Development and utilization of new O2-independent bioreporters.

Fluorescent proteins have revolutionized science since their discovery in 1962. They have enabled imaging experiments to decipher the function of proteins, cells, and organisms, as well as gene regulation. Green fluorescent protein and all its derivatives are now standard tools in cell biology, immunology, molecular biology, and microbiology laboratories around the world. A common feature of these proteins is their dioxygen (O2)-dependent maturation allowing fluorescence, which precludes their use in anoxic contexts. In this work, we report the development and in cellulo characterization of genetic circuits encoding the O2-independent KOFP-7 protein, a flavin-binding fluorescent protein. We have optimized the genetic circuit for high bacterial fluorescence at population and single-cell level, implemented this circuit in various plasmids differing in host range, and quantified their fluorescence under both aerobic and anaerobic conditions. Finally, we showed that KOFP-7-based constructions can be used to produce fluorescing cells of Vibrio diazotrophicus, a facultative anaerobe, demonstrating the usefulness of the genetic circuits for various anaerobic bacteria. These genetic circuits can thus be modified at will, both to solve basic and applied research questions, opening a highway to shed light on the obscure anaerobic world.IMPORTANCEFluorescent proteins are used for decades, and have allowed major discoveries in biology in a wide variety of fields, and are used in environmental as well as clinical contexts. Green fluorescent protein (GFP) and all its derivatives share a common feature: they rely on the presence of dioxygen (O2) for protein maturation and fluorescence. This dependency precludes their use in anoxic environments. Here, we constructed a series of genetic circuits allowing production of KOFP-7, an O2-independant flavin-binding fluorescent protein. We demonstrated that Escherichia coli cells producing KOFP-7 are fluorescent, both at the population and single-cell levels. Importantly, we showed that, unlike cells producing GFP, cells producing KOFP-7 are fluorescent in anoxia. Finally, we demonstrated that Vibrio diazotrophicus NS1, a facultative anaerobe, is fluorescent in the absence of O2 when KOFP-7 is produced. Altogether, the development of new genetic circuits allowing O2-independent fluorescence will open new perspective to study anaerobic processes.

Genetic Insights into Biofilm Formation by a Pathogenic Strain of Vibrio harveyi.

The Vibrio genus includes bacteria widely distributed in aquatic habitats and the infections caused by these bacteria can affect a wide range of hosts. They are able to adhere to numerous surfaces, which can result in biofilm formation that helps maintain them in the environment. The involvement of the biofilm lifestyle in the virulence of Vibrio pathogens of aquatic organisms remains to be investigated. Vibrio harveyi ORM4 is a pathogen responsible for an outbreak in European abalone Haliotis tuberculata populations. In the present study, we used a dynamic biofilm culture technique coupled with laser scanning microscopy to characterize the biofilm formed by V. harveyi ORM4. We furthermore used RNA-seq analysis to examine the global changes in gene expression in biofilm cells compared to planktonic bacteria, and to identify biofilm- and virulence-related genes showing altered expression. A total of 1565 genes were differentially expressed, including genes associated with motility, polysaccharide synthesis, and quorum sensing. The up-regulation of 18 genes associated with the synthesis of the type III secretion system suggests that this virulence factor is induced in V. harveyi ORM4 biofilms, providing indirect evidence of a relationship between biofilm and virulence.

Cooperation and cheating orchestrate Vibrio assemblages and polymicrobial synergy in oysters infected with OsHV-1 virus.

Polymicrobial infections threaten the health of humans and animals but remain understudied in natural systems. We recently described the Pacific Oyster Mortality Syndrome (POMS), a polymicrobial disease affecting oyster production worldwide. In the French Atlantic coast, the disease involves coinfection with ostreid herpesvirus 1 (OsHV-1) and virulent Vibrio. However, it is unknown whether consistent Vibrio populations are associated with POMS in different regions, how Vibrio contribute to POMS, and how they interact with OsHV-1 during pathogenesis. By connecting field-based approaches in a Mediterranean ecosystem, laboratory infection assays and functional genomics, we uncovered a web of interdependencies that shape the structure and function of the POMS pathobiota. We show that Vibrio harveyi and Vibrio rotiferianus are predominant in OsHV-1-diseased oysters and that OsHV-1 drives the partition of the Vibrio community observed in the field. However only V. harveyi synergizes with OsHV-1 by promoting mutual growth and accelerating oyster death. V. harveyi shows high-virulence potential and dampens oyster cellular defenses through a type 3 secretion system, making oysters a more favorable niche for microbe colonization. In addition, V. harveyi produces a key siderophore called vibrioferrin. This important resource promotes the growth of V. rotiferianus, which cooccurs with V. harveyi in diseased oysters, and behaves as a cheater by benefiting from V. harveyi metabolite sharing. Our data show that cooperative behaviors contribute to synergy between bacterial and viral coinfecting partners. Additional cheating behaviors further shape the polymicrobial consortium. Controlling cooperative behaviors or countering their effects opens avenues for mitigating polymicrobial diseases.

Genetic and physiological insights into the diazotrophic activity of a non-cyanobacterial marine diazotroph.

Nitrogen (N2 ) fixation, or diazotrophy, supports a large part of primary production in oceans. Culture-independent approaches highlighted the presence in abundance of marine non-cyanobacterial diazotrophs (NCD), but their ecophysiology remains elusive, mostly because of the low number of isolated NCD and because of the lack of available genetic tools for these isolates. Here, a dual genetic and functional approach allowed unveiling the ecophysiology of a marine NCD affiliated to the species Vibrio diazotrophicus. Physiological characterization of the first marine NCD mutant obtained so far was performed using a soft-gellan assay, demonstrating that a ΔnifH mutant is not able to grow in nitrogen-free media. Furthermore, we demonstrated that V. diazotrophicus produces a thick biofilm under diazotrophic conditions, suggesting biofilm production as an adaptive response of this NCD to cope with the inhibition of nitrogen fixation by molecular oxygen. Finally, the genomic signature of V. diazotrophicus is essentially absent from metagenomic data of Tara Ocean expeditions, despite having been isolated from various marine environments. We think that the genetically tractable V. diazotrophicus strain used in this study may serve as an ideal model to study the ecophysiology of these overlooked procaryotic group.

A bistable prokaryotic differentiation system underlying development of conjugative transfer competence.

The mechanisms and impact of horizontal gene transfer processes to distribute gene functions with potential adaptive benefit among prokaryotes have been well documented. In contrast, little is known about the life-style of mobile elements mediating horizontal gene transfer, whereas this is the ultimate determinant for their transfer fitness. Here, we investigate the life-style of an integrative and conjugative element (ICE) within the genus Pseudomonas that is a model for a widespread family transmitting genes for xenobiotic compound metabolism and antibiotic resistances. Previous work showed bimodal ICE activation, but by using single cell time-lapse microscopy coupled to combinations of chromosomally integrated single copy ICE promoter-driven fluorescence reporters, RNA sequencing and mutant analysis, we now describe the complete regulon leading to the arisal of differentiated dedicated transfer competent cells. The regulon encompasses at least three regulatory nodes and five (possibly six) further conserved gene clusters on the ICE that all become expressed under stationary phase conditions. Time-lapse microscopy indicated expression of two regulatory nodes (i.e., bisR and alpA-bisDC) to precede that of the other clusters. Notably, expression of all clusters except of bisR was confined to the same cell subpopulation, and was dependent on the same key ICE regulatory factors. The ICE thus only transfers from a small fraction of cells in a population, with an estimated proportion of between 1.7-4%, which express various components of a dedicated transfer competence program imposed by the ICE, and form the centerpiece of ICE conjugation. The components mediating transfer competence are widely conserved, underscoring their selected fitness for efficient transfer of this class of mobile elements.

Life history of oysters influences Vibrio parahaemolyticus accumulation in Pacific oysters (Crassostrea gigas).

Vibrio parahaemolyticus infection in humans is associated with raw oyster consumption. Evaluation of V. parahaemolyticus presence in oysters is of most interest because of the economic and public health issues that it represents. To explore V. parahaemolyticus accumulation and depuration in adult Crassostrea gigas, we developed a GFP-tagged V. parahaemolyticus strain (IFVp201-gfp+ ), as well as a rapid and efficient quantification method in C. gigas oysters haemolymph by flow cytometry. Impact of the life history of C. gigas on accumulation and depuration of V. parahaemolyticus IFVp201 was subsequently investigated. We found that naive oysters, i.e. grown in controlled facilities with UV treated seawater, accumulated significantly more IFVp201 than environmental oysters, i.e. grown in intertidal environment. We hypothesized that environmental oysters could have been immune primed, thus could limit V. parahaemolyticus accumulation. Meanwhile, both naive and environmental oysters had similar depuration rates.

Virulence of Vibrio harveyi ORM4 towards the European abalone Haliotis tuberculata involves both quorum sensing and a type III secretion system.

Environmental Vibrio strains represent a major threat in aquaculture, but the understanding of their virulence mechanisms heavily relies on the transposition of knowledge from human-pathogen vibrios. Here, the genetic bases of the virulence of Vibrio harveyi ORM4 towards the European abalone Haliotis tuberculata were characterized. We demonstrated that luxO, encoding a major regulator of the quorum sensing system, is crucial for the virulence of this strain, and that its deletion leads to a decrease in swimming motility, biofilm formation, and exopolysaccharide production. Furthermore, the biofilm formation by V. harveyi ORM4 was increased by abalone serum, which required LuxO. The absence of LuxO in V. harveyi ORM4 yielded opposite phenotypes compared with other Vibrio species including V. campbellii (still frequently named V. harveyi). In addition, we report a full type III secretion system (T3SS) gene cluster in the V. harveyi ORM4 genome. LuxO was shown to negatively regulate the promoter activity of exsA, encoding the major regulator of the T3SS genes, and the deletion of exsA abolished the virulence of V. harveyi ORM4. These results unveil virulence mechanisms set up by this environmentally important bacterial pathogen and pave the way for a better molecular understanding of the regulation of its pathogenicity.

Implication of the Type IV Secretion System in the Pathogenicity of Vibrio tapetis, the Etiological Agent of Brown Ring Disease Affecting the Manila Clam Ruditapes philippinarum.

Vibrio tapetis is a Gram-negative bacterium that causes infections of mollusk bivalves and fish. The Brown Ring Disease (BRD) is an infection caused by V. tapetis that primarily affects the Manila clam Ruditapes philippinarum. Recent studies have shown that a type IV secretion system (T4SS) gene cluster is exclusively found in strains of V. tapetis pathogenic to clams. However, whether the T4SS is implicated or not during the infection process remains unknown. The aim of this study was to create and characterize a V. tapetis T4SS null mutant, obtained by a near-complete deletion of the virB4 gene, in order to determine the role of T4SS in the development of BRD. This study demonstrated that the T4SS is neither responsible for the loss of hemocyte adhesion capacities, nor for the decrease of the lysosomal activity during BRD. Nevertheless, we observed a 50% decrease of the BRD prevalence and a decrease of mortality dynamics with the ΔvirB4 mutant. This work demonstrates that the T4SS of V. tapetis plays an important role in the development of BRD in the Manila clam.

An analog to digital converter controls bistable transfer competence development of a widespread bacterial integrative and conjugative element.

Conjugative transfer of the integrative and conjugative element ICEclc in Pseudomonas requires development of a transfer competence state in stationary phase, which arises only in 3-5% of individual cells. The mechanisms controlling this bistable switch between non-active and transfer competent cells have long remained enigmatic. Using a variety of genetic tools and epistasis experiments in P. putida, we uncovered an 'upstream' cascade of three consecutive transcription factor-nodes, which controls transfer competence initiation. One of the uncovered transcription factors (named BisR) is representative for a new regulator family. Initiation activates a feedback loop, controlled by a second hitherto unrecognized heteromeric transcription factor named BisDC. Stochastic modelling and experimental data demonstrated the feedback loop to act as a scalable converter of unimodal (population-wide or 'analog') input to bistable (subpopulation-specific or 'digital') output. The feedback loop further enables prolonged production of BisDC, which ensures expression of the 'downstream' functions mediating ICE transfer competence in activated cells. Phylogenetic analyses showed that the ICEclc regulatory constellation with BisR and BisDC is widespread among Gamma- and Beta-proteobacteria, including various pathogenic strains, highlighting its evolutionary conservation and prime importance to control the behaviour of this wide family of conjugative elements.

Mobile DNA elements are pieces of genetic material that can jump from one bacterium to another, and even across species. They are often useful to their host, for example carrying genes that allow bacteria to resist antibiotics. One example of bacterial mobile DNA is the ICEclc element. Usually, ICEclc sits passively within the bacterium's own DNA, but in a small number of cells, it takes over, hijacking its host to multiply and to get transferred to other bacteria. Cells that can pass on the elements cannot divide, and so this ability is ultimately harmful to individual bacteria. Carrying ICEclc can therefore be positive for a bacterium but passing it on is not in the cell's best interest. On the other hand, mobile DNAs like ICEclc have evolved to be disseminated as efficiently as possible. To shed more light on this tense relationship, Carraro et al. set out to identify the molecular mechanisms ICEclc deploys to control its host. Experiments using mutant bacteria revealed that for ICEclc to successfully take over the cell, a number of proteins needed to be produced in the correct order. In particular, a protein called BisDC triggers a mechanism to make more of itself, creating a self-reinforcing 'feedback loop'. Mathematical simulations of the feedback loop showed that it could result in two potential outcomes for the cell. In most of the 'virtual cells', ICEclc ultimately remained passive; however, in a few, ICEclc managed to take over its hosts. In this case, the feedback loop ensured that there was always enough BisDC to maintain ICEclc's control over the cell. Further analyses suggested that this feedback mechanism is also common in many other mobile DNA elements, including some that help bacteria to resist drugs. These results are an important contribution to understand how mobile DNAs manipulate their bacterial host in order to propagate and disperse. In the future, this knowledge could help develop new strategies to combat the spread of antibiotic resistance.

Transient Replication in Specialized Cells Favors Transfer of an Integrative and Conjugative Element.

Integrative and conjugative elements (ICEs) are widespread mobile DNA within bacterial genomes, whose lifestyle is relatively poorly understood. ICEs transmit vertically through donor cell chromosome replication, but in order to transfer, they have to excise from the chromosome. The excision step makes ICEs prone to loss, in case the donor cell divides and the ICE is not replicated. By adapting the system of LacI-cyan fluorescent protein (CFP) binding to lacO operator arrays, we analyze here the process of excision and transfer of the ICE for 3-chlorobenzoate degradation (ICEclc) in individual cells of the bacterium Pseudomonas putida We provide evidence that ICEclc excises exclusively in a subset of specialized transfer-competent cells. ICEclc copy numbers in transfer-competent cells were higher than in regular nontransferring cells but were reduced in mutants lacking the ICE oriT1 origin of transfer, the ICE DNA relaxase, or the excision recombination sites. Consistently, transfer-competent cells showed a higher proportion without any observable LacI-CFP foci, suggesting ICEclc loss, but this proportion was independent of the ICE relaxase or the ICE origins of transfer. Our results thus indicated that the excised ICE becomes transiently replicated in transfer-competent cells, with up to six observable copies from LacI-CFP fluorescent focus measurements. Most of the observed ICEclc transfer to ICE-free P. putida recipients occurred from donors displaying 3 to 4 ICE copies, which constitute a minority among all transfer-competent cells. This finding suggests, therefore, that replication of the excised ICEclc in donors is beneficial for transfer fitness to recipient cells.IMPORTANCE Bacterial evolution is driven to a large extent by horizontal gene transfer (HGT)-the processes that distribute genetic material between species rather than by vertical descent. The different elements and processes mediating HGT have been characterized in great molecular detail. In contrast, very little is known on adaptive features selecting HGT evolvability and fitness optimization. By studying the molecular behavior of an integrated mobile DNA of the class of integrative and conjugative elements in individual Pseudomonas putida donor bacteria, we report here how transient replication of the element after its excision from the chromosome is favorable for its transfer success. Since successful transfer into a new recipient is a measure of the element's fitness, transient replication may have been selected as an adaptive benefit for more-optimal transfer.

Rapid and efficient protocol to introduce exogenous DNA in Vibrio harveyi and Pseudoalteromonas sp.

Vibrio campbellii BAA-1116 is renowned for its bioluminescence properties, and genetic tools are available to genetically track this strain. However, many other ecologically important V. harveyi strains exist, for which only few genetic tools are available. In this study, a rapid electroporation protocol was developed to transform replicative plasmids in various environmental V. harveyi and Pseudoalteromonas strains. Moreover, a mini-Tn7 delivery system was modified to site-specifically integrate mini-transposons in the genome of V. harveyi ORM4. As a proof-of-principle, replicative plasmids carrying bioreporters were introduced by electroporation in V. harveyi ORM4 cells, and gene expression was followed at the single cell level. We could demonstrate that a flagellar gene is subjected to bimodal gene expression in V. harveyi ORM4, being highly expressed in 10% of the population in stationary phase. This study extends the possibilities to study environmental Vibrio strains and uncovers the occurrence of phenotypic heterogeneity in flagellar expression in Vibrio.

The hidden life of integrative and conjugative elements.

Integrative and conjugative elements (ICEs) are widespread mobile DNA that transmit both vertically, in a host-integrated state, and horizontally, through excision and transfer to new recipients. Different families of ICEs have been discovered with more or less restricted host ranges, which operate by similar mechanisms but differ in regulatory networks, evolutionary origin and the types of variable genes they contribute to the host. Based on reviewing recent experimental data, we propose a general model of ICE life style that explains the transition between vertical and horizontal transmission as a result of a bistable decision in the ICE-host partnership. In the large majority of cells, the ICE remains silent and integrated, but hidden at low to very low frequencies in the population specialized host cells appear in which the ICE starts its process of horizontal transmission. This bistable process leads to host cell differentiation, ICE excision and transfer, when suitable recipients are present. The ratio of ICE bistability (i.e. ratio of horizontal to vertical transmission) is the outcome of a balance between fitness costs imposed by the ICE horizontal transmission process on the host cell, and selection for ICE distribution (i.e. ICE 'fitness'). From this emerges a picture of ICEs as elements that have adapted to a mostly confined life style within their host, but with a very effective and dynamic transfer from a subpopulation of dedicated cells.

Highly variable individual donor cell fates characterize robust horizontal gene transfer of an integrative and conjugative element.

Horizontal gene transfer is an important evolutionary mechanism for bacterial adaptation. However, given the typical low transfer frequencies in a bacterial population, little is known about the fate and interplay of donor cells and the mobilized DNA during transfer. Here we study transfer of an integrative and conjugative element (ICE) among individual live bacterial cells. ICEs are widely distributed mobile DNA elements that are different than plasmids because they reside silent in the host chromosome and are maintained through vertical descent. Occasionally, ICEs become active, excise, and transmit their DNA to a new recipient, where it is reintegrated. We develop a fluorescent tool to differentiate excision, transfer, and reintegration of a model ICE named ICEclc (for carrying the clc genes for chlorocatechol metabolism) among single Pseudomonas cells by using time-lapse microscopy. We find that ICEclc activation is initiated in stationary phase cells, but excision and transfer predominantly occur only when such cells have been presented with new nutrients. Donors with activated ICE develop a number of different states, characterized by reduced cell division rates or growth arrest, persistence, or lysis, concomitant with ICE excision, and likely, ICE loss or replication. The donor cell state transitions can be described by using a stochastic model, which predicts that ICE fitness is optimal at low initiation rates in stationary phase. Despite highly variable donor cell fates, ICE transfer is remarkably robust overall, with 75% success after excision. Our results help to better understand ICE behavior and shed a new light on bacterial cellular differentiation during horizontal gene transfer.

The TetR-type MfsR protein of the integrative and conjugative element (ICE) ICEclc controls both a putative efflux system and initiation of ICE transfer.

Integrative and conjugating elements (ICE) are self-transferable DNAs widely present in bacterial genomes, which often carry a variety of auxiliary genes of potential adaptive benefit. One of the model ICE is ICEclc, an element originally found in Pseudomonas knackmussii B13 and known for its propensity to provide its host with the capacity to metabolize chlorocatechols and 2-aminophenol. In this work, we studied the mechanism and target of regulation of MfsR, a TetR-type repressor previously found to exert global control on ICEclc horizontal transfer. By using a combination of ICEclc mutant and transcriptome analysis, gene reporter fusions, and DNA binding assays, we found that MfsR is a repressor of both its own expression and that of a gene cluster putatively coding for a major facilitator superfamily efflux system on ICEclc (named mfsABC). Phylogenetic analysis suggests that mfsR was originally located immediately adjacent to the efflux pump genes but became displaced from its original cis target DNA by a gene insertion. This resulted in divergence of the original bidirectional promoters into two separated individual regulatory units. Deletion of mfsABC did not result in a strong phenotype, and despite screening a large number of compounds and conditions, we were unable to define the precise current function or target of the putative efflux pump. Our data reconstruct how the separation of an ancestor mfsR-mfsABC system led to global control of ICEclc transfer by MfsR.

An operon of three transcriptional regulators controls horizontal gene transfer of the integrative and conjugative element ICEclc in Pseudomonas knackmussii B13.

The integrative and conjugative element ICEclc is a mobile genetic element in Pseudomonas knackmussii B13, and an experimental model for a widely distributed group of elements in Proteobacteria. ICEclc is transferred from specialized transfer competent cells, which arise at a frequency of 3-5% in a population at stationary phase. Very little is known about the different factors that control the transfer frequency of this ICE family. Here we report the discovery of a three-gene operon encoded by ICEclc, which exerts global control on transfer initiation. The operon consists of three consecutive regulatory genes, encoding a TetR-type repressor MfsR, a MarR-type regulator and a LysR-type activator TciR. We show that MfsR autoregulates expression of the operon, whereas TciR is a global activator of ICEclc gene expression, but no clear role was yet found for MarR. Deletion of mfsR increases expression of tciR and marR, causing the proportion of transfer competent cells to reach almost 100% and transfer frequencies to approach 1 per donor. mfsR deletion also caused a two orders of magnitude loss in population viability, individual cell growth arrest and loss of ICEclc. This indicates that autoregulation is an important feature maintaining ICE transfer but avoiding fitness loss. Bioinformatic analysis showed that the mfsR-marR-tciR operon is unique for ICEclc and a few highly related ICE, whereas tciR orthologues occur more widely in a large variety of suspected ICE among Proteobacteria.

Yeast and bacterial diversity along a transect in an acidic, As-Fe rich environment revealed by cultural approaches.

Acid mine drainages (AMDs) are often thought to harbour low biodiversity, yet little is known about the diversity distribution along the drainages. Using culture-dependent approaches, the microbial diversity from the Carnoulès AMD sediment was investigated for the first time along a transect showing progressive environmental stringency decrease. In total, 20 bacterial genera were detected, highlighting a higher bacterial diversity than previously thought. Moreover, this approach led to the discovery of 16 yeast species, demonstrating for the first time the presence of this important phylogenetic group in this AMD. All in all, the location of the microbes along the transect helps to better understand their distribution in a pollution gradient.

Novel and unexpected bacterial diversity in an arsenic-rich ecosystem revealed by culture-dependent approaches.

BACKGROUND: Acid Mine Drainages (AMDs) are extreme environments characterized by very acid conditions and heavy metal contaminations. In these ecosystems, the bacterial diversity is considered to be low. Previous culture-independent approaches performed in the AMD of Carnoulès (France) confirmed this low species richness. However, very little is known about the cultured bacteria in this ecosystem. The aims of the study were firstly to apply novel culture methods in order to access to the largest cultured bacterial diversity, and secondly to better define the robustness of the community for 3 important functions: As(III) oxidation, cellulose degradation and cobalamine biosynthesis. RESULTS: Despite the oligotrophic and acidic conditions found in AMDs, the newly designed media covered a large range of nutrient concentrations and a pH range from 3.5 to 9.8, in order to target also non-acidophilic bacteria. These approaches generated 49 isolates representing 19 genera belonging to 4 different phyla. Importantly, overall diversity gained 16 extra genera never detected in Carnoulès. Among the 19 genera, 3 were previously uncultured, one of them being novel in databases. This strategy increased the overall diversity in the Carnoulès sediment by 70% when compared with previous culture-independent approaches, as specific phylogenetic groups (e.g. the subclass Actinobacteridae or the order Rhizobiales) were only detected by culture. Cobalamin auxotrophy, cellulose degradation and As(III)-oxidation are 3 crucial functions in this ecosystem, and a previous meta- and proteo-genomic work attributed each function to only one taxon. Here, we demonstrate that other members of this community can also assume these functions, thus increasing the overall community robustness. CONCLUSIONS: This work highlights that bacterial diversity in AMDs is much higher than previously envisaged, thus pointing out that the AMD system is functionally more robust than expected. The isolated bacteria may be part of the rare biosphere which remained previously undetected due to molecular biases. No matter their current ecological relevance, the exploration of the full diversity remains crucial to decipher the function and dynamic of any community. This work also underlines the importance to associate culture-dependent and -independent approaches to gain an integrative view of the community function.

Amylases without known homologues discovered in an acid mine drainage: significance and impact.

Acid Mine Drainages (AMDs) are extreme environments characterized by acidic and oligotrophic conditions and by metal contaminations. A function-based screening of an AMD-derived metagenomic library led to the discovery and partial characterization of two non-homologous endo-acting amylases sharing no sequence similarity with any known amylase nor glycosidase. None carried known amylolytic domains, nor could be assigned to any GH-family. One amylase displayed no similarity with any known protein, whereas the second one was similar to TraC proteins involved in the bacterial type IV secretion system. According to the scarce similarities with known proteins, 3D-structure modelling using I-TASSER was unsuccessful. This study underlined the utility of a function-driven metagenomic approach to obtain a clearer image of the bacterial community enzymatic landscape. More generally, this work points out that screening for microorganisms or biomolecules in a priori incongruous environments could provide unconventional and new exciting ways for bioprospecting.

Deciphering the role of Paenibacillus strain Q8 in the organic matter recycling in the acid mine drainage of Carnoulès.

BACKGROUND: The recycling of the organic matter is a crucial function in any environment, especially in oligotrophic environments such as Acid Mine Drainages (AMDs). Polymer-degrading bacteria might play an important role in such ecosystem, at least by releasing by-products useful for the rest of the community. In this study, physiological, molecular and biochemical experiments were performed to decipher the role of a Paenibacillus strain isolated from the sediment of Carnoulès AMD. RESULTS: Even though Paenibacillus sp. strain Q8 was isolated from an oligotrophic AMD showing an acidic pH, it developed under both acidic and alkaline conditions and showed a heterotrophic metabolism based on the utilization of a broad range of organic compounds. It resisted to numerous metallic stresses, particularly high arsenite (As(III)) concentrations (> 1,800 mg/L). Q8 was also able to efficiently degrade polymers such as cellulose, xylan and starch. Function-based screening of a Q8 DNA-library allowed the detection of 15 clones with starch-degrading activity and 3 clones with xylan-degrading activity. One clone positive for starch degradation carried a single gene encoding a "protein of unknown function". Amylolytic and xylanolytic activities were measured both in growing cells and with acellular extracts of Q8. The results showed the ability of Q8 to degrade both polymers under a broad pH range and high As(III) and As(V) concentrations. Activity measurements allowed to point out the constitutive expression of the amylase genes and the mainly inducible expression of the xylanase genes. PACE demonstrated the endo-acting activity of the amylases and the exo-acting activity of the xylanases. CONCLUSIONS: AMDs have been studied for years especially with regard to interactions between bacteria and the inorganic compartment hosting them. To date, no study reported the role of microorganisms in the recycling of the organic matter. The present work suggests that the strain Q8 might play an important role in the community by recycling the scarce organic matter (cellulose, hemicellulose, starch...), especially when the conditions change. Furthermore, function-based screening of a Q8 DNA library allowed to assign an amylolytic function to a gene previously unknown. AMDs could be considered as a reservoir of genes with potential biotechnological properties.