant-chip platforms [START_REF] Aufrecht | Quantifying the spatiotemporal dynamics of plant root colonization by beneficial bacteria in a microfluidic habitat[END_REF], toward validating the utility of ready-to-use packaging to overcome common implementation barriers for environmental science research and for new users of microfluidics. We believe that this approach is applicable for a variety of different platform architectures and a wide range of biological studies. The result of our fabrication process is a sterile, ready-to-use microfluidic system that can be implemented anywhere. We demonstrate initial results from international collaborations focusing on the biology of filamentous fungi and include other applications for branching samples such as plants and neurons. In these biological systems, microfluidics enable real-time visualization and quantification of fungi, plants and multispecies interactions.

Methods

Methods for the fabrication and assembly of microfluidics are detailed in Additional file 1. Figure 1 summarizes the fabrication and assembly process used to prepare 'ready-to-use microfluidics. '

Vacuum packaging

Fully assembled microfluidic channels on glass slides, or in glass-bottom dishes, were first equilibrated under vacuum (90 kPa, 30 min) in a sealed glass desiccator. Then, channel assemblies were immediately removed from the vacuum chamber and transferred to thermal-sealable pouches and immediately vacuum-sealed (82 kPa, Food Saver model #V3240) for transport and until the time of use. To determine the 'best if used by date' , 35 microfluidic channel samples were vacuum packed and marked with the test date. To measure the time for the microfluidics to fill, channels were removed from vacuum-sealed pouches and loaded with water containing food color dye; the time from dye addition to channel fill was documented and plotted (Fig. 2).

Microfluidic design and operation

Spoke-wheel microfluidics This novel chip architecture (new and not previously published) consists of a central circular chamber (8 mm) with the roof supported by pillars (n = 118 dia. = 100 µm). Sixteen primary radiating channels (l = 1.5 mm, w = 500 µm) emanate from the central chamber to terminate at a primary concentric channel (r = 6 mm, w = 500 µm). Secondary radial channels (n = 123, dia. = 4 mm) connect the primary and secondary concentric channels. The depth of the entire channel system is different for fungal cultures (h = 11 µm) and neuronal cultures (h = 50 µm).

Two ports at the ends of the peripheral channel permit fluidic exchange. The middle 'accessory' port between the center well and the peripheral ports allows for efficient priming of the microfluidic space; for bacterial-fungal interaction studies, this port is for loading agar into the central chamber. For bacterial-fungal interactions, the center well is used for inoculating fungi, the primary concentric channel is used for observing bacterial-fungal interactions, and the peripheral channel is used for inoculating bacteria. For neuronal cultures, the center well is used for the introduction of dissociated neurons, the peripheral channel ports are used for media exchange.

Fig. 1 An overview of the fabrication and assembly protocol for creating ready-to-use microfluidics. Standard photolithography is used to create microfluidic masters, conventional replicate molding with PDMS is used to produce microfluidic structures that are annealed to glass slides or coverslips. UV light exposure or autoclaving processes are used to sterilize the microfluidic platforms. Equilibration under vacuum is the conditioning step that immediately precedes vacuum packaging to produce a ready-to-use microfluidic platform "ORNL" chambers This novel architecture (new and not previously published) provides two parallel boundary channels that connect two separate ports. Parallel boundary channels are connected through a grid of microfluidic channels (w = 15 µm, h = 17 µm). Within the grid between the boundary channels are four chambers formed of the letters "ORNL. " Pitch of the 15-µm channel grid is ~ 500 µm for the smaller array. This design enables fungal growth in chambers for hyphal isolation and bacterial fungal interaction studies.

Root chip microfluidic This microfluidic architecture was previously designed for Arabidopsis seed germination and growth rate measures, and has been published for root-bacteria interaction studies [START_REF] Aufrecht | Quantifying the spatiotemporal dynamics of plant root colonization by beneficial bacteria in a microfluidic habitat[END_REF][START_REF] Aufrecht | Imaging the root hair morphology of Arabidopsis; seedlings in a two-layer microfluidic platform[END_REF]. Here, this device is used to demonstrate that prior art, such as the seed-in-chip system, can be incorporated into this fabrication process for decreasing barriers to implementing microfluidics in collaborations. Channel dimensions include a central channel (4 mm l, 200 µm w, 200 µm h) guides the primary root shaft down a larger (20 µm tall, 1 µL) rectangular chamber for bathing the growing root hairs. The PDMS microfluidic chip was exposed to air plasma prior to conformal contact and thermal annealing (70 °C). The channel-on-slide structures were cleaned by autoclave sterilization prior to vacuum-packaging of Arabidopsis seeds.

Cell cultures

Protocols for preparations and cell cultures of eukaryotic and prokaryotic cells (fungi, plant, bacteria, and neuronal cultures) are modified from prior reports and detailed in supplementary information [START_REF] Michelsen | Nonribosomal peptides, key biocontrol components for Pseudomonas fluorescens In5, isolated from a Greenlandic suppressive soil[END_REF][START_REF] Kachar | Asymmetric illumination contrast: a method of image formation for video light microscopy[END_REF][START_REF] Cuong | Hyphae-colonizing Burkholderia sp.: a new source of biological control agents against sheath blight disease (Rhizoctonia solani AG1-IA) in rice[END_REF][START_REF] Nevill | Vacuum soft lithography to direct neuronal polarization[END_REF][START_REF] Tero | Rules for biologically inspired adaptive network design[END_REF][START_REF] Rayner | The challenge of the individualistic mycelium[END_REF][START_REF] Dhamgaye | The influence of bacterial interaction on the virulence of Cryptococcus neoformans[END_REF][START_REF] Peleg | Medically important bacterial-fungal interactions[END_REF][START_REF] Artursson | Interactions between arbuscular mycorrhizal fungi and bacteria and their potential for stimulating plant growth[END_REF][START_REF] Bonfante | Mechanisms underlying beneficial plant-fungus interactions in mycorrhizal symbiosis[END_REF][START_REF] Uehling | Comparative genomics of Mortierella elongata and its bacterial endosymbiont Mycoavidus cysteinexigens[END_REF][START_REF] Müller | Growing poplars for research with and without mycorrhizas[END_REF][START_REF] Teal | Spatiometabolic stratification of Shewanella oneidensis biofilms[END_REF][START_REF] Chen | NS21: redefined and modified supplement B27 for neuronal cultures[END_REF].

Results

The fabrication and assembly of PDMS-based microfluidics for fungi, roots, and neurons was achieved through conventional photolithographic processes yielding Fig. 2 Sterile pre-packaged ready-to-use microfluidics. a Design of the spoke-wheel device used for characterizing the vacuum-packaging and use process, and for quantifying the 'best if used by date. ' b Spoke wheel microfluidic chamber with integrated PDMS tubing in a glass-bottom dish, vacuum-equilibrated (20 min), then vacuum-sealed for storage, transport, or direct use. c Autoclaved microscope slide with spoke-wheel microfluidic culture chamber in a vacuum-sealed pouch. d The spoke wheel microfluidic device in a coverslip-bottom dish filled with water containing green food-dye. e Vacuum packaged microfluidics on microscope slides or in coverslip-bottom dishes maintain ability to fill (< 2 weeks) within 13 min ◂ PDMS-channels on microscope slides or coverslip-bottomed dishes. UV-treatment (for glass-bottomed dishes), or autoclave sterilization (microscope slides), produces a sterile microfluidic platform that is amenable for cell culture studies (Fig. 1). Figure 2a-d shows that microfluidics on glass-bottomed dishes (with or without lids) or microscope slides can be vacuum sealed in plastic pouches for shipping, and later, fluidic priming. Low viscosity solutions (e.g. culture media or water) completely fill the entire channel network within minutes after removing the PDMS spoke-wheel microfluidics from the vacuum chamber. Figure 2e shows that vacuum-packaged microfluidics continue to fill with culture media toward completion, if used within 14 days of the vacuum sealing date.

Prior to distributing vacuum-packed microfluidics to collaborators, we characterized the feasibility of our spoke-wheel microfluidic design to permit the observation of bacterial-fungal interactions for high resolution imaging studies. Figure 3a, b shows a spoke-wheel microfluidic architecture that promotes fungal-bacterial interaction studies in microfluidics. The agar-filled central chamber promotes fungal growth while stabilizing the fungal inoculum during culture and shipping (Fig. 3b). The peripheral concentric channel enables the introduction of bacteria at the periphery of the microenvironment. Center to edge, the radiating channels maintain a similar culture space and volume in the channels while providing increasingly narrow passages for the navigation of the fungal hyphae. The central chamber (without the culture well) has a volume of 0.04 µL, the 16 primary radiating channels (large channels) with the primary concentric channel retains 0.03 µL, and the sum of the 63 spokes (small channels) is 0.04 µL. Figure 3c shows a summary of the number of hyphae that colonize the intersection of the spoke-wheel of the platform following 2 weeks in culture. In Fig. 3c, an asterisk and hash symbol in the inset and data bar demarcate the corresponding values for regions of the device quantified as entering and exciting the large microfluidic channel prior to entering the small microfluidic spoke channel. Figure 3d, e is an example of hyphae in the large microfluidic channel, on average two hyphae occupy the intersection of the primary concentric circle and the hyphal spoke to permit a low-density of hyphae for resolving bacteria-fungal interactions. As the fungal cultures continue to grow and mature, the fungal hyphae reproducibly fill the device.

After 3 days of co-culturing bacteria in the microfluidic environment, individual hyphae and bacterial colonizations can be observed in the 15 µm-wide radiating (spoke) channel (Additional file 2: Figure S1). After 4 weeks in culture, fungal hyphae navigate the entire fluidic architecture with bacteria to intersect in open places and align in confined spaces. Probiotic biofilms of the mutualistic Pseudomonas fluorescens GM41 become established on the surface of Laccaria bicolor hyphae [START_REF] Guennoc | Bacterial biofilm formation on the hyphae of ectomycorrhizal fungi: a widespread ability under controls?[END_REF][START_REF] Labbe | Newly identified helper bacteria stimulate ectomycorrhizal formation in Populus[END_REF]. We observed that L. bicolor hyphae show preference for the PDMS surface over the glass surface (Fig. 3f(iii)), which is softer and more gas permeable. The bacterial colonies form at either glass or PDMS interfaces, but colony prevalence is substantially reduced at the glassfluid interface (Fig. 3f(i)) as compared to the PDMS-fluid interface (Fig. 3f(ii)). Through collaboration, high-resolution images of fluorescent P. fluorescens BBc6 colonization of fungal hyphae were observed (Fig. 3g).

In minimal media, P. fluorescens GM41 cells navigate along the surface of Laccaria bicolor S238N hyphae in vitro prior to colonization on or adjacent to the hyphae (Additional file 3: Figure S2). A standard deviation image of a an image stack (25 frames) from a time-lapse image series (6 s) shows a range of densities of bacteria (white borders on fungi against a black background) moving along Laccaria hyphae (Additional file 3: Figure S2). Kymographs (line traces over time displayed in a twodimension image) clarify the range of densities for which bacterial transport occurs on adjacent segments of the hypha (Additional file 3: Figure S2). But the biological question remains to be answered exactly how bacteria select colonization sites on fungal hyphae. While fungal (See figure on next page.) Fig. 3 Microfluidics for bacterial-fungal interaction studies. a The spoke-wheel microfluidic design incorporates ports for separate, spatially-defined inoculations of fungi and bacteria. b Shown are green dye-filled channels (loaded with vacuum-assisted filling) in a glass-bottomed dish. An agar plug fills the center of the chamber introduced through the center culture well and aspirated through the agar exchange port. The agar plug holds the fungal inoculum in place and minimizes dehydration during growth. For scale, the open glass surface (between black arrows) is 30 mm. c Average number of hyphae per primary and secondary radiating channels from a single dish (15 DIV). The inset image shows channel locations that correspond to the graphed data. d, e Bacterial-fungal interactions are conveniently established and imaged with microfluidic systems. Image d and inset e of Pseudomonas fluorescens GM41 navigating the Laccaria bicolor fungal highway within a microfluidic chamber (3 DIV bacteria co-culture). f(i, ii) P. fluorescens GM41 bacterial communities accumulate where fungi contact the glass (i) or PDMS (ii) surface, preferentially forming at the PDMS-hyphae surface d over the glass-hyphae interface (30 DIV co-culture). g In the same device architecture, Pseudomonas fluorescens BBc6 biofilm-like accumulation on ectomycorrhizal fungi (L. bicolor S238N) 16 h after bacterial inoculation. h Vacuum-packed spoke-wheel microfluidics are permissive for even the most sensitive cell cultures, neurons. Here, neurons (DIV 4) were transfected for molecular imaging studies (VAMP2, magenta; PSD-95, green) highway and bacterial-fungal interactions can be imaged outside of microfluidics, these microsystems make the process more convenient by preventing drying and enabling confinement over long culture periods to image and map bacterial motility and colonization on hyphal outgrowths.

With this vacuum packaging process being amenable for coverslip-bottomed dishes, with or without lids, we prepared 22 dishes capped with lids in vacuum-sealed pouches. Nineteen dishes were shipped to collaborators for culturing cortical neurons for transfection and molecular imaging of neuronal connections; three dishes were retained in-house. Of the 22 coverslip-bottomed dishes, three coverslips buckled due to the vacuum pressure and pouch pressing on the back of the fragile coverslip window. Placing the glass-bottomed dish within the lid of a 50 mm Petri dish stabilized the glass and solved the issue of the collapsing cover glass. With vacuumpackaged microfluidics, collaborators that are new to microfluidics were able to achieve neuronal cultures in challenging culture volumes and microfluidic dimensions (Fig. 3h).

Figure 4a-d shows seed germination results for Arabidopsis thaliana seeds cultivated in a root-chip system. In prior work, this system supported bacterial-plant interaction studies; here, we vacuum packed A. thaliana seeds in the system to determine if the packaging process negatively influenced the seed germination process. After 7 (n = 3) and 14 (n = 7) days of storage in the vacuum packaging (kept in the dark), 100% of the seeds germinated within 3 days of opening the package and filling the chamber with Murashige-Skoog medium (Fig. 4d). Of these germinated seedlings, all of the roots from the 7-day storage and two of the roots from the 14-day storage test grew into the large microfluidic channel. By the second and third days in vitro, the growth rate of the roots for the vacuum-stored seedlings in the channel were not significantly different from the growth rate of non-vacuumed control (n = 3) seedlings (Fig. 4d), although initial growth rates at day 1 were significantly different (p = 0.0227), unpaired t test, p < 0.05.

To improve access to microfluidics for fungal-bacterial interaction studies by microbiologists, we developed a universal chamber-grid architecture amenable for culturing fungi, bacteria, or bacterial-fungal co-cultures.

▸

We vacuum-packed sterile PDMS-based microfluidics for distribution to potential collaborators through a workshop on bacterial-fungal interactions. The ORNL microfluidic system is designed to contain two outer boundary channels and four central chambers in the formation of letters, "ORNL" (Fig. 5a). Upon removal of the microfluidic chamber from the vacuum-sealed pouch, the channel array is primed by adding fluid to all ports of the microfluidic channel system (Fig. 5b,c). 28 sample chambers were distributed to 18 fungal biologists within the United States (Michigan, New York, North Carolina) and throughout Europe (France, Germany, Netherlands, Denmark, Hungary, Sweden, and Switzerland) (Fig. 5d). Of the responses, at least 13 samples from 9 collaborators yielded 6 successful cultures. Figure 5e-h demonstrates a sample of the range of results achieved from successful cultures in microfluidics primed after removal from the vacuum package. Of the 7 unsuccessful reports, three samples only partially filled, and three containers reportedly lost vacuum by handling, and one sample was neglected until long after the 'best use by date. ' A discussion on the contributing factors competing with complete successful deployment is contained in the following discussion section.

Successful implementation of ORNL-chamber microfluidics to visualize fungal-bacterial and fungalplant interactions was achieved through collaborative exchanges (Fig. 5f-h). Bacterial-fungal interactions can occur between Neurospora crassa and P. fluorescens In5 (Fig. 5f ). The bacterium was isolated from Greenlandic soil which is naturally suppressive to plant infections by fungal pathogens [START_REF] Michelsen | Nonribosomal peptides, key biocontrol components for Pseudomonas fluorescens In5, isolated from a Greenlandic suppressive soil[END_REF]. Since some antagonistic bacteria are known to attach to fungal hyphae and parasitize the fungus without penetrating the hyphae [START_REF] Cuong | Hyphae-colonizing Burkholderia sp.: a new source of biological control agents against sheath blight disease (Rhizoctonia solani AG1-IA) in rice[END_REF] it was of interest to investigate if the In5 isolate attach to fungi. Neurospora crassa was selected for growth in the chamber for its fast growth and wide hyphae. As can be seen in Fig. 5f the bacteria may attach to, or highly associate with, the fungus. Further studies are warranted to determine if N. crassa forms microcolonies along the growing hypha.

With chambers and boundary channels available for high-resolution imaging, the ORNL microfluidic devices were used to visualize fine-scale interactions between a plant growth promoting bacterium Paraburkholderia caribensis and Mortierella elongata NVP64+, a fungus that naturally hosts endobacteria belonging to Burkholderiaceae. We were specifically interested in whether Paraburkholderia caribensis is able to enter the mycelium of M. elongata. A co-culture of M. elongata and a GFPtransformed strain of P. caribensis were initiated in an ORNL chamber, each inoculated in their own port. For this microbial pair we did not see a strong interaction between partners, under these conditions at 10 days in culture; however, a number of fluorescent cells characterized by single long sinuous shape were observed (Fig. 5g) when co-cultured with M. elongata, in addition to the typical bacilli and diplobacilli morphologies prevalent with this bacterium. This was repeated and confirmed by fluorescence microscopy. This morphology of P. caribensis has not been observed previously, and it is not known what triggers this morphological switch in the bacterium.

To begin probing mechanisms of endophytic growth in M. elongata, we introduced germinated Nicotiana attenuata seeds into one end of the microfluidic wells of ORNL vacuum-packed microfluidics. The other of the two ports was inoculated with M. elongata NVP64. Within 1 week of growth we observed fungal hypha and chlamydospores, or vesicle-like structures, of M. elongata within the plant roots (Fig. 5h). Fungal growth within the root hairs was not observed in this sample suggesting that M. elongata may enter roots through epidermal cells.

To better visualize and quantify the rate of cytoplasmic streaming in M. elongata NVP64+, we used ORNL microfluidic devices. Reverse cytoplasmic streaming was observed in M. elongata NVP64+ using these microfluidic chambers, with cellular vesicles and contents moving in opposite directions (Additional file 4: Figure S3). Consistent with prior reports for cytoplasmic streaming in fungi [START_REF] Roper | Nuclear dynamics in a fungal chimera[END_REF][START_REF] Schmieder | Bidirectional propagation of signals and nutrients in fungal networks via specialized hyphae[END_REF], clear cytoplasmic paths containing relatively fast velocity streaming and slow velocity streaming were observed, as evidenced by the black streaks running in straight diagonal and curved lines (Additional file 4: Figure S3).

Discussion

In this work we sought to overcome the common frustrations present with adopting microfluidic technology that limits the successful incorporation of microfluidics into traditional biology. The highlight of this work extends beyond a mere description of our approach to improving the accessibility to microfluidics, but also includes results from successful tests of microfluidics from collaborative works. First, we briefly discuss our approach to overcome these barriers including helpful tips for new users of microfluidics. In the remaining discussion we delineate the benefits and implications of ready-to-use microfluidics. We also highlight the examples and illuminate future directions of research possibilities that can be realized by microfluidics made easier.

Barriers to microfluidics in biology

Barriers to implementing microfluidics in science and research settings span a range of categories, from operational barriers (protocols and the degree of subjectmatter or device sophistication) to technical barriers (facilities and equipment) to individual barriers (experience and access to collaborators). We introduce a process that aids in removing technical and operational barriers through our design and packaging process. Nevertheless, individual experience and repetition is still required to maximize the use of microfluidic systems in biological science.

Fluidic priming is a common operational barrier to implementing microfluidics. The process of loading the microfluidic channel with fluid without introducing bubbles is a common hurdle in the implementation of microfluidics in biological labs. Filling the channels with fluid while eliminating bubbles may seem to be a trivial problem to one trained in the field, but it can be a tremendous barrier to entry for the microfluidic novice. Other technical barriers such as available equipment and facilities (for example, house vacuum) can put new collaborators, beginning to work with microfluidics, at a significant disadvantage or discourage collaboration. Syringe pumps are not typically available within biological labs; therefore, other means must be employed to prime the channels with fluid, or significant time and finances are a needed to invest in new pumping systems. A reliable alternative to priming microfluidics without syringe or pneumatic pumps is to equilibrate the PDMS channels in a vacuum chamber, or desiccator, then upon releasing the vacuum, immediately supply fluid to the channels (e.g. water, cell culture media, food color dye, oil, solvents) [START_REF] Nevill | Vacuum soft lithography to direct neuronal polarization[END_REF][START_REF] Millet | Modular microfluidics for point-of-care protein purifications[END_REF]. As the PDMS equilibrates with the local atmosphere, the material efficiently absorbs the air from the channels and is displaced by fluid from the fluid-filled ports. However, even for this simple approach, much time is spent solving this limitation on a lab-to-lab basis-regardless of distance between labs-due to the lack of vacuum supplies and equipment, and the variability in vacuum quality. House vacuum supplies, if available for degassing PDMS, are typically low vacuum (~ 70 kPa) and require long vacuum equilibration times to achieve enough drawing capacity to completely fill the channels with fluid. Vacuum pumps (> 80 kPa) may be available or can be purchased; however expensive equipment investments are typically unwarranted until the feasibility and promise of the microfluidic technologies have been established for collaborative investigators.

The design and architecture of a microfluidic system can potentially be a barrier to this enabling technology. While a detailed discussion on effective architectural elements for easily implementing microfluidic platforms is beyond the scope of this work, it should be noted that depending on the volume of the microfluidic architecture, house vacuum may be vastly insufficient in providing enough drawing capacity to completely prime microfluidic channels with fluid. As an alternative, inert gasses can be connected through specialized connections to push the fluid into the channels to displace bubbles through the material, here again, specialized supplies are needed.

To overcome common technical barriers that hinder new microfluidics experimentation, we sought to simplify the fluidic priming process by using a common vacuumbased approach that fills-on-demand. This inexpensive and commercially available packaging process allows for the retention of a vacuum potential within PDMS-based microfluidics for up to 2 weeks when stored under vacuum, thus preserving the ability to prime the microfluidics when returning the PDMS material to atmospheric conditions (Fig. 2). We also eliminated the need for the end-user to determine and incorporate sanitization processes for an unfamiliar technology; we pre-cleaned the microfluidics and included sanitization into the assembly, vacuum, and packaging process. We increased the accessibility to microfluidics by demonstrating a process that decreases the cost-of-investment and minimizes the steep learning curve. We disseminated microfluidics to potential collaborators for testing and use. Through our simple, inexpensive, effective, and easy-to-use process, academic labs can deliver a valuable microfluidic product for biologists to use for resolving the spatial and temporal dynamics of biological interactions (e.g. bacterial-fungal, bacterial-plant, and fungal-plant interactions).

Benefits and implications

Microfluidics are ideally suited to benefit studies of branched biological systems and mixed community interactions, for they enable physically and chemically controlled microenvironments. In nature, branched structures are capable of extending into multiple spatial domains for resource exploitation (plants and fungi), signal transduction and integration (neurons and fungi), mechanical support (plants and fungi), mass transport (plants, fungi, neurons), and cellular/organismal adaptation and regulation [START_REF] Tero | Rules for biologically inspired adaptive network design[END_REF][START_REF] Rayner | The challenge of the individualistic mycelium[END_REF].

The robust and inert construction of microfluidics enables pre-priming, filling, and shipping of hydrated microfluidics, with or without cultures. Preconditioning of microfluidics may be useful for initiating cultures prior to shipment, or in collecting and preserving field samples.

Incorporating agar plugs (Fig. 3b) into microfluidics substantially delays drying and provides an anchor for fungal cultures. Additionally, agar can be poured around the periphery of the microfluidics within the dish to aid in hydrating the PDMS and moistening the chamber for long-term growth and observation without fluid spillage and shearing cells.

With our approach, potential users need only open and fill the microfluidic platform to prepare samples for high-resolution imaging and can forego learning the device fabrication, assembly, sanitizing, and other preparation processes. For example, we tested our vacuum preconditioning process before deploying to collaborators, we packaged and germinated Arabidopsis seeds on chip. The channels filled with fluid, the majority of seeds germinated down the channel, and the cultures remained sterile, all without an overall impact to the root length. From these tests, we learned that the ability to vacuum seal and viably store seeds in a microfluidic platform has the potential to greatly enhance collaborations within and across disciplines. The low cost and portability of the Food Saver system also allows for field researchers to sow collected seeds or spores directly into a microfluidic platform and vacuum package for later use, thus preserving the seed with native microbes.

The process of preparing inoculated cultures in microfluidics saves time and eliminates initial barriers to implementing microfluidics. For example, Laccaria bicolor is a slow growing fungus and the culture can begin to be established before or during shipment. To confront this challenge, we prepared microfluidics on glass-bottomed dishes, then primed with culture media and agar, inoculated the central compartment with Laccaria fungi prior to sealing the dishes and shipping the samples to collaborators for bacterial inoculation and observation of bacterial-fungal interactions. After sustained development of Laccaria in the microfluidics during shipment and extended culture, transgenic bacteria were introduced and documented with microscopy (Fig. 3g). Biofilms formed on both bacterial-fungal co-cultures within 1 month and improve access to imaging bacterial-fungal interactions. While beneficial for some samples, this process is not suitable for all experimental conditions, cells, or organisms. Indeed, most biological samples cannot be maintained so easily during transport, (mammalian cells for example); therefore, a more ready-to-use approach is needed to expedite collaborative exchanges by overcoming initial barriers to implementing microfluidics in biology.

As an alternative to preparing cultures for transport, we propose a ready-to-use approach where microfluidics can be preconditioned dry, with vacuum, or hydrated and conditioned with agar to prevent drying and to provide an immediate-use product for the recipient to inoculate.

Deployment of vacuum-packed microfluidics

After characterizing the 'ready-to-use microfluidics, ' we prepared spoke-wheel microfluidics and characterized the filling time as a function of post-packaging storage time. From these samples, we determined a 2-week window for a 'best-use by' date. To deploy ready-to-usemicrofluidics with this preparation process, we fabricated an "ORNL" microfluidic architecture for testing and use by potential domestic and international collaborators. From our experience, the best process for loading chambers and successful culture is to initiate microfluidic priming within a 2-day shipping window. Longer shipments or travel may increase the time to fill, or rate of failure for this microfluidic preparation. One possible explanation for increased fill times or incomplete filling after 2 weeks in vacuum storage includes handling-induced loss of vacuum to the packaged system. Another possible explanation includes, age-and environmental-related (temperature) aging or stiffening of the elastomeric material. For example, twenty-eight sample chambers were deployed in response to interests communicated at a bacterial-fungal interactions workshop. Factors influencing the rate of successfully obtained cultures include, travel time, sample handling and storage during transit, and the learning curve for handling and filling microfluidics. Notwithstanding these possible limitations, some users reported that the devices were simple and easy to use, while others needed more replicates to successfully implement microfluidics in their experimental system. In this regard, we cannot overlook the procedural requirements of the biological system being integrated into the microfluidic chip. Culturing cells at room temperature without specialized gaseous environmental culture conditions (e.g. fungal cultures) may be easier and more forgiving, thus providing a higher apparent 'success rate' than trying to implement microfluidics with a biological protocol were time, temperature, and gas concentrations are all critical (e.g. anaerobic cultures and mammalian cell cultures). By extension, learning to use microfluidics in a biological safety cabinet is more challenging than working with them on a benchtop. Overall, we were pleased with the ability of this packaging processes to accelerate collaborative research and to quickly achieve productive results. It is our goal that this vacuum-packing process can be adopted and decrease the barriers to implementing microfluidics by providing a process to get microfluidics in-hand to end-users for testing and implementing in biological science.

Focus areas and future directions

Future research on fungal-bacterial interactions with bacteria could include fluorescent vitality stains for assessing fungi and bacteria, the use of fluorescence reporters for monitoring gene expression activity in microbes, or the use of chamber doors for sampling cells, nucleotides or metabolites from interacting tissues. Of special interest would be to investigate (1) if the architecture of the chamber can influence the outcome of a biological interaction related to previous studies, (2) how bacteria and fungi enter and grow within plant roots, (3) how environmental conditions influence cytoplasmic flow of cellular contents, or the colonization and community structure of the rhizosphere.

Microfluidic platforms have proven to be indispensable tools for probing processes of cellular function, organismal behavior, and environmental interactions. While biomedical disciplines have greatly benefited from the discoveries enabled by microfluidics [START_REF] Millet | New perspectives on neuronal development via microfluidic environments[END_REF], botany and mycology are ripe for microfluidic-enabled discoveries and solutions. Focus areas include, but are not limited to, hyphal chemotropism, fungal pathogenecity, tripartite interactions of the rhizosphere. More specifically, bacterial-fungal interaction studies are also in their infancy, as evidenced by much higher resolution studies [START_REF] Kohlmeier | Taking the fungal highway: mobilization of pollutant-degrading bacteria by fungi[END_REF][START_REF] Berthold | Mycelia as a focal point for horizontal gene transfer among soil bacteria[END_REF][START_REF] Worrich | Mycelium-mediated transfer of water and nutrients stimulates bacterial activity in dry and oligotrophic environments[END_REF]. While microfluidics offer the opportunity to engineer microcosms for probing the physical, chemical and biologicals aspects of multispecies interactions, these devices are convenient (but not required) for such imaging studies in general. Plant-fungal mutualism studies offer ample promise, and plant-bacteria interactions are valuable as well in medicine as for bioenergy, food crop production, and purifying natural resources [START_REF] Dhamgaye | The influence of bacterial interaction on the virulence of Cryptococcus neoformans[END_REF][START_REF] Peleg | Medically important bacterial-fungal interactions[END_REF][START_REF] Artursson | Interactions between arbuscular mycorrhizal fungi and bacteria and their potential for stimulating plant growth[END_REF][START_REF] Bonfante | Mechanisms underlying beneficial plant-fungus interactions in mycorrhizal symbiosis[END_REF].

Prior to deploying vacuum-equilibrated microfluidics for studying the biology of branching structures, we developed an architecture that enables low-density, high resolution access to branched hyphae for fungalbacterial interaction studies. Spoke-wheel microfluidics for ectomycorrhizal bacterial-fungal interactions were tested in-house before shipping to international colleagues for implementation (Fig. 3). The purpose of our microfluidic design was to restrict the hyphal growth region to a large fluidic area while maintaining a low microchannel ceiling (11 µm) that confines the biological observations to a stationary fluidic microenvironment for imaging. This work demonstrates the ability to accelerate studies of defined microcosms within microfluidic platforms by incorporating fabrication and sterilization processes into the packing process for shipping and sharing.

Conclusions

We have characterized and deployed an affordable, readily available, and inexpensive process that solves the priming challenge, for new collaborators, by storing sterile dry vacuum-equilibrated microfluidics in a vacuum-sealed pouch for easy fluid priming, anytime, anywhere. This preparation and packaging process allow the end user to achieve fluid-primed microfluidics without the need for pumping systems. The need for fluidic priming at the fabrication source is eliminated, the shipment of fluids is avoided, and the ease of priming microfluidics is achieved. This process requires the fabrication source to have access to equipment for sterilizing (UV, plasma, autoclave), generating relatively high vacuum (< 81 kPa), and maintaining sterility during the packaging process (biosafety cabinet). The end user specifies and supplies the fluids, thereby alleviating the supplier from the need to fluid-match recipes, requirements (pH), or conditions (prevent fluid aging or contamination).

We present a process that accelerates the adoption of microfluidics in labs lacking experience with the technique. The adaptation of challenging microtechnological methods across discipline boundaries can further accelerate the development and implementation of defined microcosms and engineered niches to better resolve fungal, bacterial, and plant interactions. By extension, microfluidics could also allow for the extraction or injection of biological contents for biochemical analyses and genome regulation studies, respectively. Breaking down scientific barriers through technological simplifications enables studies that have the power to move science forward toward resolving mechanisms of complex and pressing biological problems (e.g. microbial symbiosis or pathogen-host interactions) [START_REF] Deveau | Bacterial-fungal interactions: ecology, mechanisms and challenges[END_REF].

Fig. 4

 4 Fig. 4 Root-chip microfluidic channel for packaging, storing, culturing, and imaging Arabidopsis roots. a The chip design contains a single root culture channel and accessory ports for sampling and delivery of media and microbes. b A vacuum-packaged microfluidic root-chip shown with the same orientation. c Arabidopsis root growing in the central microfluidic channel with marks for measuring growth. d Growth data for vacuum-packed and control seeds. The packaging process does not attenuate growth conditions of stored roots (< 14 days at room temperature)

Fig. 5

 5 Fig. 5 Ready-to-use microfluidics for studying the biology of branched structures. a Schematic design of a four-chamber (O R N L) microfluidic architecture with two end-wells for cultures (6 mm circles). The interconnecting channel grid dimensions are shown, 15 µm wide channels spaced by 500 µm gap. Boundary channels framing the four chambers and grid are 500 µm wide. b A sterile vacuum-packaged microfluidic chip ready for shipment, and ready to use. c An ORNL microfluidic chip with green dye in channels. d Map showing distribution of 28 ready-to-use ORNL chambers to 18 potential collaborators for fungal and other branched biology research (United States: Michigan, New York, North Carolina, and California. Europe: France, Germany, Netherlands, Denmark, Hungary, Sweden, and Switzerland0. e-h Results from implementing ready-to-use microfluidics in collaborative labs. e Rapidly growing M. elongata AG77 navigates the peripheral channels to fill the ORNL chambers of the device. f Microfluidic co-culture of Neurospora crassa and Pseudomonas fluorescens in microfluidic cultures (bacteria 1 μm, hypha 3 μm). g Merged image of bright field and fluorescence image of Paraburkholderia caribensis and M. elongata. h Nicotiana attenuata root grown in the presence of endophytic fungi, M. elongata NVP64+ ; fungal hypha and chlamydospores are observed within the plant root

Acknowledgements

This research was performed at Oak Ridge National Laboratory (ORNL). ORNL is managed by UT-Battelle, LLC, for the U.S. Department of Energy Under Contract No. DE-AC05-00OR22725. The fabrication of the microfluidic platforms was carried out in the Nanofabrication Research Laboratory at the Center for Nanophase Materials Sciences, which is a DOE Office of Science User Facility. The views expressed are those of the authors and do not reflect the official policy or position of the Department of Energy or the U.S. Government. Collaborative contributions by Natalie Vande Pol, James Kremer, and David Rhodes are part of the Greg Bonito Group at Michigan State University.

under Contract No. DE-AC05-00OR22725 with the U.S. the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (http://energ y.gov/downl oads/doe-publi c-acces s-plan).

Funding

This work was supported in part by the Genomic Science Program, U.S. Department of Energy, Office of Science, Biological and Environmental Research, as part of the Plant Microbe Interfaces Scientific Focus Area (http:// pmi.ornl.gov). AD's lab was supported by the Laboratory of Excellence Advanced Research on the Biology of Tree and Forest Ecosystems (ARBRE; ANR-11-LABX 0002 01).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Additional files

Additional file 1. Supporting methods.

Additional file 2. Maturation of bacterial-fungal interactions in microfluidics.

Additional file 3. Dynamics of bacteria along the 'hyphal-highway' and cytoplasmic streaming within the mycelium.

Additional file 4. Bidirectional cytoplasmic streaming in fungal hyphae within microfluidic culture.

Authors' contributions

LJM and JA designed and characterized the microfluidic preparation and packaging process, performed cultures and data analyses, and prepared manuscript. JL and JU tested and implemented microfluidics with fungal cultures and contributed to manuscript preparation. NVP, JK, DR, implemented microfluidics with fungal cultures. MLE implemented microfluidics for neuronal cultures. CMG, AD, and SO implemented microfluidics for bacterial fungal interaction studies and contributed to manuscript preparation. GB implemented microfluidics through mentoring (NVP, JK, DR) and contributed to manuscript preparation. RV, MJD, and STR supervised this work. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Kanchana Chathoth

Bénédicte Martin

Pierre Cornelis

Stéven Yvenou

Martine Bonnaure-Mallet

Christine Baysse
email: christine.baysse@univ-rennes1.fr

The

Keywords: oral biofilm, iron, hydrogen sulfide, hydrogen peroxide, oxygen One-sentence summary

teaching and research institutions in France or abroad, or from public or private research centers.

Introduction

Unlike the first billion years on earth, oxygen is now abundant in most environments and it shapes the ecology and microbial behaviour. Amongst several effects, oxygen oxidizes soluble ferrous iron into insoluble ferric hydroxides. Oxygen also decomposes Fe-S clusters which are the essential cofactors of redox processes and metabolisms and generates reactive oxygen species (ROS). Most microorganisms rely on Fe-S clusters inherited from ancient proteins which have evolved in anoxic conditions rich in iron and sulfide [START_REF] Imlay | Iron-sulfur clusters and the problem with oxygen[END_REF].

Because of their Fe-S dependency, most microorganisms are sensitive to high levels of oxygen and iron deficiency, but also to excess in ferrous iron which generates hydroxyl radical, the most harmful ROS, in presence of the oxygen derivative H2O2. The so-called anaerobes have restricted their habitat to hypoxic niches or created local hypoxic niche by producing reductive volatile compounds such as hydrogen sulfide H2S. However, constant hypoxic conditions are rare and these communities are likely to be exposed to changing oxygen levels, which would modify their behaviour. The focus of this review is to describe the potential interplay between oxygen, iron and sulfide in the oral ecosystem, especially in the subgingival plaque, which consists of hundreds of bacterial species. This ecosystem exposed to ambient oxygen, but composed of both aerobic and anaerobic bacteria, some of them sulfidogenic, is particularly relevant for the topic.

The oral microbiota

Over 500 species have been identified in the oral ecosystem by metagenomic analyses but a large proportion remains uncharacterized [START_REF] Ai | Integrated metagenomic data analysis demonstrates that a loss of diversity in oral microbiota is associated with periodontitis[END_REF]. The oral bacterial composition varies from a commensal biofilm mainly composed of Gram-positive bacteria to a pathogenic biofilm dominated by Gram-negative anaerobic bacteria which can lead to periodontitis, a chronic infection and inflammation of teeth-supporting tissues. Periodontitis is predominantly caused by the pathogens of the red (Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia) and orange complex (Fusobacterium nucleatum, Prevotella intermedia, Peptostreptococcus micros, Eubacterium nodatum) as described by Socransky et al. [START_REF] Socransky | Microbial complexes in subgingival plaque[END_REF] P. gingivalis is a keystone pathogen because of its ability to subvert the immune response [START_REF] Maekawa | Porphyromonas gingivalis manipulates complement and TLR signaling to uncouple bacterial clearance from inflammation and promote dysbiosis[END_REF] and to activate the virulence of pathobionts [START_REF] Hajishengallis | The keystone-pathogen hypothesis[END_REF]. The optimal growth rate of P. gingivalis takes place in anaerobic condition, but it possesses an O2 consuming cytochrome bd oxidase that contributes to its aerotolerant phenotype [START_REF] Leclerc | The Cytochrome bd Oxidase of Porphyromonas gingivalis contributes to oxidative stress resistance and dioxygen tolerance[END_REF] as well as an O2-generating SOD (superoxide dismutase) instead of a non-O2-generating SOR (superoxide reductase) [START_REF] Lynch | Role of superoxide dismutase activity in the physiology of Porphyromonas gingivalis[END_REF].

Therefore, like the genotypically-related Bacteroides fragilis, P. gingivalis may be considered as a nanaerobe which means an organism that can grow with nanomolar concentrations of oxygen and benefit from it although it does not require oxygen for growth [START_REF] Morris | Shallow breathing: bacterial life at low O(2)[END_REF]. A species-specific co-aggregation of P. gingivalis with oral streptococci such as S. gordonii promotes initial colonization [START_REF] Lamont | Characterization of the adherence of Porphyromonas gingivalis to oral streptococci[END_REF]. P. gingivalis and T. denticola exibit a symbiotic relationship in nutrient utilization, growth promotion, and synergetic biofilm formation [START_REF] Ng | Bacterial interactions in pathogenic subgingival plaque[END_REF][START_REF] Meuric | Treponema denticola improves adhesive capacities of Porphyromonas gingivalis[END_REF]. It was also suggested that P. gingivalis may benefit from a mutual colonization with Candida albicans, a fungal species commonly encountered in the oral ecosystem. The growth of the filamentous form of the fungi under oxic conditions may offer a protective environment for the growth of P. gingivalis by depleting oxygen [START_REF] Bartnicka | Adhesive protein-mediated cross-talk between Candida albicans and Porphyromonas gingivalis in dual species biofilm protects the anaerobic bacterium in unfavorable oxic environment[END_REF]. As described for other nanaerobes such as B. fragilis the genomes of the pathogens P. gingivalis, Prevotella intermedia and Tannerella forsythia, all from the family of the bacteroidetes, possess a typical cytochrome bd oxidase with a long Q-loop, an hydrophilic region facing the outside of the cells and connecting transmembrane helices of subunit I [START_REF] Borisov | The cytochrome bd respiratory oxygen reductases[END_REF]. Some microaerophilic organisms are also part of the oral microbiota, such as Campylobacter concisus [START_REF] Tanner | Wolinella gen. nov., Wolinella succinogenes (Vibrio succinogenes Wolin et al.) comb. nov., and Description of Bacteroides gracilis sp. nov., Wolinella recta sp. nov., Campylobacter concisus sp. nov., and Eikenella corrodens from Humans with periodontal disease[END_REF].

Several studies showed C. concisus to be associated with bleeding in young adults with rapidly progressive periodontitis (Kamma, Nakou andManti 1994, 1995). The presence of O2-dependent or O2-tolerant microorganisms in the oral biofilm of periodontal patients suggests that the anaerobic-strict picture of the pathogenic subgingival biofilm is controversial and that changing pO2 (partial pressure of oxygen) may modulate the behaviour of the community.

A c c e p t e d M a n u s c r i p t [1] Oxygen in the oral ecosystem

Oxygen is abundant in the oral cavity. The primary source of oxygen is the free entry of atmospheric oxygen through the mouth. Another oxygen supplier is the gingival crevicular fluid (GCF) from the tissues surrounding the gingival crevice or periodontal pocket where the subgingival biofilm generally develops [START_REF] Loesche | Relationship between oxygen tension and subgingival bacterial flora in untreated human periodontal pockets[END_REF]. GCF is formed by the passage of fluid from capillaries into the tissues and then in the gingival sulcus. The pO2 in the periodontal pockets, which are the pathologically deepened gingival sulci, is low but not negligible. During the development of oral biofilms, the redox potential decreases: electrodes in place for up to 7 days in patients showed a fall in oxidation-reduction potential as plaque bacteria accumulated [START_REF] Kenney | Oxidation reduction potential of developing plaque, periodontal pockets and gingival sulci[END_REF]. For reference, under an atmospheric pressure of 760 mm Hg (sea level), the partial pressure of oxygen is 160 mm Hg. Deep periodontal pockets (7-10 mm) have a pO2 of 11.6 mmHg and the moderately deep periodontal pockets

(5-6 mm) have a pO2 of 15 mmHg. The pO2 of healthy sites is unavailable for comparison due to the limitation posed by length of the sensor used. The mean pO2 of untreated periodontal pockets (13.2 mmHg) marks the upper limit of hypoxia [START_REF] Mettraux | Oxygen tension (pO2) in untreated human periodontal pockets[END_REF].

Moreover, under inflammatory condition, oxygen levels rise due to elevated blood flow and haemoglobin. The pocket pO2 showed correlation with probing depth and with oxygen saturation of haemoglobin [START_REF] Tanaka | Association of oxygen tension in human periodontal pockets with gingival inflammation[END_REF].

[2] Production of H2O2

If not the starting event, oxidative stress contributes to the snowball effect due to the interdependence between oxidative stress and inflammation. Streptococci from the mitis and sanguinis group dominate the initial colonization of the tooth surface [START_REF] Rosan | Dental plaque formation[END_REF][START_REF] Welch | Biogeography of a human oral microbiome at the micron scale[END_REF][START_REF] Wake | Temporal dynamics of bacterial microbiota in the human oral cavity determined using an in situ model of dental biofilms[END_REF]. They are facultative anaerobes and the main producers of H2O2 in response to oxygen. This metabolite was first considered beneficial for oral health by maintaining a symbiotic microbiota and preventing pathogens [START_REF] Kreth | Streptococcal antagonism in oral biofilms: Streptococcus sanguinis and Streptococcus gordonii interference with Streptococcus mutans[END_REF][START_REF] Redanz | Live and let die: Hydrogen peroxide production by the commensal flora and its role in maintaining a symbiotic microbiome[END_REF].

The sources of microbial H2O2 in the oral biofilm have been reviewed by Redanz et al. [START_REF] Redanz | Live and let die: Hydrogen peroxide production by the commensal flora and its role in maintaining a symbiotic microbiome[END_REF]. Briefly, the main enzyme involved in hydrogen peroxide production is the streptococcal protein SpxB, a pyruvate oxidase that converts pyruvate to acetyl phosphate and H2O2 and consumes oxygen (Fig. 1). Minor synthesis of H2O2 may arise from detoxifying A c c e p t e d M a n u s c r i p t enzymes from various species such as SOD (superoxide dismutase) and SOR (superoxide reductase) that reduce superoxide anions into less reactive H2O2. Another streptococcal enzyme capable of generating H2O2 is the FMN-dependent lactate oxidase (LctO) which performs the aerobic oxidation of lactate into pyruvate (Fig. 1). Finally, under conditions where peptone and amino acid are the only available nutrients, such as in saliva, H2O2 together with ammonia and α-ketoacid may be produced by the oxidation of amino acid by the L-amino acid oxidase (LAAO) (Fig. 1) [START_REF] Tong | SO-LAAO, a novel L-amino acid oxidase that enables Streptococcus oligofermentans to outcompete Streptococcus mutans by generating H2O2 from peptone[END_REF]. The generation of H2O2 is also a constant process in host eukaryotic cells, which increases during inflammation in response to S. sanguinis and S. gordonii are often called "oral health compatible organisms", due to their H2O2-related antimicrobial properties against anaerobic pathogens. However, the effect of H2O2 on the microbiota cannot be limited to the inhibition of pathogen viability or a global beneficial input on oral health. H2O2 is a non-radical product with low reactivity with organic molecules. Its longer life span (1ms) compared to free radicals such as O2 °-(1 µs) and OH -(1 ns) makes it a good signalling molecule [START_REF] Dahiya | Reactive oxygen species in periodontitis[END_REF]).

[3] The Fenton chemistry and the sources of Iron

The toxicity of H2O2 mainly depends on transition metal concentration in the surrounding, especially ferrous iron or copper, because of the Fenton reaction [H2O2 + Fe 2+ → •OH + Fe 3+ + - OH] using both substrates to generate the highly reactive hydroxyl radical (OH-). H2O2, via the production of OH-, may be toxic not only for other bacteria including potential pathogens but also for host cells. The effect of H2O2 on eukaryotic oral host cells is either beneficial or deleterious depending on its concentration. At low levels (28.9 µM maximum concentration for 4 h and reduced up to 13.5 µM for 24h), it can stimulate the proliferation of human periodontal ligament fibroblasts in vitro, but it also displays cytotoxic effect at higher concentrations (1mM), including on endothelial cells [START_REF] Choe | generated H2O2 stimulates the proliferation and osteoblastic differentiation of human periodontal ligament fibroblasts[END_REF]. Some results suggest that streptococcal H2O2 is not only cytotoxic to epithelial cells, but also promotes bacterial evasion of the host defence systems in the oral cavity [START_REF] Okahashi | Hydrogen Peroxide contributes to the epithelial cell death induced by the oral mitis group of Streptococci[END_REF][START_REF] Liu | The role of reactive oxygen species and autophagy in periodontitis and their potential linkage[END_REF][START_REF] Erttmann | Hydrogen peroxide release by bacteria suppresses inflammasomedependent innate immunity[END_REF]. Moreover, H2O2 produced by S. gordonii can serve as an electron acceptor to increase the persistence of Aggregatibacter actinomycetemcomitans by a A c c e p t e d M a n u s c r i p t shift from an anaerobic to an aerobic mode of growth, an interaction termed "crossrespiration". A. actinomycetemcomitans is associated with aggressive periodontitis, presumably due to the production of the leukotoxin A [START_REF] Stacy | Bacterial fight-and-flight responses enhance virulence in a polymicrobial infection[END_REF][START_REF] Stacy | A commensal bacterium promotes virulence of an opportunistic pathogen via cross-respiration[END_REF].Therefore, the balance between good and bad for oral health may be, amongst several factors, a matter of concentration (entry, release and turnover) of free ferrous iron and H2O2 inside target cells.

Oxygen-derived free radical resulting from Fenton reaction serves to amplify the inflammatory response, by favouring the expression of cytokines in endothelial cells, promoting the recruitment of neutrophils and inflammatory cells. As a result of tissue damage, mitochondria, xanthine oxidase and neutrophils produce more reactive oxygen species [START_REF] Closa | Oxygen free radicals and the systemic inflammatory response[END_REF][START_REF] Mittal | Reactive oxygen species in inflammation and tissue injury[END_REF]).

In the oral environment, iron may come from the gingival crevicular fluid (GCF), saliva and dietary sources. GCF is an inflammatory exudate derived from the periodontal tissues and is composed of serum, tissue breakdown products, inflammatory mediators, and sometimes antibodies. During the development of gingivitis, the flow rate of GCF increases [START_REF] Goodson | Gingival crevice fluid flow[END_REF]). In the host, iron is bound in complexes such as lactoferrin, haemoglobin and transferrin. Lactoferrin is a glycoprotein with iron-binding properties, secreted by exocrine glands and neutrophils. It binds to two atoms of iron and retains iron at a much lower pH (up to pH 3.0) than the main systemic transporter transferrin (up to pH 5.5) [START_REF] Baker | Molecular structure, binding properties and dynamics of lactoferrin[END_REF]. Lactoferrin is an important defence factor against Streptococcus mutans and periodontopathic bacteria. It has the ability not only to inhibit bacterial growth and biofilm development, but also to limit free-iron overload, and therefore reduce reactive oxygen formation and inflammatory processes [START_REF] Berlutti | Lactoferrin and oral diseases: current status and perspective in periodontitis[END_REF]. A correlation between lactoferrin concentration in the GCF and the number of PMNs in GCF was reported [START_REF] Adonogianaki | Lactoferrin in the gingival crevice as a marker of polymorphonuclear leucocytes in periodontal diseases[END_REF]. Although lactoferrin can be used as an iron source for some pathogens such as Neisseria gonorrhoeae [START_REF] Blanton | Genetic evidence that Neisseria gonorrhoeae produces specific receptors for transferrin and lactoferrin[END_REF] or Campylobacter jejuni [START_REF] Miller | Utilization of lactoferrin-bound and transferrin-bound iron by Campylobacter jejuni[END_REF], its role as an iron source for periodontal bacteria is not yet proven.

Hemic iron is mainly provided by haemoglobin (Hb). Haemoglobin derived from microbleeding in gingival sulci was detected in GCF samples from periodontal sites [START_REF] Ito | Correlation between gingival crevicular fluid hemoglobin content and periodontal clinical parameters[END_REF]. Iron acquisition from Hb by oral/periodontal bacteria may be facilitated by the H2O2 produced by the streptococci: the initial oxidation of oxyHb into metHb, a form of haemoglobin that contains the ferric [Fe 3+ ] form of iron is carried out by H2O2. This further promoted the activity of the gingipains, proteases of P. gingivalis, which cleave such iron A c c e p t e d M a n u s c r i p t proteins as the first step of heme uptake. Also, the dissociation of the ferric iron from heme protoporphyrin is easier than in the case of ferrous iron [START_REF] Brown | Potential role for Streptococcus gordonii-derived hydrogen peroxide in heme acquisition by Porphyromonas gingivalis[END_REF].

Transferrin is a glycoprotein with 2 iron-binding sites (composed of 39 cysteines), found in the periodontal pocket together with haemoglobin and lactoferrin. Transferrin and its ironsaturated form, holotransferrin support the growth of several oral bacteria such as Campylobacter rectus [START_REF] Grenier | Transferrin as a source of iron for Campylobacter rectus[END_REF] and P. gingivalis [START_REF] Brochu | Acquisition of iron from human transferrin by Porphyromonas gingivalis: a role for Arg-and Lys-gingipain activities[END_REF][START_REF] Goulet | Cleavage of human transferrin by Porphyromonas gingivalis gingipains promotes growth and formation of hydroxyl radicals[END_REF]. Increased transferrin saturation is generally due to genetic haemochromatosis that provokes iron egress from macrophages and enterocytes in relation with hepcidin deficiency. Increased transferrin saturation is not only associated with increased mortality in patients with sepsis [START_REF] Lan | High serum iron level is associated with increased mortality in patients with sepsis[END_REF]) but also with dysbiosis and severe periodontitis [START_REF] Boyer | Increased transferrin saturation is associated with subgingival microbiota dysbiosis and severe periodontitis in genetic haemochromatosis[END_REF]. Moreover, as previously stated, iron saturation and transferrin concentration increase in the GCF during periodontitis [START_REF] Adonogianaki | Lactoferrin in the gingival crevice as a marker of polymorphonuclear leucocytes in periodontal diseases[END_REF].

Along with the external sources of iron in the GCF, oxygen radicals, especially superoxide anions, destroy the ubiquitous 4Fe-4S clusters of the host and proteins of the microbiota, releasing free ferrous iron in the surrounding. The fate of the dissolved ferrous iron depends on the properties of the environment such as oxidative status and pH. As the pH increases, the speed of the oxidation of ferrous iron increases and produces ferric oxides and hydroxides.

Altogether free iron overload enhanced by oxygen and combined with oxygen derivatives may be one of the triggers that favour the dysbiosis and inflammatory processes, provided some bacteria (pathogens and H2O2 producers) can protect themselves against O2/H2O2 toxicity in the presence of ferrous iron.

[4] The role of reductive compounds, H2S and glutathione Peroxygenic streptococci limit their reliance to Fe-S cluster protein and use manganese in place of Fe to avoid H2O2 self-toxicity [START_REF] Jakubovics | Oxidative stress tolerance is manganese (Mn(2+)) regulated in Streptococcus gordonii[END_REF][START_REF] Tseng | Virulence of Streptococcus pneumoniae: psaA mutants are hypersensitive to oxidative stress[END_REF]. However, the sensitivity of anaerobes to oxidative stress mainly relies on the sensitivity of the Fe-S cluster of their pyruvate-ferredoxin oxidoreductase (PFOR). PFOR is a major metabolic enzyme involved in energy generation through oxidative decarboxylation of pyruvate, as demonstrated with the model Bacteroides thetaiotaomicron [START_REF] Pan | How does oxygen inhibit central metabolism in the obligate anaerobe Bacteroides thetaiotaomicron[END_REF][START_REF] Lu | Endogenous superoxide is a key effector of the oxygen sensitivity of a model obligate anaerobe[END_REF]. The periodontal pathogens P. gingivalis, P. intermedia and T. forsythia use PFOR which allowed the development of a new antimicrobial compound, amixicile, specifically targeting the anaerobic pathogen via the inhibition of PFOR A c c e p t e d M a n u s c r i p t [START_REF] Hutcherson | Amixicile, a novel strategy for targeting oral anaerobic pathogens[END_REF]. Anaerobes need reduced environment rather than low oxygen concentration for growth. PFOR help bacteria to thrive in a reduced environment because of low-potential electron transfer pathway, in which PFOR passes low-potential electrons toward hydrogen formation and/or NAD reduction. The redox potential measured in saliva was +158 to 542 mV while it was lower in periodontal pockets (-300 mV) and gingival crevices (-200 mV). Indeed, the redox potential is reduced by microbial metabolism during the development of oral biofilm [START_REF] Kenney | Oxidation reduction potential of developing plaque, periodontal pockets and gingival sulci[END_REF]. Hydrogen sulfide is a metabolite which is produced in high amount by microorganisms in the periodontal environment [START_REF] Horowitz | Hydrogen sulfide production in the periodontal environment[END_REF]. H2S production add to oral malodour: halitosis is caused by various volatile sulfur compounds (VSCs), including H2S but also ethanethiol, S-ethyl thioacetate, diethyl disulfide, dimethyl sulfide and methyl mercaptan. The amount of VSCs has been shown to significantly increase in patients with periodontal disease. H2S produced in subgingival pocket probably contributes to halitosis even though the predominance of methyl mercaptan produced by the microbiota of the tongue has been shown [START_REF] Yaegaki | Biochemical and clinical factors influencing oral malodor in periodontal patients[END_REF]. Such metabolites with low redox potential (E0 +0.17 V), potentially contribute to the reductive and protective environment for anaerobes and nanaerobes in subgingival pockets. The effect of H2S greatly varies with environmental conditions. H2S molecules dissolve in water and dissociate into H + , HS -and S 2-ions. HS -and to a lower extent, H2S are chemical reductants capable of scavenging ROS and inducing defence response in eukaryotic cells [START_REF] Xie | Sulfhydration of p66Shc at cysteine59 mediates the antioxidant effect of hydrogen sulfide[END_REF]). H2S can be as toxic as cyanide due to its ability to bind with and precipitate metal cations. Likewise, for some bacterial species, exogenous H2S can suppress H2O2-mediated oxidative stress. It was shown that H2S suppressed the oxidative stress in E. coli by sequestering free iron that reacted with H2O2 in Fenton chemistry [START_REF] Mironov | Mechanism of H2S-mediated protection against oxidative stress in Escherichia coli[END_REF]. Another study on Streptococci showed that H2S reduced intracellular bound ferric iron to form unbound ferrous iron that can act as a substrate for Fenton chemistry [START_REF] Dunning | Anaerobic killing of oral streptococci by reduced, transition metal cations[END_REF]. Therefore, H2S may be beneficial to oral anaerobes [START_REF] Lai | Novel mechanism for conditional aerobic growth of the anaerobic bacterium Treponema denticola[END_REF] but exerts a toxic effect on other microorganisms, by inhibition of dismutase, catalase, and sequestration of trace metals in the surrounding or increase of free cytosolic ferrous iron. The toxic effect of H2S when studied on streptococci, was iron-dependent. The addition of the iron chelator deferoxamine obliterated the bactericidal effect of H2S [START_REF] Ooi | Reduced glutathione mediates resistance to H2S toxicity in oral streptococci. Nojiri H (ed.)[END_REF]. In Shewanella oneidensis, the coordinated effect of H2S and H2O2 was described as being time-dependent. On simultaneous addition of H2O2 and H2S to cultures, H2S promoted H2O2 toxicity via catalase inactivation. However, on later supplementation of H2O2, H2S promoted H2O2 resistance, probably by lowering external iron concentrations, activating the influx of iron and OxyR-dependent stress defences (Wu et denticola resulted in a dose-dependent removal of dissolved O2 in the growth cultures [START_REF] Lai | Novel mechanism for conditional aerobic growth of the anaerobic bacterium Treponema denticola[END_REF].

H2S also increases intracellular glutathione (GSH) synthesis in streptococci. GSH protects streptococci against both their H2O2 [START_REF] Zheng | Involvement of gshAB in the interspecies competition within oral biofilm[END_REF]) and H2S produced by neighbouring bacteria [START_REF] Ooi | Reduced glutathione mediates resistance to H2S toxicity in oral streptococci. Nojiri H (ed.)[END_REF]. GSH (γ -L-glutamyl-L-cysteinyl-glycine) is an abundant nonprotein thiol present in many bacteria and all eukaryotes [START_REF] Masip | The many faces of glutathione in Bacteria[END_REF]. Glutathione biosynthesis generally occurs via a conserved two-step mechanism in which a γ-glutamylcysteine ligase (GshA) uses l-glutamate and l-cysteine to form γglutamylcysteine and then, a glutathione synthetase (GshB) condenses γ-glutamylcysteine with glycine to give glutathione (Fig. 2). H2S reduces cystine into cysteine in extracellular space and increases the expression of glutamate-cysteine ligase that converts cysteine into γglutamyl cysteine, the direct precursor of GSH. The susceptibility of oral streptococci to H2S is dependent on intracellular glutathione levels, which increase in the presence of the H2S donor NaHS in vitro [START_REF] Ooi | Reduced glutathione mediates resistance to H2S toxicity in oral streptococci. Nojiri H (ed.)[END_REF].

GSH synthesis by eukaryotic and prokaryotic cells may promote H2S production by sulfidogenic bacteria if released in the surrounding. H2S is produced by several anaerobic bacteria of the oral microbiota mainly from cysteine, which originated from the catabolism of cysteine-containing proteins/peptides and glutathione, via cysteine desulfhydration. The possible pathway for production of H2S from the degradation of GSH is depicted in Figure .2.

As the initial step, the γ-glutamyltransferase GGT breaks down glutathione into cysteinyl glycine (Cys-Gly) and glutamate. Next, the cysteinyl glycinase CGase degrades Cys-Gly into glycine and l-cysteine. Finally, the βC-S lyases, pyridoxal-5′-phosphate (PLP)-dependent enzyme (L-cysteine desulfhydrase LCD for cysteine) catalyse the α, β-elimination of sulfur amino acids (l-cysteine, l-cystathionine, l-cystine, S-(2-aminoethyl)-l-cysteine, and S-methyll-cysteine) to the corresponding sulfur-containing molecules (H2S in case of cysteine), pyruvate and ammonia. The NH3 produced by this reaction may contribute to the alkaline pH encountered in periodontal pockets. This pathway is certainly relevant in the oral biofilm in vivo since glutathione was identified as a suitable substrate for H2S production by the major pathogen T. denticola [START_REF] Chu | Role of Glutathione Metabolism of Treponema denticola in bacterial growth and virulence expression[END_REF][START_REF] Chu | Role for recombinant -Glutamyltransferase from Treponema denticola in glutathione metabolism[END_REF][START_REF] Chu | A 52-kDa Leucyl Aminopeptidase from Treponema denticola Is a Cysteinylglycinase that mediates the second step of glutathione metabolism[END_REF]. Recently a T. denticola mutant lacking the gamma-glutamyltransferase enzyme GGT was constructed [START_REF] Chu | Glutathione catabolism by Treponema denticola impacts its pathogenic potential[END_REF]. The mutant cannot produce H2S from glutathione. Interestingly, the mutant has also lost the ability to A c c e p t e d M a n u s c r i p t grow in the presence of oxygen when incubated with glutathione and showed a reduced virulence in vitro against human gingival fibroblasts. The data also pointed out that the levels of cell death correlated with the amounts of H2S produced. P. gingivalis does not possess GGT, but it is capable of producing H2S from cysteinyl glycine, therefore, possessing CGase activity [START_REF] Tang-Larsen | Competition for peptides and amino acids among periodontal bacteria[END_REF]. Neither of the two pathogens possesses the gshA and gshB genes for GSH synthesis. On the contrary, in silico genome analysis (unpublished analysis) indicates that the primary coloniser S. gordonii possesses a GshF-homologue gene. The product of this gene is a bifunctional enzyme that may carry out glutathione biosynthesis by catalysing both reactions on its own. Several studies pointed out that other pathogenic and free-living bacteria possess a single enzyme to catalyse the γ-glutamylcysteine ligase reaction and the condensation of γ-glutamylcysteine with glycine to give glutathione [START_REF] Gopal | A multidomain fusion protein in Listeria monocytogenes catalyzes the two primary activities for glutathione biosynthesis[END_REF][START_REF] Janowiak | Glutathione synthesis in Streptococcus agalactiae. One protein accounts for gamma-glutamylcysteine synthetase and glutathione synthetase activities[END_REF][START_REF] Vergauwen | Characterization of the bifunctional gammaglutamate-cysteine ligase/glutathione synthetase (GshF) of Pasteurella multocida[END_REF][START_REF] Stout | Glutathione biosynthesis in bacteria by bifunctional GshF is driven by a modular structure featuring a novel hybrid ATP-grasp fold[END_REF]. Indeed, the SGO-1990 gene product of S. gordonii showed 100% identity with the bifunctional GshF enzyme of Streptococcus sanguinis [START_REF] Cui | Efficient glutathione production in metabolically engineered Escherichia coli strains using constitutive promoters[END_REF]. What remains to be investigated is whether reactive oxygen species enhance GSH synthesis in streptococci. Such activation was described in different bacteria and is as per the role of GSH in oxidative stress responses. For example, in Pseudomonas aeruginosa, hydrogen peroxide, cumene hydroperoxide, and t-butyl hydroperoxide increase the expression of gshA and gshB involved in glutathione synthesis [START_REF] Wongsaroj | Pseudomonas aeruginosa glutathione biosynthesis genes play multiple roles in stress protection, bacterial virulence and biofilm formation[END_REF]. If ROS promotes GSH synthesis in the oral microbiota, oxidative stress conditions from the presence of H2O2 and ferrous iron could indirectly trigger H2S synthesis by pathogenic oral bacteria via GSH cross-feeding and initiate a vicious circle leading to an H2S-mediated reductive environment where anaerobic bacteria would flourish. The use of GSH by H2S-producing bacteria may also explain why in GCF, the amount of GSH is lower in cases of chronic periodontal disease compared to healthier sites [START_REF] Chapple | Glutathione in gingival crevicular fluid and its relation to local antioxidant capacity in periodontal health and disease[END_REF][START_REF] Grant | Crevicular fluid glutathione levels in periodontitis and the effect of non-surgical therapy[END_REF][START_REF] Savita | Evaluation of glutathione level in gingival crevicular fluid in periodontal health, in chronic periodontitis and after nonsurgical periodontal therapy: A clinicobiochemical study[END_REF].

The cysteine-rich transferrin may also play a role in the oral dysbiosis not only by providing iron but also by favouring the synthesis of volatile sulfur compounds. The transferrin concentration increases in the gingival crevicular fluid during periodontitis [START_REF] Adonogianaki | Lactoferrin in the gingival crevice as a marker of polymorphonuclear leucocytes in periodontal diseases[END_REF]. As previously said, some bacteria can use it as both iron and nutrient source in the host [START_REF] Goulet | Cleavage of human transferrin by Porphyromonas gingivalis gingipains promotes growth and formation of hydroxyl radicals[END_REF]. In vitro, iron-saturated transferrin supported the growth of P. gingivalis via degradation of the protein [START_REF] Brochu | Acquisition of iron from human transferrin by Porphyromonas gingivalis: a role for Arg-and Lys-gingipain activities[END_REF]. [START_REF] Boyer | Increased transferrin saturation is associated with subgingival microbiota dysbiosis and severe periodontitis in genetic haemochromatosis[END_REF] observed a significant link between the periodontitis severity and increased transferrin saturation in patients with iron-overload disease (haemochromatosis). However, the levels of A c c e p t e d M a n u s c r i p t transferrin in the GCF of haemochromatosis patients compared to healthy patients are not known. The use of transferrin as a suitable substrate for H2S synthesis by oral bacteria remains to be investigated as well. Finally, following an initial observation that mutants of S. sanguinis and S. gordonii depleted of the pleiotropic carbon catabolite regulator CcpA lost their ability to inhibit H2O2-sensitive strains of S. mutans, the authors identified pyruvate as the compound, overproduced by these mutants and responsible for the protective effect. This study highlighted a new mechanism for regulating H2O2 homeostasis within microbiota, which would depend on pyruvate secretion and H2O2 scavenging by some members of the community and which will enrich studies on interactions and dysbioses related to oxidative stress [START_REF] Redanz | Live and let die: Hydrogen peroxide production by the commensal flora and its role in maintaining a symbiotic microbiome[END_REF].

Conclusion

This review combines separate data, mainly obtained in vitro, to propose hypotheses that will need to be tested in vivo or in a relevant context such as multispecies biofilms. Oxygen always being present in oral sites, even at low levels, allows H2O2 production by peroxygenic bacteria, mainly streptococci. Combined with H2O2, the levels of free ferrous iron may be one of the signals that will promote dysbiosis. Free iron along with the H2O2 in the oral sites can give rise to the Fenton reaction and thus produce ROS, which in turn generates more free iron via Fe-S degradation. The generation of toxic hydroxyl radicals may induce the production of GSH by some bacteria, and then indirectly of H2S since GSH can be used as a substrate. H2S protects anaerobes and may trigger more GSH production by H2O2 producing organisms. H2S can also reduce ferric iron to promote the Fenton reaction or chelate ferrous iron and decrease the Fenton chemistry in vitro. The major points raised by this speculation is whether oxidative stress in a first step and then H2S can significantly increase the production of glutathione by oral bacteria so that it can serve as a substrate for the production of H2S. The whole picture is depicted in Figure 3. Ultimately, a treatment containing both an oxygen donor and a nonmetabolizable iron chelator could be of interest to treat and prevent periodontal diseases as it may favour the development of commensal aerobic organisms without inducing deleterious oxidative stress. Besides, inhibitors of enzymes involved in the production of volatile sulfur compounds may be worth investigating especially on periodontal diseases in relation with increased levels of cysteine-containing proteins such as transferrin (hemochromatosis-related periodontitis). An in vitro study has identified propargylglycine as a potent inhibitor of volatile sulfur compound synthesis in P. gingivalis. The inhibitor hampers the activity of methionine gamma-lyase deaminase [START_REF] Kandalam | Inhibition of methionine gamma lyase deaminase and the growth of Porphyromonas gingivalis: A therapeutic target for halitosis/periodontitis[END_REF]. Further detailed and focused

  . The green complex (Clostridium gingivalis, C. sputigena, C. ochracea, Campylobacter concisus, Eikenella corrodens, and Actinobacillus actinomycetemcomitans serotype a) is also associated with periodontal disease. The purple (Veillonella parvula, Actinomyces odontolyticus) and the yellow (Streptococcus gordonii,

  Streptococcus intermedius, Streptococcus mitis, Streptococcus oralis, Streptococcus sanguis)complexes mainly consist of facultative anaerobic and Gram-positive bacteria associated with oral health. They are also involved in cooperative metabolisms in the periodontal biofilm. In the subgingival plaque, facultative anaerobic or microaerophilic microorganisms predominate.

  pathogens. When stimulated by pathogens, host cells release cytokines, recruit polymorphonuclear leukocytes which synthesize proteolytic enzymes and ROS. The primary sources of ROS are the NADPH oxidase (Nox), the mitochondrial respiratory chain and SOD (Sculley and Langley-Evans 2002; Sies 2017).

  s c r i p t al. 2015). For anaerobic bacteria, H2S production helps to maintain a reduced environment and to decrease dissolved O2. A study of Lai and Chu has revealed that H2S production by T.

  Figure 1. Possible H2O2-producing enzymes in oral microbiota.

Figure 2 .

 2 Figure 2. Common pathway of cysteine degradation to H2S and GSH synthesis.

Figure 3 .

 3 Figure 3. Hypothetical cascade of events leading to dysbiosis in periodontitis: the possible role of oxygen, iron and H2S in the multifactorial process.

  s c r i p t present in iron-sulfur cluster containing proteins, to generate insoluble precipitates (FeS).Altogether, this vicious circle starting from O2 and ferrous iron may promote anaerobic pathogens and inflammation.