Patrick Tschopp

Emma Sherratt

Thomas J Sanger

Anna C Groner

Ariel C Aspiras

Jimmy K Hu

Olivier Pourquié

Jérôme Gros
email: jgros@pasteur.fr..

Clifford J Tabin
email: tabin@genetics.med.harvard.edu

A relative shift in cloacal location repositions external genitalia in amniote evolution

 , yet without providing any underlying developmental mechanism. Here, we have reexamined this question using micro-Computed Tomography (μCT), lineage tracing in three amniote clades and RNA-Seq transcriptional profiling. We show that the developmental origin of external genitalia has shifted through evolution, and in some taxa limbs and genitals share a common primordium. In squamates, the genitalia develop directly from the budding hindlimbs, or the remnants thereof, whereas in mice the genital tubercle originates from Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.

the ventral and tail bud mesenchyme. The recruitment of different cell populations for genital outgrowth follows a change in the relative position of the cloaca, the genitalia organizing center. Ectopic grafting of the cloaca demonstrates the conserved ability of different mesenchymal cells to respond to these genitalia-inducing signals. Our results support a limb-like developmental origin of external genitalia as the ancestral condition. Moreover, it suggests that a change in the relative position of the cloacal signaling center during evolution has led to an altered developmental route of external genitalia in mammals, while preserving parts of the ancestral limb molecular circuitry due to a common evolutionary origin.

To investigate potential interdependencies in the development of limbs and external genitalia, we first determined the location of the two structures during embryogenesis. We focused on mouse [START_REF] Yamada | Molecular genetic cascades for external genitalia formation: An emerging organogenesis program[END_REF][START_REF] Cohn | Development of the external genitalia: Conserved and divergent mechanisms of appendage patterning[END_REF] and squamates (lizard and snakes), which show progressive limbreduction [START_REF] Greer | Limb reduction in squamates: identification of the lineages and discussion of the trends[END_REF] , yet maintain their external genitalia, the hemipenes [START_REF] Cope | On the hemipenes of the Sauria[END_REF] . μCT-reconstructions of mouse, anole lizard (Anolis), python and house snake embryos revealed different anteriorposterior locations of the developing external genitalia, relative to limbs. In mice, the genital tubercle is positioned caudal to the hindlimbs (Fig. 1a), whereas in squamates the paired hemipenes bud from the limbs, or remnants thereof (Fig. 1b-d). The cloaca, a signaling center important for genitalia development [START_REF] Lin | Delineating a conserved genetic cassette promoting outgrowth of body appendages[END_REF][START_REF] Haraguchi | Unique functions of Sonic hedgehog signaling during external genitalia development[END_REF][START_REF] Perriton | Sonic hedgehog signaling from the urethral epithelium controls external genital development[END_REF] , is similarly located within the limb-field of squamates (Fig. 1f-h) and expresses Shh (Fig. 1j-l). Thus in squamates all three anatomical structures, limb, hemipenis and cloaca, align at the same anterior-posterior position.

We decided to investigate whether these positional differences would reflect distinct developmental origins of external genitalia. While the cells of the vertebrate limb bud arise through an epithelial-to-mesenchymal transition (EMT) of an epithelial lateral plate mesoderm (LPM) population lining the coelomic cavity [START_REF] Gros | Vertebrate limb bud formation is initiated by localized epithelial-tomesenchymal transition[END_REF] , the developmental origin of external genitalia in vertebrates is still unclear. We developed a lenti-viral lineage tracing approach (see Methods), to systematically follow the two sources previously proposed, the LPM and tail bud [START_REF] Perriton | Sonic hedgehog signaling from the urethral epithelium controls external genital development[END_REF][START_REF] Ohta | Cessation of gastrulation is mediated by suppression of epithelial-mesenchymal transition at the ventral ectodermal ridge[END_REF][START_REF] Suzuki | New horizons at the caudal embryos: coordinated urogenital/reproductive organ formation by growth factor signaling[END_REF] , in three amniote species, mouse, chicken and anole. Injections into the coelom of embryonic day 9.5 (E9.5) mouse embryos label cells surrounding the coelom as well as the developing hindlimb (Fig. 2a). However, no GFP-positive cells are observed in the genital tubercle, with a sharp boundary of labeled cells extending from the coelomic cavity (Fig. 2a,b). In contrast, injection into the posterior mesenchyme of mouse embryos labels the genital tubercle (Fig. 2c). Tailbud injections label the posterior half, whereas the infra-umbilical mesenchyme gives rise to its anterior part [START_REF] Ohta | Cessation of gastrulation is mediated by suppression of epithelial-mesenchymal transition at the ventral ectodermal ridge[END_REF][START_REF] Suzuki | New horizons at the caudal embryos: coordinated urogenital/reproductive organ formation by growth factor signaling[END_REF][START_REF] Matsumaru | Genetic analysis of the role of Alx4 in the coordination of lower body and external genitalia formation[END_REF] (Extended Data Fig. 1). In chicken, coelomic injection into stage HH14 embryos labels cells in both limb and genital tubercle (Fig. 2d,e), without any obvious boundary. Tailbud infection also results in GFPpositive genitalia cells, mostly in the posterior tubercle, suggesting that multiple lineages contribute to this species' genitalia (Fig. 2f). Similar conclusions were reached in a parallel study (Embryonic origin and compartmental organization of the external genitalia, Herrera A. M. & Cohn M. J.). In Anolis, coelomic injections at stage 2-3 result in GFP-positive cells in the limb and the developing genitalia (Fig. 2g,h), whereas no labeled cells are seen in the hemipenes following tailbud injections (Fig. 2i).

As for chicken and mouse limbs [START_REF] Gros | Vertebrate limb bud formation is initiated by localized epithelial-tomesenchymal transition[END_REF] , cells of the Anolis limb, but also the hemipenis, originate via an EMT of the coelomic epithelium (Extended Data Fig. 2a,b). In snakes, we find evidence for similar cellular dynamics. The hemipenes emerge as small buds at a "limblike" lateral position, juxtaposed to the coelomic cavity (Extended Data Fig. 2c-f). A concomitant basement membrane breakdown, consistent with an EMT of the LPM, is seen in the budding of both mouse limbs and snake hemipenes (Extended Data Fig. 2d,f). Moreover, we find that Tbx4, a gene important for hindlimb development [START_REF] Naiche | Loss of Tbx4 blocks hindlimb development and affects vascularization and fusion of the allantois[END_REF] is expressed from early on, in both the coelomic epithelium and the mesenchyme of the developing hemipenis (Extended Data Fig. 2g-i). Its forelimb counterpart Tbx5 is expressed later, in the mesenchyme only, in agreement with its genitalia-expression in mammals [START_REF] Chapman | Expression of the T-box family genes, Tbx1-Tbx5, during early mouse development[END_REF] (Extended Data Fig. 2j-l). This suggests that squamate external genitalia initiate with limb-like cellular dynamics, the resulting mesenchymal cell population in modern snakes being converted to a genital fate [START_REF] Raynaud | Biology of the Reptilia[END_REF] . Thus, important differences exist in the developmental origins of external genitalia in amniotes: chicken genitals originate from both LPM and the tailbud, whereas the mouse genital tubercle consists of infra-umbilical mesenchyme and tailbud-descendant cells. In contrast, the hemipenis shares a developmental route with hindlimbs, either through secondary budding as in lizards, or by entirely recruiting the mesenchymal cell population to a genital fate in modern snakes. Given the impact of developmental lineage on an organ's molecular architecture, we next explored the transcriptomes of emerging genitalia, in the two opposing routes of mammals and squamates.

Gene expression profiling has successfully been applied to questions of developmental and evolutionary origin, of cell types and entire morphological features [START_REF] Arendt | Genes and homology in nervous system evolution: comparing gene functions, expression patterns, and cell type molecular fingerprints[END_REF][START_REF] Wagner | The developmental genetics of homology[END_REF][START_REF] Shubin | Deep homology and the origins of evolutionary novelty[END_REF][START_REF] Wang | Transcriptomic analysis of avian digits reveals conserved and derived digit identities in birds[END_REF] . We thus dissected early and late stages of developing limbs and genitalia from mouse and anole embryos (Fig. 3a), for comparative RNA-seq analyses. Overall transcriptome similarities were assessed using multidimensional scaling (MDS; Fig. 3b). The transcriptomes dominantly resolve along dimension 1 in a species-dependent manner, as expected for similar tissues in evolutionary distant species [START_REF] Merkin | Evolutionary Dynamics of Gene and Isoform Regulation in Mammalian Tissues[END_REF] . Dimension 2, however, contains a clear organ identity signal, i.e. limb versus genitalia. This separation is virtually absent in Anolis samples, compared to mouse, reflecting similar transcriptional programs due to a common developmental origin for genitalia and hindlimbs. For hierarchical clustering, we included the early tail bud, as outgroup of the primary body axis, and forelimbs, to account for anterior-posterior differences in the two species' genitalia (Fig. 3c,d; see Methods). In Anolis, the early hemipenis transcriptome falls within the limb clade, indicative of an almost generic limb molecular architecture, and only later differentiates into a more organ-specific signature (Fig. 3c and Extended Data Fig. 3a). In contrast, mouse genitalia transcriptomes are clearly distinct from limbs, from early on, highlighting the separate developmental origins of the two organs (Fig. 3d and Extended Data Fig. 3b).

To identify genes driving hindlimb-and genitalia-specific transcriptome separation, we used principal component analysis. Principal component 1 (PC1) correlates with species differences, while organ specificity is resolved along PC2 (Fig. 3e), allowing us to identify organ-specific 'driver' genes in a species-independent manner. We assessed the contribution of orthologous genes to PC2, according to their absolute loading values (Fig. 3f). Gene Ontology (GO) analysis [START_REF] Ashburner | The Gene Ontology Consortium. Gene ontology: tool for the unification of biology[END_REF] of the top500 genes (Fig. 3f and Supplementary Table 1) identified GO-terms related to transcription factors (TFs) and signaling molecules (Fig. 3g). Gene regulatory networks thus determine limb versus genital organ transcriptomes, but also mirror their developmental origin. Strikingly, TFs/signaling molecule data is sufficient to reproduce the clustering seen with whole-transcriptomes (Fig. 3h,i and Extended Data Fig. 4a,b). Collectively, we find a clear distinction between mouse genitalia and limb transcriptomes, during early and late organogenesis. Such genitalia-specific separation is only seen in late Anolis hemipenes, arguing for a developmental repurposing to a copulatory structure. Importantly, we find transcriptional similarities between early hemipenes and hindlimbs in squamates, illustrating a common developmental origin.

An attractive model for the varied developmental origins of amniote external genitalia would be a repositioning of the cloacal signaling center with respect to different mesenchymal cell populations with progenitor potential. Hence, bringing either hindlimb or tailbud close to the cloaca would allow these lineages to contribute to genital outgrowth. We tested this hypothesis by grafting GFP-transgenic chicken or quail cloacae into the hindlimb bud of wild-type embryos (Fig. 4a and Extended Data Fig. 5a-c). After 1-2 days of incubation, limbs showed ectopic, secondary buds (Fig. 4b,c). GFP-negative cells suggest an inductive effect of the cloaca on the surrounding mesenchyme, rather than simple overproliferation of the graft itself (Fig. 4c). Similar buds could be induced by grafting beads soaked in known cloacal signaling molecules Shh and Fgf 9,10,24,25 (Extended Data Fig. 5dg). To assess the fate of responding cells, we re-analyzed our RNA-seq data for potential genital versus limb markers. For both species, we performed stage-and organ-specific differential expression analyses. Of the 2003 genes showing an absolute log2 fold-change greater than 1.5 (p-value < 0.05) (Extended Data Fig. 6), we identified 27 that are altered in all four comparisons, 25 of which in the same direction (Fig. 4d). Hierarchical clustering of normalized mouse expression values reveals four stage-and organ-specific signatures, which are largely conserved in Anolis (Fig. 4e). Based on expression patterns and levels (Extended Data Fig. 6 and7), we chose marker genes to assess transcriptional changes due to ectopic cloacal signals. Indeed, limb cells close to GFP-positive cloacal grafts downregulate limb markers Lhx9 and Tbx18 (Fig. 4f,g), and ectopically express genital markers Isl1 26 , Gata2 and Runx1 (Fig. 4h and Extended Data Fig. 5j-m). Accordingly, when we graft cloacal tissue into the tailbud mesenchyme, ectopic budding and genital marker expression are equally induced (Fig. 4i-k and Extended Data Fig. 5l-n and8). Hence, mesenchymal cells from different developmental origins can respond to inductive cloacal signals, generating outgrowths and genitalia-like marker gene expression. Importantly, these results support the idea that changing the relative anterior-posterior position of the cloaca could generate external genitalia with distinct developmental origins during the course of amniote evolution.

In summary, we show substantial variation in external genitalia development in extant amniote species. We propose that repositioning the cloaca can recruit different mesenchymal cell populations, either through spatial or heterochronic changes in the dynamics of their emergence. In squamates, the hindlimb is the dominant source, with modern snakes entirely repurposing a mesenchymal bud to a genital fate. In mice, limbs and genitals have discrete developmental origins, the LPM and the ventral and tailbud mesenchyme, respectively, with chicken showing an intermediate state. Moreover, we find that similarities in limb and genitalia transcriptomes are dependent on the cellular source of the primordia from which they emerge. Specifically, there is a higher degree of early transcriptome congruence in species deriving their intromittent organs from limb anlagen. Strikingly, the ability of different mesenchymal cell populations to respond to cloacal, genitalia-inducing signals seems conserved in extant species. It is therefore tempting to speculate that a limb-derived state could represent the ancestral condition in the evolution of external genitalia, as suggested by their position relative to limbs during turtle development [START_REF] Raynaud | Biology of the Reptilia[END_REF][START_REF] Larkins | Phallus Development in the Turtle Trachemys scripta[END_REF] and the bifid genitalia of basal mammals [START_REF] Griffiths | The Biology of the Monotremes[END_REF][START_REF] De Barros | Marsupial morphology of reproduction: South America opossum male model[END_REF] . As such, a developmental continuity between limbs and genitalia could have turned into an 'evolutionary continuity' in mammals, as the two organs spatially separated due to a relative repositioning of the cloaca [START_REF] Jurberg | Switching Axial Progenitors from Producing Trunk to Tail Tissues in Vertebrate Embryos[END_REF] . Once shared developmental trajectories could thus help to explain molecular similarities still noticeable in species that develop the two organs from distinct cellular sources [START_REF] Kondo | Of fingers, toes and penises[END_REF][START_REF] Yamada | Molecular genetic cascades for external genitalia formation: An emerging organogenesis program[END_REF][START_REF] Wagner | The developmental genetics of homology[END_REF] .

METHODS SUMMARY

μCT-scan reconstructions were used to visualize overall embryonic morphology. For lineage tracing, GFP-expressing lentiviruses were injected into the coelomic cavity or tailbud of mouse, chicken and Anolis embryos. RNA-seq was performed on mouse and Anolis embryonic tissue, using an Illumina HiSeq 2000. Normalized gene read-counts were used for downstream analyses. For grafting experiments, GFP-transgenic chicken or quail cloacal tissue was grafted ectopically into hindlimbs or tailbuds of wild-type recipient chicken. Marker gene expression and grafted tissue were assessed with in situ hybridization and immunohistochemistry. See full Methods for details and any associated references.

METHODS

Tissue sample collection

All embryos were collected in accordance with the appropriate Institutional Animal Care and Use Committee guidelines. Timed-pregnant CD1 and C57BL/6 females were purchased from Charles River. Gravid Anolis females were purchased from Candy Quality Reptiles, La Place, LA (IACUC #26-11 and #28-14). Anole housing and egg incubation were done as previously described [START_REF] Sanger | A developmental staging series for the lizard genusAnolis: A new system for the integration of evolution, development, and ecology[END_REF] . To collect early stage pre-oviposition embryos, females were euthanized by intraperitoneal Euthasol injection and eggs dissected from the oviduct. Snake husbandry and egg collection have been described before [START_REF] Gomez | Control of segment number in vertebrate embryos[END_REF] . Fertilized white leghorns chicken eggs were obtained from Charles River Laboratories and incubated at 38°C. For staging of mouse embryos, noon on the day of the vaginal plug was considered as embryonic day 0.5 (E0.5). For chicken and anole embryos, staging was performed according to Hamburger and Hamilton [START_REF] Hamburger | A Series of Normal Stages in the Development of the Chick Embryo[END_REF] or Sanger and colleagues [START_REF] Sanger | A developmental staging series for the lizard genusAnolis: A new system for the integration of evolution, development, and ecology[END_REF] , respectively. Embryonic tissue was dissected in cold PBS and either fixed in 4% PFA and processed for cryo-embedding or CT scanning, or else directly processed for RNA extraction or stored in RNAlater (Qiagen).

CT scanning and image processing

Embryos were fixed in 4% PFA and stored in 100% ethanol. Staining was done for 48h in 30% PTA (1% (w/v) phosphotungstic acid in water) and 70% ethanol [START_REF] Metscher | MicroCT for developmental biology: A versatile tool for high-contrast 3D imaging at histological resolutions[END_REF] . Specimens were rinsed and stored in 70% ethanol until image acquisition. CT scans were carried out on a Bruker Skyscan 1173 or a Nikon (Metris) X-tek HMXST225, at 50-57kV, 115-145uA and 810-1000ms exposure time. Voxel sizes ranged from 0.0023 to 0.0056, with 1500 to 2400 total projections. Post-processing of scan data was done in VGStudio MAX 2.2 (Volume Graphics). For 3D re-construction of the cloaca, serial TIFF stacks produced by VGStudio were read into the Imaris software package (Bitplane) and the endodermal epithelium was used as a guide to manually outline the extent of the cloacal volume.

Immunohistochemistry and In Situ Hybridization

Fixed embryos were embedded in 7.5% gelatin / 15% sucrose or dehydrated in sucrose gradients and embedded in OCT. Sectioning was performed on a Leica CM3000 cryostat. For immunohistochemistry, sections were incubated with primary antibodies in PBST (PBS/BSA 0.2%, Triton 0,1% / SDS 0,02%) overnight, washed 2×10min in PBST and incubated for 1h with secondary antibodies. To detect genital tubercle and ectopic limb Isl1 expression, as well as Lmx1b, signal was amplified using the TSA Plus Cy3 kit (Perkin Elmer). Primary antibodies used were anti β (Santa Cruz), Laminin (Sigma), GFP (Abcam), QCPN, Lmx1b and Isl1 (all Developmental Studies Hybridoma Bank). In Situ Hybridization was performed using standard protocols [START_REF] Gomez | Control of segment number in vertebrate embryos[END_REF][START_REF] Mcglinn | Detection of gene expression in mouse embryos and tissue sections[END_REF] . Fluorescent images were acquired on a Zeiss LSM10 inverted confocal miscroscope. Bright-field images were acquired on a Nikon Eclipse E1000. Whole-mount images were acquired on a Leica MZ FL III. Images were globally processed for color balance and brightness using Adobe Photoshop.

Lineage tracing analyses

For lentiviral lineage tracing, viral particles harboring ubiquitously expressing GFP cassettes (UbiC-GFP or hPGK-GFP) were produced by transient transfection in 293T cells as described elsewhere [START_REF] Barde | Production and Titration of Lentiviral Vectors[END_REF] . Viral particles were then injected into either the coelomic cavitiy, or the tailbud mesenchyme of mouse E9.5 embryos, chicken HH14 or Anolis st. 2-3. For mouse experiments, timed-pregnant CD1 females were anaesthetized using isoflurane and surgery, in utero visualization of embryos and virus injection was done as previously outlined [START_REF] Punzo | Ultrasound-guided in utero injections allow studies of the development and function of the eye[END_REF] . For chicken embryos, eggs were lowered and windowed and virus injected using a pressure injector. For Anolis lineage tracing, we developed a novel whole-embryo ex ovo culturing system, using media conditions previously described for squamate organ cultures [START_REF] Buchtová | Initiation and patterning of the snake dentition are dependent on Sonic Hedgehog signaling[END_REF] . Briefly, we prepared culture dishes with an indentation, by pouring 1% Agar Noble (BD Difco) dissolved in culture medium into cell culture dishes containing a modeling clay, egg-shaped casting mold. Once solidified, the mold was removed and st. 2-3 Anolis embryos, dissected in 2× PBS, were placed with their yolk intact in the resulting cavity and covered with culture medium. 10% ink in 1× PBS/PenStrep was mouth-pipetted underneath the embryo, for better visualization, and lentiviral particles were injected using a pressure injector. To increase viral infection rate, embryo plates were kept for 12-16h at 37°C in a humidified chamber, before switching them to 28°C. Additional tailbud injections were performed using DiI. Embryos survived for up to 12 days. Only live specimens showing overall normal morphology were considered for further analysis.

To assess GFP+ cell contribution, embryos were dissected, fixed in 4% PFA, gelatinembedded and cryo-sectioned. Sections were stained for GFP and imaged on a Zeiss LSM10 inverted confocal miscroscope. For quantifications, 4-5 embryos per condition and species were imaged on multiple sections spanning the respective organs. GFP+ cell counting was performed in ImageJ, using the "ITCN" plugin written by Thomas Kuo (UCSB). A total of 59331 GFP+ cells were counted (mouse: 33853; chicken: 23710, anole: 1768). Counts were averaged over multiple sections and normalized on tissue area measured. The resulting ratios of the two tissues are given as a percentage of total GFP+ cells per area.

RNA sample preparation and sequencing

Total RNA was extracted from freshly dissected or RNAlater-preserved tissue using the Arcturus PicoPure RNA Isolation Kit (Life Technologies) and enriched for mRNA fraction using MPG mRNA Purification Kit (PureBiotech). Multiplexed RNA-seq libraries were produced using the SPIA cDNA synthesis kit and the Ovation Ultralow DR Multiplex system (NuGen). Sequencing was done on an Illumina HiSeq 2000, with eight samples multiplexed per lane. Base calling was performed using the Illumina software. Over 561M 50bp reads were generated, with an average of 23.4 reads per sample (median = 22.0M reads). For all tissues, biological duplicates were sequenced.

Read mapping and transcriptome analyses

Initial read mapping was performed with the RNA-Seq unified mapper (RUM) [START_REF] Grant | Comparative Analysis of RNA-Seq Alignment Algorithms and the RNA-Seq Unified Mapper (RUM)[END_REF] , using mouse NCBI37/mm9 and Anolis AnoCar2.0 genome assemblies, with UCSC mm9 refseq and ASU_Acar v2.1 [START_REF] Eckalbar | Genome reannotation of the lizard Anolis carolinensis based on 14 adult and embryonic deep transcriptomes[END_REF] annotation files, respectively. This resulted in 10265 orthologuos genes between mouse and Anolis. An in-house improved annotation for Anolis, generated prior to the publication of Eckalbar et al., yielded concordant results in all downstream analyses. To account for species-specific differences in non-uniquely mapping (NU) reads, we redistributed NU reads based on the number of uniquely mapping (U) reads mapping to the respective loci, following a logic outlined before [START_REF] Brawand | The evolution of gene expression levels in mammalian organs[END_REF] . Multidimensional scaling analysis was carried out on normalized read counts using the 'edgeR' bioconductor package [START_REF] Robinson | edgeR: a Bioconductor package for differential expression analysis of digital gene expression data[END_REF] . Genes differentially expressed between early fore-and hindlimb samples were determined in 'edgeR', and genes showing consistent changes in mouse and Anolis were excluded from further analyses, to dampen potential anterior-posterior differences between the organs. For hierarchical clustering, correlation coefficient and principal component analyses, we calculated 'transcripts per million' (TPM) [START_REF] Wagner | Measurement of mRNA abundance using RNA-seq data: RPKM measure is inconsistent among samples[END_REF] values, which were then log2-transformed. Hierarchical clustering was done using the 'pvclust' R package [START_REF] Suzuki | Pvclust: an R package for assessing the uncertainty in hierarchical clustering[END_REF] , with 1000 iterations of multi-scale bootstrap re-sampling, and approximately unbiased (AU) p-values are provided in the graph. Heat maps of correlation coefficients were plotted with the 'lattice' R package. Principal component analysis was done with the 'prcomp' function in the 'stats' R package and 'GOseq' [START_REF] Young | Gene ontology analysis for RNA-seq: accounting for selection bias[END_REF] was used for GO-term enrichment analysis. Pair-wise differential expression analysis between early and late budding stages, in mouse and Anolis limbs and genitalia, was done in 'edgeR', and Venn diagram of genes with an absolute log(fold-change) > 1.5 and p-value < 0.05 was visualized using 'VennDiagram' [START_REF] Chen | VennDiagram: a package for the generation of highly-customizable Venn and Euler diagrams in R[END_REF] .

Grafting experiments

For heteroptopic, homochronic cloacal grafts, donor and recipient embryos were incubated to reach stage HH17-20. Donor embryos were either GFP-transgenic chicken [START_REF] Mcgrew | Localised axial progenitor cell populations in the avian tailbud are not committed to a posterior Hox identity[END_REF] , purchased from Clemson University, or quail, purchased from Strickland GameBird Farm. Cloacas were dissected in ice-cold PBS and grafted using tungsten needles to a proximal-ventral position, to mimic the squamate configuration, and removed from the apical ectodermal ridge to avoid Shh-induced digit duplications [START_REF] Perriton | Sonic hedgehog signaling from the urethral epithelium controls external genital development[END_REF] , or the tailbud in wild-type recipient chicken. Successful grafts were incubated for 1-3 additional days, dissected and screened for the appearance of ectopic outgrowths. Donor versus recipient tissue was discriminated using either GFP or QCPN antibody staining on cryo-sections, or GFP fluorescence for wholemount embryos. Sham surgery or grafting of GFP-positive limb mesenchyme did not cause any comparable outgrowths. Cloaca-induced outgrowths never stained positive for Alcian blue at later stages, indicating that they were not digit duplications (data not shown). For bead experiments, Affi-Gel Blue Gel beads (150-300μm; Bio-Rad) were washed in PBS and incubated for 1-2h at room temperature, in PBS with recombinant proteins (SHH, FGF2, FGF8; all R&D Systems) at concentrations of 0.1-1μg/μl. Soaked beads were briefly washed in PBS and grafted to limb and tail buds, as outlined for the cloacal grafts. Control grafts using beads soaked in PBS with bovine serum albumin (BSA) did not yield any observable outgrowths.

Extended Data

Extended Data Figure 1.

Two separable ventral cell populations give rise to the murine genital tubercle. a,b) Injection into the most distal ventral part of the embryo, the tailbud, marks cells posterior/ventral to the phallic part of the urethra (a, arrow; n=7), whereas injection closer to the allantois, into the infra-umbilical mesenchyme, labels cells anterior/dorsal to the phallic part of the urethra (b, arrow; n=4). Cells lining the peritoneal cavity are also being marked (arrowhead), due to accidental piercing of the coelom. Scale bars, 200 μm. gt: genital tubercle, ur: urethra.

Extended Data Figure 2.

The squamate hemipenis mesenchyme initiates with limb-like cellular dynamics from the coelomic epithelium through a epithelial-to-mesenchymal transition (EMT) a) Injection of GFP-expressing lentiviruses into the coelom of chicken embryos at HH14 labels cells emerging from the epithelium that contribute to the hindlimb mesenchyme (arrowhead). b) In lizards, labeled cells leaving the coelomic epithelium contribute to the hemipenis mesenchyme (arrowheads). c) Dorsal view of the hindlimb region of an E10.0 mouse embryo. d) Transversal section of a limb bud, showing EMT of the coelomic epithelium (diffuse Laminin staining, empty arrowhead), as cells contribute to the limb-bud mesenchyme. e) Dorsal view of the budding hemipenis of a snake embryo, one day after egg deposition. f) Transversal section of the hemipenis region. The basement membrane of the coelomic epithelium is breaking down (empty arrowhead), while it is intact for both the nephric duct and the surface ectoderm (arrowheads). g-o) Expression of genitalia and limb genes during hemipenis initation. g-i) Tbx4 is expressed early and late during hemipenis initiation, in both the coelomic epithelium (arrowhead) and the hemipenis mesenchyme (arrow). j-l) Tbx5 is only expressed later, in the mesenchyme (arrow), but is absent from the coelomic epithelium (empty arrowheads). m-o) Limb marker gene Lhx9 (see also Fig. 4e) is absent from both epithelium (empty arrowhead) and mesenchyme (empty arrow), but can de detected in dI1 neurons (asterisk). All gene expression assessed in at least n=3. Scale bars, 50 μm. co: coelom; lb: limb; hp: hemipenis; nd: nephric duct; cl: cloaca. Comparative marker gene expression analysis in mouse and squamate embryos. Genitalia markers Isl1 (a-d), Runx1 (e,f), Gata2 (g-j), Eya (k,l), Tbx5 (m-p) and Dkk2 (q,r). Gata2 only becomes visibly expressed at later stages of house snake hemipenis development (j, inset). Limb markers Lhx9 (s-v), Tbx18 (w,x) and Lmx1b (y-zi).

Extended Data Figure 3 .Extended Data Figure 4 .Extended Data Figure 5 .

 345 Heat maps of Pearson's and Spearman's rank correlation coefficients and cluster analysis of whole transcriptome data. a,b) Hierarchical clustering on pairwise correlation coefficients, for whole transcriptome data of anole (a) and mouse (b) samples. Numbers at nodes represent approximately unbiased p-values obtained by multiscale bootstrap resampling. Sample identifiers; a: anole; m: mouse; LB: limb; HP: hemipenis; GT: genital tubercle; e: early; l: late. Heat maps of Pearson's and Spearman's rank correlation coefficients and cluster analysis of transcription factor and signaling pathway data. a,b) Hierarchical clustering on pairwise correlation coefficients, of transcription factor and signaling pathway data of anole (a) and mouse (b) samples. Numbers at nodes represent approximately unbiased p-values obtained by multiscale bootstrap resampling. Sample identifiers; a: anole; m: mouse; LB: limb; HP: hemipenis; GT: genital tubercle; e: early; l: late. Heterotopic grafting of the cloacal signaling center leads to ectopic outgrowths and genitalia-like transcriptional changes. a-c) Schematics and close-up images of the cloacal grafting procedure. a) The cloaca of a stage HH17-19 GFP-transgenic chicken embryo (red rectangle) is transplanted into the proximal-ventral portion of the limb of a wild-type embryo. b,c) Only the ventral-most part of the cloaca, including the cloacal membrane, is dissected out (b, red box), and subsequently cleared of excess mesenchymal cells attached to the Shh-expressing endoderm (c, red outline). d-g) Grafting of beads soaked in Shh and Fgf can induce ectopic outgrowths on both limbs (e, n=6/48) and tail (g, n=3/31). h-k) Ectopic expression of genital markers Gata2 (h,i; arrowheads) and Runx1 (j,k; arrowhead) in limb buds, following cloaca-to-limb grafts. l-n) Ectopic expression of genital marker Gata2 (m; arrowheads) and Runx1 (n; arrowheads) in the tail region, following cloaca-to-tail grafts. All gene expression assessed in at least n=3. Scale bar, 200 μm. lb: limb; cl: cloaca; al: allantois. Extended Data Figure 6. Pairwise differential expression analysis of limb and genitalia transcriptomes. a-d) Smear plot visualization of differential expression analyses of early anole (a), late anole (b), early mouse (c) and late mouse (d) limb versus genitalia transcriptomes. Genes used for the Venn diagram in Fig. 4d (|log2(fold-change)| > 1.5; p-value < 0.05) are highlighted in red, core 25 marker genes (see Fig. 4e and text) are highlighted and labeled in blue. e,f) Heat map of Zscore normalized expression values for all genes fulfilling Venn diagram criteria (n=2003), for anole (e) and mouse (f) data. Row-based hierarchical clustering was employed; core 25 marker genes are indicated on the right. Extended Data Figure 7.

Extended Data Figure 8 .

 8 All gene expression assessed in at least n=3. Scale bar, 200 μm. lb: limb; cl: cloaca; gt: genital tubercle; hp: hemipenis. Pairwise differential expression analysis of tailbud and genitalia transcriptomes. a,b) Smear plot visualization of differential expression analyses of early anole (a) and early mouse (b) tailbud versus genitalia transcriptomes. Genes used as input for the Venn diagram in (c) (| log2(fold-change)| > 1.5; p-value < 0.05) are highlighted in red, overlapping 257 marker genes are highlighted in blue. Top 25 genes in the two species, based on logCPM and logFC, are labeled. c) Venn diagram showing overlap of pairwise differential expression analysis results (log(fold change) > 1.5, p-value < 0.05) of tailbud versus genital tissues, for early budding stages in both anole and mouse.

Figure 1 .

 1 Figure 1. A relative positional shift of limbs, genitalia and the cloaca in squamates. μCT-scans of mouse (a), anole (b), python (c) and house snake (d) lumbo-sacral regions (highlighted in sketch in white) at embryonic stages, illustrating the position of the developing external genitalia. (e-h) 3D-reconstructions of cloacal volumes. The cloaca is located at the same anterior-posterior position as the limb in squamates (f-h), however, is positioned more posteriorly in the mouse (e). (i-l) Transversal sections stained for β-catenin and Shh, indicating the conservation of a cloacal signaling center in all four species. Scale bars, 200 μm. gt: genital tubercle; hp: hemipenis; lb: limb; cl: cloaca.

Figure 2 .

 2 Figure 2. Differential developmental origins of external genitalia in amniotes. (a-i) Transversal and sagittal views of GFP lentivirus injected embryos. Relative contribution of GFP-positive cells to respective organs is quantified on the right, normalized on tissue area. Error bars represent standard deviation in at least n=4 biological replicates. (a,b) Injection into the coelom at mouse E9.5 (n=48) labels the limb at E13.5, but excludes the genital tubercle (arrows). Only cells lining the peritoneal cavity are labeled (b, arrowhead), but none in the genital tubercle proper. c) Injection into the tailbud (n=101) labels cells in the genital tubercle. Accidental piercing of the coelom labels cells of the peritoneal cavity (arrowhead). (d,e) Coelom injection in HH14 chicken embryos (n=81) labels the limb and the genital tubercle at HH30. e) Sagittal and transversal close-up (inset) views. f) Sagittal and transversal close-up (inset) views of tailbud injected chick embryos (n=77), showing labeling in the genital tubercle. (g,h) Anole embryos injected into the coelom at stage 2-3 (n=94) show GFP labeling of both limb and hemipenis at stage 6-7. i) No hemipenis cells are labeled following tailbud injection (n=57), even though there are GFP-positive cells in the tail (inset, arrowhead). Scale bars, 200 μm, 50 μm (h,i). lb: limb; gt: genital tubercle; gf: genital fold; hp: hemipenis; tl: tail.

Figure 3 .

 3 Figure 3. Molecular architecture of limbs and external genitalia in lizards and mice. a) Microdissected tissues for RNA-seq analysis, highlighted by color code and four-letter sample identifier. Early and late limb-and genitalia-buds were analyzed, for anole lizard and mouse embryos (n=2). b) Multidimensional scaling (MDS) analysis reveals greater overall transcriptome similarities in anole limb and genitalia datasets (triangles) than in their mouse counterparts (circles). (c,d) Hierarchical clustering of pairwise Pearson's correlation coefficients, for whole transcriptome data of anole (c) and mouse (d) samples. Additional datasets are stage 2-3 anole and E9.5 mouse forelimb (turquoise) and tailbud (yellow). Numbers at nodes represent approximately unbiased p-values, obtained by multiscale bootstrap resampling. e) Principal component analysis (PCA). Species transcriptomes separate along principal component 1 (PC1), whereas organs are resolved along PC2. f)Absolute loading values for PC2, as shown in (e). g) GO term enrichment analysis using the top500 genes (red bar, f). Top hits include transcription factor-and signaling pathwayrelated terms. (h,i) Hierarchical clustering analysis of pairwise Pearson's correlation coefficients, for transcription factors and signaling pathways data of anole (h) and mouse (i) samples. Sample identifiers; a: anole; m: mouse; LB: limb; HP: hemipenis; GT: genital tubercle; e: early; l: late.

Figure 4 .

 4 Figure 4. The cloacal signaling center can recruit different mesenchymal cell populations for the outgrowth of external genitalia. a) Schematic of the hindlimb grafting procedure in chicken embryos. GFP-transgenic cloacae are transplanted into the proximal-ventral portion wildtype hindlimbs. b,c) Ectopic outgrowth (arrowheads) on limbs with cloacal grafts (n=30/118). d) Venn diagram of pairwise differential expression analysis results (log(fold change) > 1.5, p-value < 0.05) of limbs versus genital tissues, for early and late budding stages in anole and mouse. e) Heat map of Z-score normalized values of core 25 genes showing consistent differential expression between limbs and genitalia. Mouse row-based hierarchical clustering was re-used for anole samples. f-h) Analysis of limb-and genitalspecific markers. Expression of limb markers Lhx9 (f) and Tbx18 (g) is down-regulated near the GFP-positive cloacal graft (empty arrowheads), while genital marker Isl1 (h) is expressed ectopically (arrowhead). i) Schematic of the tail bud grafting procedure in chicken embryos. GFP-transgenic cloacae are transplanted into ventral wild-type tailbuds. j) Ectopic outgrowth on tails with cloacal grafts (n=16/87). k) Genital marker Isl1 is up-regulated ectopically in the tail, close to the GFP-positive cloacal graft (arrowhead). All gene expression assessed in at least n=3. Scale bars, 200 μm.

Nature. Author manuscript; available in PMC 2015 June 18.

Acknowledgements

The authors thank D. Duboule, H. Kaessmann, A. Necsulea, B. Okaty, G. Rey and G.P. Wagner for discussions, M.A. de Bakker for the snake Tbx5 probe and A.M. Herrera and M.J. Cohn for discussing and sharing unpublished results. μCT scans were performed at the Center for Nanoscale Systems, Harvard University (supported by NSF award ECS-0335765) and at the Museum of Comparative Zoology. Next-Generation Sequencing was performed at the HMS Biopolymers Facility and computational analyses were run on the Orchestra Cluster, HMS Research Computing. P.T. was supported by post-doctoral fellowships from the Swiss National Science Foundation, EMBO and the Human Frontiers Science Program. A.C.G. was supported by a post-doctoral fellowship from the Swiss National Science Foundation. This work was supported by NIH grant R37-HD032443 to C.J.T.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.


Khaled Fadhlaoui

Ben Wagdi

Fabrice Hania

Manon Armougom

Marie-Laure Joseph

Gaël Fardeau

Gael Erauso

Corinne Brasseur

Moktar Aubert

Céline Hamdi

Brochier-Armanet

Wagdi Ben Hania

Fabrice Armougom

Manon Bartoli

Marie-Laure Fardeau

Ga € El Erauso

Ga € El Brasseur

Corinne Aubert

Moktar Hamdi

C Eline Brochier-Armanet

Alain Dolla
email: dolla@imm.cnrs.fr

Bernard Ollivier
email: bernard.ollivier@mio.osupytheas.fr

Obligate sugar oxidation in Mesotoga spp., phylum Thermotogae, in the presence of either elemental sulfur or hydrogenotrophic sulfate-reducers as electron acceptor

Introduction

For a long time, cultivated members of Thermotogae, a deep-branching phylum within Bacteria [START_REF] Huber | Thermotogales. In The Prokaryotes[END_REF][START_REF] Cappelletti | Members of the order Thermotogales: from microbiology to hydrogen production[END_REF], were known to be essentially thermophilic to hyperthermophilic. This situation changed when 16S rRNA gene sequences were reported in many mesothermic environments, suggesting that mesophilic (Mesotoga) Thermotogae exist [START_REF] Nesbø | Evidence for existence of "mesotogas", members of the order Thermotogales adapted to low-temperature environments[END_REF]. The first mesophilic representative of Thermotogae (Mesotoga strain PhosAc3) was cultivated in 2011 [START_REF] Ben Hania | Cultivation of the first mesophilic representative ("mesotoga") within the order Thermotogales[END_REF]. Thereafter, two Mesotoga species, M. prima strain MesG1.Ag.4.2 T [START_REF] Nesbø | Mesotoga prima gen. nov., sp. nov., the first described mesophilic species of the Thermotogales[END_REF] and M. infera strain VNs100 T (Ben Hania et al., 2013) were characterized while Mesotoga strain PhosAc3 was recently recognized as a M. prima strain [START_REF] Ben Hania | Draft genome sequence of Mesotoga strain PhosAC3, a mesophilic member of the bacterial order Thermotogales, isolated from a digestor treating phosphogypsum in Tunisia[END_REF]. Phylogenetic analyses of 16S rRNA genes revealed that several distinct uncultured lineages of mesophilic Thermotogae may exist and thus that adaptation to mesothermic environments occurred several times independently during the diversification of Thermotogae [START_REF] Nesbø | Searching for mesophilic Thermotogales bacteria: "mesotogas" in the wild[END_REF][START_REF] Ben Hania | Cultivation of the first mesophilic representative ("mesotoga") within the order Thermotogales[END_REF]. Therefore, Mesotoga spp. are of noticeable interest to understand bacterial evolution from thermophily to mesophily. To date, beside the genus Mesotoga, Thermotogae comprise 12 other genera including Athalassotoga, Defluviitoga, Fervidobacterium, Geotoga, Kosmotoga, Marinitoga, Mesoaciditoga, Oceanotoga, Petrotoga, Pseudothermotoga, Thermosipho and Thermotoga [START_REF] Di Pippo | Kosmotoga olearia gen. nov., sp. nov., a thermophilic, anaerobic heterotroph isolated from an oil production fluid[END_REF][START_REF] Jayasinghearachchi | Oceanotoga teriensis gen. nov., sp. nov., a thermophilic, bacterium isolated from off-shore oil-producing wells[END_REF][START_REF] Ben Hania | Defluviitoga tunisiensis gen. nov., sp. nov., a thermophilic bacterium isolated from a mesothermic and anaerobic whey digester[END_REF][START_REF] Reysenbach | Mesoaciditoga lauensis gen. nov., sp. nov., a moderately thermoacidophilic member of the order Thermotogales from a deepsea hydrothermal vent[END_REF][START_REF] Bhandari | Molecular signatures for the phylum (class) Thermotogae and a proposal for its division into three orders (Thermotogales, Kosmotogales ord. nov. and Petrotogales ord. nov.) containing four families (Thermotogaceae, Fervidobacteriaceae fam. nov., Kosmotogaceae fam. nov. and Petrotogaceae fam. nov.) and a new genus Pseudothermotoga gen. nov. with five new combinations[END_REF][START_REF] Cappelletti | Members of the order Thermotogales: from microbiology to hydrogen production[END_REF][START_REF] Itoh | Athalassotoga saccharophila gen. nov. sp. nov. isolated from an acidic terrestrial hot spring of Japan, and proposal of Mesoaciditogales ord. nov., Mesoaciditogaceae fam. nov. in the phylum Thermotogae[END_REF]. They all share an outer sheath-like structure called a 'toga' ballooning over the ends of the cell (e.g. Thermotoga and Thermosipho spp.) [START_REF] Huber | Thermotoga maritima sp. nov. represents a new genus of unique extremely thermophilic eubacteria growing up to 908C[END_REF][START_REF] Antoine | Thermosipho Obligate sugar oxidation by Mesotoga spp[END_REF]. Members of the phylum Thermotogae including the orders Thermotogales, Kosmotogales, Petrotogales and Mesoaciditogales [START_REF] Bhandari | Molecular signatures for the phylum (class) Thermotogae and a proposal for its division into three orders (Thermotogales, Kosmotogales ord. nov. and Petrotogales ord. nov.) containing four families (Thermotogaceae, Fervidobacteriaceae fam. nov., Kosmotogaceae fam. nov. and Petrotogaceae fam. nov.) and a new genus Pseudothermotoga gen. nov. with five new combinations[END_REF][START_REF] Itoh | Athalassotoga saccharophila gen. nov. sp. nov. isolated from an acidic terrestrial hot spring of Japan, and proposal of Mesoaciditogales ord. nov., Mesoaciditogaceae fam. nov. in the phylum Thermotogae[END_REF] are usually considered as heterotrophic fermentative microorganisms able to use sugars, polysaccharides or complex organic substrates such as peptone and yeast extract. However, in contrast to all Thermotogae and to M. prima strain MesG1.Ag.4.2 T in particular, and despite a very close phylogenetic relatedness with the latter, M. prima strain PhosAc3 and M. infera strain VNs100 T are able to use sugars only in the presence of elemental sulfur as terminal electron acceptor leading to the production of acetate, CO 2 and sulfide (around 2 moles of acetate and 4 moles of sulfide produced per mole of glucose consumed) with no or only traces of hydrogen production (less than 1 mM) [START_REF] Ben Hania | Mesotoga infera sp. nov., a novel mesophilic member of the order Thermotogales, isolated from an underground gas storage in France[END_REF]2015;[START_REF] Cappelletti | Members of the order Thermotogales: from microbiology to hydrogen production[END_REF]. This sulfur-dependent metabolism discovered in these two closely related Mesotoga species does not concern exclusively sugars but also applies to other substrates tested (e.g. lactate and pyruvate) [START_REF] Ben Hania | Cultivation of the first mesophilic representative ("mesotoga") within the order Thermotogales[END_REF]2015) clearly contrasting with typical metabolic features of Thermotogae well known as efficient H 2producing bacteria (over 1 mM) [START_REF] Cappelletti | Members of the order Thermotogales: from microbiology to hydrogen production[END_REF]. However, determining if sulfur reduction is an ATPyielding reaction in Mesotoga spp. has not been established so far.

Here we show that co-culturing M. prima strain PhosAc3 with hydrogenotrophic sulfate-reducing partners (Desulfovibrio and Desulfotomaculum spp.) in the presence of sulfate as terminal electron acceptors significantly improved glucose oxidation by the former. Such metabolic process was extended to other Mesotoga spp. isolated so far including both M. prima strain MesG1.Ag.4.2 T and M. infera strain VNs100 T . We demonstrate two major points regarding Mesotoga species: (i) their dependence on the presence of elemental sulfur as terminal electron acceptor to use sugar, thus indicating that sugars are oxidized rather than fermented and (ii) the efficient syntrophic association with hydrogenotrophic partners able to replace more efficiently elemental sulfur as biological electron acceptor for sugar oxidation. Our data point out an unusual prokaryotic metabolism within the Thermotogae, the syntrophic association with a hydrogenotrophic partner in the absence of elemental sulfur to degrade easily fermentable substrates such as carbohydrates and highlight its possible ecological significance in nature.

Results

Growth of Mesotoga spp. in pure culture and in co-culture with either sulfate-reducing bacteria or methanogens When M. prima strain PhosAc3 was cultured with glucose as energy source, only a slight glucose consumption was observed even after 250 days of incubation at 378C only (Fig. 1A, Table 1). When elemental sulfur was added in the medium, a slow linear degradation of glucose was measured with 6.57 6 0.19 mM of the glucose consumed (around 33%) after 250 days of incubation (Fig. 1A, Table 1). Accordingly, only a slight acetate production was measured in the absence of elemental sulfur while 9.21 6 0.13 mM acetate was produced in its presence after 250 days. The only detected end-products of glucose metabolism were acetate (Fig. 1B), CO 2 and sulfide (Table 1). No other volatile fatty acid (e.g. formate, butyrate, etc.) nor organic compound (e.g. lactate, ethanol) was detected in any growth conditions. Surprisingly, only trace amount of hydrogen (around 1 mM) were detected in the gas phase during glucose consumption whatever the presence or the absence of elemental sulfur.

These data clearly showed that M. prima strain PhosAc3 was unable to ferment glucose while, in the presence of an external electron acceptor (elemental sulfur), it was able to oxidize it although with a low efficiency. The slight glucose consumption in the absence of elemental sulfur was probably due to the presence of an electron acceptor available in yeast extract since hydrogen was detected only in minor quantities (around 1 mM) in the gas phase.

Because Mesotoga species are often detected in environments where sulfate-reducing bacteria are present [START_REF] Nesbø | Searching for mesophilic Thermotogales bacteria: "mesotogas" in the wild[END_REF], the capability of M. prima strain PhosAc3 to grow in syntrophic association with a sulfatereducing bacterium (SRB), Desulfovibrio vulgaris subsp. vulgaris, was tested. As with many other SRB, D. vulgaris gains energy for biosynthesis and growth by coupling oxidation of organic compounds or molecular hydrogen to reduction of sulfate to sulfide [START_REF] Muyzer | The ecology and biotechnology of sulphate-reducing bacteria[END_REF].

When either M. prima strain PhosAc3 or D. vulgaris subsp. vulgaris alone was cultured in a glucose/sulfate medium, no growth was observed (Fig. 2A), confirming that this Desulfovibrio species is unable to metabolize sugar. However, when the two bacteria were co-cultured, growth occurred as evidenced by a substantial increase of the optical density (OD) at 580 nm (Fig. 2A). After only 20 days of incubation, 80% of the initial glucose was consumed (12 mM) with the concomitant acetate production of 25 mM (Fig. 1A andB). The rate of glucose consumption was 0.8 mM glucose consumed/day. After 250 days of incubation, glucose consumption and acetate production only slightly progressed to give 13 mM glucose consumed and 28 mM acetate produced (Fig. 1, Table 1). At this time, around 10 mM sulfide had been produced (Table 1). Electron recovery values were 77% and 85% when M. prima strain PhosAc3 was grown in the presence of elemental sulfur and D. vulgaris as electron acceptors respectively. These values are in agreement with those expected for an anaerobic sugar-oxidizing metabolism leading to acetate as the only produced fatty acid, where about 20% of the sugar-derived reducing equivalents have been shown to be incorporated into the cells [START_REF] Thauer | Energy conservation in chemotrophic anaerobic bacteria[END_REF][START_REF] Cord-Ruwish | Fructose degradation by Desulfovibrio sp. in pure culture and in coculture with Methanospirillum hungatei[END_REF]. In contrast, when M. prima strain PhosAc3 was grown in the absence of elemental sulfur, electron recovery was lower (60%). In this case however, minor amounts of glucose used by M. prima strain PhosAc3 made this value less accurate (Table 1). When another SRB, Desulfotomaculum gibsoniae was used instead of D. vulgaris in the co-culture, a similar growth was obtained (data not shown). M. infera strain VNs100 T which was already reported to use elemental sulfur as electron acceptor for oxidizing glucose [START_REF] Ben Hania | Mesotoga infera sp. nov., a novel mesophilic member of the order Thermotogales, isolated from an underground gas storage in France[END_REF] was also co-cultured successfully with D. vulgaris subsp. vulgaris as confirmed by the increase of the OD at 580 nm (from 0.08 to 0.19) after 23 days of incubation.

The same kind of experiment was performed with the marine Mesotoga genus member, M. prima strain MesG1.Ag.4.2 T . However, as glucose was reported to be weakly used by this bacterium, it was replaced by fructose [START_REF] Nesbø | Mesotoga prima gen. nov., sp. nov., the first described mesophilic species of the Thermotogales[END_REF]. Similarly to M. prima strain PhosAc3, no growth was observed in fructose/sulfate medium and only a poor growth was obtained in the presence of S8 (Fig. 2B). When M. prima strain MesG1.Ag.4.2 T was cocultured with Desulfovibrio salexigens which was isolated from a marine environment as well, the OD at 580 nm increased during the first 9 days to reach around 0.4 OD 580nm unit, thus demonstrating the establishment of the co-culture when growing on fructose. At that time, around 13 mM acetate and 14 mM H 2 S were produced (Table 1). A lower amount of acetate was produced in mono-culture in the presence of S8, in agreement with a lower growth under this condition (Table 1, Fig. 2B). Whatever the growth condition, the measured hydrogen concentration in the gas phase was around 1 mM.

To ensure that both M. prima strain PhosAc3 and D. vulgaris subsp. vulgaris actually grew under co-culture conditions, each cell-type was counted by epifluorescence microscopy after DAPI staining. This was made possible since the two bacteria have distinct cell morphology, easily recognizable: small vibrios for D. vulgaris and pleomorphic rods surrounded with the sheath-like structure known as toga for M. prima strain PhosAc3 (Fig. 3). It revealed that four and five times more M. prima strain PhosAc3 and D. vulgaris cells, respectively, were present after 10 days of incubation in comparison to the inoculation time (time 0). At time 0, a Mesotoga/Desulfovibrio cell number ratio of 5.16 6 1.06 was counted. This ratio changed very little during the growth time (5.54 6 1.11, 3.97 6 0.5 and 3.72 6 0.91 after 5, 10 and 20 days of incubation respectively), showing that in a well-established co-culture, Mesotoga cells were about four times more abundant than Desulfovibrio cells.

Our data contrast with that previously obtained on M. prima strain MesG1.Ag4.2 T which has been described as a sugar fermentative bacterium producing acetate as major soluble fermentation product [START_REF] Zhaxybayeva | On the chimeric nature, thermophilic origin, and phylogenetic placement of the Thermotogales[END_REF][START_REF] Nesbø | Mesotoga prima gen. nov., sp. nov., the first described mesophilic species of the Thermotogales[END_REF]. However, the authors did not provide any information on hydrogen production from sugars but noticed a slight stimulation of growth in the presence of thiosulfate, sulfite or elemental sulfur. In addition, they used a high concentration of yeast extract (5 g l 21 ) in their culture media [START_REF] Nesbø | Mesotoga prima gen. nov., sp. nov., the first described mesophilic species of the Thermotogales[END_REF]. Here, we show that M. prima strain PhosAc3 and M. prima strain MesG1.Ag4.2 T are able to degrade sugar albeit poorly, in the presence of yeast extract (1 g l 21 ) and in the absence of elemental sulfur (Table 1). However, due to the production of only traces of hydrogen in such conditions, we can assume that an unknown mineral or organic compound present in the yeast extract serves as electron acceptor as already reported for the sulfur-respiring archaeon Pyrococcus woesei [START_REF] Zillig | Pyrococcus woesei, sp. nov., an ultra-thermophilic marine archaebacterium, representing a novel order, Thermococcales[END_REF]. Under the growth condition used for the co-cultures (sugar/sulfate), Desulfovibrio spp. were unable to grow. This indicated that, in the co-cultures, Desulfovibrio spp. required metabolite(s) or end-product(s) of sugar metabolism released by Mesotoga strains. To test this hypothesis and to check whether cell-to-cell contacts were required, M. prima strain PhosAc3 and D. vulgaris subsp. vulgaris were co-cultured using a special set-up where the two bacteria were grown into two compartments separated with a dialysis membrane (Supporting Information Fig. S1). Under these conditions, after 18 days of incubation at 378C, the OD 580nm increased from 0.02 6 0.01 to 0.31 6 0.03 in the M. prima compartment and from 0.07 6 0.01 to 0.34 6 0.03 in the D. vulgaris compartment. This clearly indicated that in co-cultures, both bacteria species were able to grow without the requirement of any cellto-cell contact between them and suggested that Mesotoga and Desulfovibrio exchanged metabolites, required for their respective growth, able to diffuse across a dialysis membrane. Several attempts to co-culture Mesotoga with hydrogenotrophic methanogens as H 2 scavenger (Methanospirillum hungatei, Methanobacterium congolense or Methanobacterium aarhusense) were only successful using M. hungatei; after 67 days of incubation at 378C, increase of the OD 580nm (0.135 6 0.03) and microscopic observations (data not shown) indicated a growth of both microorganisms while no growth was observed when only one partner was present in the culture medium.

All the data presented above suggested that M. prima strain PhosAc3 and D. vulgaris growths were tightly coupled through a mutualistic/syntrophic association. Several examples of syntrophic associations involving the exchange of hydrogen or formate between the partners have been documented [START_REF] Stams | Electron transfer in syntrophic communities of anaerobic bacteria and archaea[END_REF]. To test the involvement of hydrogen as a possible metabolic intermediate, Desulfovibrio and Mesotoga were cultured in two separate serum bottles whose headspaces communicated via tubing (Supporting Information Fig. S1), allowing only gas to be exchanged. After 24 days of incubation at 378C, the OD 580nm increased from 0.02 6 0.01 to 0.24 6 0.03 in the M. prima compartment, and from 0.06 6 0.01 to 0.22 6 0.02 in the D. vulgaris compartment. These results indicated that (a) gaseous substance(s) exchanged between the two partners permitted syntrophic growth between Mesotoga and Desulfovibrio. Given the previous examples of syntrophic associations, our knowledge of the metabolism of M. prima and D. vulgaris and the fact that we did detect only traces of H 2 but not that of formate, we suggest that the best candidate as exchanged metabolite was hydrogen. As hydrogen metabolism requires hydrogenases, hydrogenase activity and glucose-dependent H 2 production from M. prima strain PhosAc3 cells were thus tested.

Hydrogenase activity and hydrogen production in M. prima strain PhosAc3

Glucose-dependent hydrogen production by M. prima strain PhosAc3, was evaluated using a Clark-type hydrogen microsensor on concentrated cells from either pure culture in the presence of elemental sulfur or co-culture with D. vulgaris in dialysis membrane-separated compartments (Fig. 4). A hydrogen production of 1.13 6 0.25 nmol H 2 /min/mg protein using glucose as substrate was measured by M. prima strain PhosAc3 when co-cultured with D. vulgaris while only 0.42 6 0.06 nmol H 2 /min/mg protein were produced when it was cultured alone in the presence of S8.

Spectrophotometric hydrogenase activity assays using methyl-viologen revealed hydrogenase activities of 33 6 3 nmol H 2 produced/min/mg total protein and 5920 6 930 nmol H 2 consumed/min/mg total protein when M. prima strain PhosAc3 was co-cultured with D. vulgaris while no hydrogenase activity could be detected when it was cultured alone in the presence of elemental sulfur. These data showed that hydrogen production and hydrogenase activity were higher when M. prima was cultured in the presence of D. vulgaris than in its absence, suggesting that M. prima was able to regulate the amount of hydrogenase in the cells, depending on the presence of a H 2 scavenger such as D. vulgaris.

Overall, these experimental data clearly showed that M. prima strain PhosAc3 was able to oxidize efficiently glucose through an interspecies H 2 transfer with D. vulgaris, Hydrogen evolution was measured on Mesotoga whole washed cells grown either in dialysis tubing co-cultured with Desulfovibrio vulgaris subsp. vulgaris in glucose/sulfate medium (black line) or grown in pure culture on glucose in the presence of elemental sulfur (grey line). Experiments were carried out in a respirometry chamber (1.9 ml) in Tris-HCl 0.1M NaCl 0.15M buffer (pH 7.5) at 378C using a specific H 2 microsensor. Addition of cells (1.6 mg total protein for both samples) and glucose (50 mM final concentration) are indicated by arrows. the latter acting as a hydrogen scavenger. It is thus capable of syntrophic association with Desulfovibrio that leads to a more efficient growth, as evidenced by a significant improvement of biomass production and growth rate, than when cultured alone in the presence of elemental sulfur as terminal electron acceptor.

Mesotoga spp. genome mining regarding hydrogen production and sugar oxidation

Because hydrogen metabolism is a key pathway for this syntrophic association, the occurrence of genes encoding hydrogenases in the M. prima strain PhosAc3 genome was explored. No blast hits were found when using [NiFe]hydrogenase sequences as input (data not shown), while one [FeFe]-hydrogenase homologue was detected in M. prima strain PhosAc3 (MESO_PHOSAC3V1_90124). Close homologues were found in the two other Mesotoga members (i.e. M. prima strain MesG1.Ag4.2 T (Theba_0443) and M. infera strain VNs100 (MESINFAv2 2088)). The phylogenetic analysis of Thermotogae [FeFe]hydrogenases showed that the sequences of Mesotoga branched specifically with the ferredoxin-dependent [FeFe]-hydrogenase TM0201 of Thermotoga maritima strain MSB8 (Supporting Information Fig. S2). Interestingly, no homologue of the bifurcating trimeric [FeFe]hydrogenase (TM1424-1426) from T. maritima strain MSB8 [START_REF] Schut | The iron-hydrogenase of Thermotoga maritima utilizes ferredoxin and NADH synergistically: a new perspective on anaerobic hydrogen production[END_REF] was found in any Mesotoga strains so far sequenced (Table 2). Bifurcating [FeFe]hydrogenases play an important function in the link between the carbohydrates oxidation via a classical Embden-Meyerhof pathway and the production of hydrogen in T. maritima by using the exergonic oxidation of ferredoxin to drive the endergonic oxidation of NADH to produce H 2 [START_REF] Schut | The iron-hydrogenase of Thermotoga maritima utilizes ferredoxin and NADH synergistically: a new perspective on anaerobic hydrogen production[END_REF][START_REF] Buckel | Energy conservation via electron bifurcating ferredoxin reduction and proton/Na1 translocating ferredoxin oxidation[END_REF]. It thus provides a mechanism to regenerate NAD from NADH by producing H 2 , even if this reaction is thermodynamically unfavourable. In contrast with T. maritima, M. prima strain PhosAc3 is unable to ferment glucose; we propose that this metabolic difference is due to the absence of the bifurcating [FeFe]-hydrogenase in M. prima preventing the efficient re-oxidation by this enzyme of NADH generated by the NAD-dependent glyceraldehyde 3-phosphate dehydrogenase during glucose oxidation.

A search in M. prima strain PhosAc3 genome revealed the presence of two candidates for NADH re-oxidation: the Na 1 -translocating NADH-quinone oxidoreductase (Na 1 -NQR) complex (MESO_PHOSAC3V1_90501-MES-O_PHOSAC3V1_90504) and the type 2 Rnf complex (MESO_PHOSAC3V1_120288-MESO_PHOSAC3V1_ 120293). It has been proposed that both Na 1 -NQR and Rnf complexes would allow the re-oxidation of the NADH by transferring electrons to quinone and ferredoxin, coupled to Na 1 and Na 1 /H 1 translocation through the membrane respectively [START_REF] Hayashi | Recent progress in the Na1-translocating NADH-quinone reductase from the marine Vibrio alginolyticus[END_REF][START_REF] Biegel | Bacterial Na1-translocating ferredoxin: NAD1 oxidoreductase[END_REF][START_REF] Biegel | Biochemistry, evolution and physiological function of the Rnf complex, a novel ion-motive electron transport complex in prokaryotes[END_REF][START_REF] Barquera | The sodium pumping NADH: quinone oxidoreductase (Na1-NQR), a unique redox-driven ion pump[END_REF]. While the Rnf complex of type 2 is present in all Thermotogae, the Na 1 -NQR complex is absent in Kosmotoga olearia, Marinitoga piezophila and Thermotoga species (Table 2). Phylogenetic analysis of the genes encoding Rnf complex of type 2 indicated that this complex was probably present in the ancestor of Thermotogae (Supporting Information Fig. S3). In contrast, the Na 1 -NQR complex is patchy distributed in Thermotogae and the corresponding sequences appeared intermixed with sequences from Spirochaetes, Clostridia and Fusobacteria (Supporting Information Fig. S4), indicating that horizontal gene transfers (HGT) occurred among these lineages and that it could have been secondarily acquired in Thermotogae.

Sequences alignment of RnfB subunit from Thermotogae

Table 2. Enzymes occurrence in Thermotogae.

Bifurcating

[FeFe] hydrogenase Fd-dependent [FeFe] hydrogenase Na1-NQR complex Rnf complex Fervidobacterium nodosum Rt17-B1 1 1 1 1 /Short RnfB Fervidobacterium pennivorans DSM 9078 1 1 1 1 /Short RnfB Pseudothermotoga thermarum DSM 5069 1 1 1 1 /Short RnfB Pseudothermotoga lettingae TMO 1 1 1 1 /Short RnfB Thermosipho africanus TCF52B 1 1 1 1 /Short RnfB Thermosipho melanesiensis BI429 1 1 1 1 /Short RnfB Thermotoga neapolitana DSM 4359 1 1 2 1 /Short RnfB Thermotoga maritima MSB8 1 1 2 1 /Short RnfB Thermotoga petrophila RKU-1 1 1 2 1 /Short RnfB Thermotoga naphthophila RKU-10 1 1 2 1 /Short RnfB Marinitoga piezophila KA3 2 1 2 1 /Short RnfB Petrotoga mobilis SJ95 2 1 1 1 /Short RnfB Kosmotoga olearia TBF 19.5.1 2 1 2 1 /Long RnfB Mesotoga infera VNs100 2 1 1 1 /Long RnfB Mesotoga prima MesG1 Ag 4 2 2 1 1 1 /Long RnfB Mesotoga prima PhosAc3 2 1 1 1 /Long RnfB
revealed that the subunit from T. maritima was shorter than that from M. prima strain PhosAc3. This shorter sequence bound only one FeS cluster compared to at least two FeS clusters in the larger ones (Supporting Information Fig. S5). This characteristic was shared by all Thermotogae sequence so far, except members of the Mesotoga and Kosmotoga genera (Supporting Information Fig. S5). Interestingly, the presence of a shorter RnfB subunit correlated very well with the presence of a bifurcating [FeFe] hydrogenase in the organism, suggesting that this peculiar sequence feature could be involved in the specific interaction between the two proteins (Table 2).

Discussion

M. prima strain PhosAc3 was the first mesophilic cultivated member within the phylum Thermotogae [START_REF] Ben Hania | Cultivation of the first mesophilic representative ("mesotoga") within the order Thermotogales[END_REF] which comprised only fermentative thermophilic or hyperthermophilic bacteria [START_REF] Huber | Thermotogales. In The Prokaryotes[END_REF][START_REF] Zhaxybayeva | On the chimeric nature, thermophilic origin, and phylogenetic placement of the Thermotogales[END_REF]. Here we show that this bacterium is able to consume glucose only in the presence of elemental sulfur, thus clearly indicating that its sugar metabolism is based on an obligatory oxidative pathway, the elemental sulfur being essential to eliminate excess reducing equivalents generated from sugar oxidation. However, because M. prima strain PhosAc3 oxidizes very slowly sugar in the presence of elemental sulfur, one could expect that sulfur reduction is not a true respiratory process linked to oxidative phosphorylation. In this respect, elemental sulfur should serve as electron sink. Nevertheless, further experiments are needed to clarify this peculiar point regarding sulfur reduction. It is noteworthy that hydrogen was detected only as traces (around 1 mM in the gas phase) in any tested growth condition. The same type of sugar metabolism was demonstrated for M. prima strain MesG1.Ag.4.2 T and M. infera strain VNs100 T , the two other known members of the Mesotoga genus so far. All these data contrast with energy metabolism of thermophilic and hyperthermophilic Thermotogae known to (i) ferment sugars with substantial production of hydrogen (over 1 mM) [START_REF] Schut | The iron-hydrogenase of Thermotoga maritima utilizes ferredoxin and NADH synergistically: a new perspective on anaerobic hydrogen production[END_REF][START_REF] Cappelletti | Members of the order Thermotogales: from microbiology to hydrogen production[END_REF] and (ii) reduce elemental sulfur facultatively (Huber and Hannif, 2006;[START_REF] Cappelletti | Members of the order Thermotogales: from microbiology to hydrogen production[END_REF]. Strictly following the definition of fermentation as 'an energy yielding process in which an energy substrate is metabolized without the involvement of an exogenous electron acceptor' [START_REF] Singleton | Dictionary of Microbiology and Molecular Biology[END_REF], Mesotoga spp. should not be considered as sugar-fermenting but rather as sugar-oxidizing microorganisms.

We demonstrate also that M. prima strain PhosAc3, M. prima strain MesG1.Ag.4.2 T and M. infera strain VNs100 T grow more efficiently in syntrophic association with a hydrogenotrophic SRB, which serves as a terminal biological electron acceptor, than as pure culture in the presence of sulfur. Our data strongly suggest the existence of an interspecies hydrogen transfer between these Mesotoga species and hydrogenotrophic SRB. It should be noted that while we succeeded to co-culture M. prima strain PhosAc3 with all hydrogenotrophic SRB tested, attempts to co-culture it with hydrogenotrophic methanogens were only successful with M. hungatei only after several months of incubation. Because sulfate-reducing bacteria are known to have higher affinity for hydrogen than methanogens [START_REF] Kristjansson | Different Ks values for hydrogen of methanogenic bacteria and sulfate reducing bacteria: an explanation for the apparent inhibition of methanogenesis by sulfate[END_REF][START_REF] Robinson | Competition between sulfate-reducing and methanogenic bacteria for H 2 under resting and growing conditions[END_REF], it may indicate that the oxidation of substrates by Mesotoga requires a very low partial hydrogen pressure that could be better established by SRB than methanogens, with the exception of M. hungatei which is known to have the highest affinity for H 2 among methanogens [START_REF] Robinson | Competition between sulfate-reducing and methanogenic bacteria for H 2 under resting and growing conditions[END_REF]. In this respect, here we provide evidence for Thermotogae members to perform an efficient obligatory sugar oxidation through a syntrophic association with a hydrogenotrophic microbial partner.

We propose that the metabolic difference between Thermotoga spp. and M. prima strain PhosAc3 regarding sugar degradation is related to the absence of a bifurcating [FeFe]-hydrogenase in the latter, preventing it to ferment sugar. In contrast with T. maritima, the absence of this enzyme in M. prima would prevent the efficient re-oxidation of NADH, generated during glucose oxidation, linked to H 2 production. In the absence of an external electron acceptor, NADH accumulation would thus lead to growth inhibition. During the syntrophic association with a hydrogenotrophic partner, the ferredoxin-dependent [FeFe]hydrogenase (locus tag MESO_PHOSAC3V1_90124) would produce hydrogen that would be, in turn, metabolized by the hydrogenotrophic partner. It has been proposed that the Rnf complex in T. maritima is involved in maintaining the appropriate ferredoxin/NADH ratio for the bifurcating [FeFe]-hydrogenase and other cell processes [START_REF] Schut | The iron-hydrogenase of Thermotoga maritima utilizes ferredoxin and NADH synergistically: a new perspective on anaerobic hydrogen production[END_REF]. Similarly, we advance that the M. prima strain PhosAc3 Rnf complex (MESO_PHO-SAC3V1_120288-MESO_PHOSAC3V1_120293) would participate in hydrogen metabolism by driving reverse electron flow from NADH, generated by glucose oxidation, to the reduction of ferredoxin which then delivers electrons to the ferredoxin-dependent [FeFe]-hydrogenase. The Na 1 -NQR complex (MESO_PHOSAC3V1_90501-MESO_ PHOSAC3V1_90504) would also participate in re-oxidation of the reduced NADH generated from glucose oxidation.

Because M. prima strain PhosAc3, M. prima strain Mes-G1.Ag4.2 T and M. infera strain VNs100 T are all able to oxidize sugars, one can hypothesize that this capacity was inherited from their common ancestor. Actually, like M. prima strain PhosAc3, M. prima strain MesG1.Ag4. (Theba_0829-Theba_0833) and Rnf (Theba_1343-Theba_1348) complexes but no bifurcating [FeFe]-hydrogenase. We thus propose that the efficient sugar-oxidizing metabolism linked to a syntrophic association with a hydrogenotrophic bacterium can be extrapolated to all members of the Mesotoga genus.

The importance of obligate interspecies hydrogen transfer for substrate oxidation has been documented in many occasions with a peculiar emphasis for fatty acids, ethanol as well as aromatic and alicyclic compounds [START_REF] Sieber | The importance of hydrogen and formate transfer for syntrophic fatty, aromatic and alicyclic metabolism[END_REF][START_REF] Schmidt | Peat: home to novel syntrophic species that feed acetate-and hydrogen-scavenging methanogens[END_REF]. In contrast, such process involving a hydrogenotrophic partner to degrade easily fermentable substrates as carbohydrates has been reported only a few times, as for Syntrophococcus sucromutans, isolated from rumen of steer [START_REF] Krumholz | Syntrophococcus sucromutans sp. nov. gen. nov. uses carbohydrates as electron donors and formate, methoxymonobenzenoids or Methanobrevibacter as electron acceptor systems[END_REF]) and Bacillus spp., isolated from Lake Constance sediment (M€ uller et al., 2008). The latter authors, by direct dilution of Lake Constance sediment in mineral agar medium containing Methanosprillum hungatei, provided evidence that obligate sugar-degrading Bacillus sp. using a hydrogenscavenging methanogen as terminal electron acceptor outnumbered those fermenting easily these substrates. These Bacillus spp. were thus believed to be of high ecological significance in these sediments (M€ uller et al., 2008). The same conclusion may be drawn for M. prima strain PhosAc3 since, similarly as for M€ uller et al. (2008), it has been isolated after high dilution steps from anaerobic digester treating phosphogypsum [START_REF] Ben Hania | Cultivation of the first mesophilic representative ("mesotoga") within the order Thermotogales[END_REF]2015). It may be extended to other Mesotoga strains which have been detected by molecular approaches in various ecosystems including those contaminated by toxic compounds [START_REF] Nesbø | Evidence for existence of "mesotogas", members of the order Thermotogales adapted to low-temperature environments[END_REF]2010). Association with hydrogenotrophic SRB and possibly with high hydrogen affinity hydrogenotrophic methanogens such as M. hungatei would thus be of primary ecological importance for growth of Mesotoga spp. and other obligate-oxidizing bacteria in their natural habitats. Interestingly, all these bacteria (S. sucromutans, Bacillus and Mesotoga spp.) produce acetate as the sole organic acid end-product of sugar metabolism. Their ecological role as saccharolytic acetogenic bacteria in association with anaerobic hydrogen scavengers (e.g. sulfate-reducing bacteria), might have been underestimated so far. It thus deserves further investigations especially in sugar containing environments, to enlarge the current view of the microbiology of anaerobic digestion of organic matter (e.g. carbohydrates) that typically gives a predominant role to classical sugar-fermenting bacteria over syntrophic sugar-oxidizing associations.

Experimental procedures

Media and culture conditions M. prima strain PhosAc3 and M. infera strain VNs100 T were grown with glucose (20 mM) as carbon and energy sources at 378C in medium containing per litre 0.3 g KH 2 PO 4 ; 0.3 g K 2 HPO 4 ; 1.0 g NH 4 Cl; 2.0 g NaCl; 0.1 g KCl; 0.1 g CaCl 2 .2H 2 O; 0.5 g MgCl 2 .6H 2 O; 1 g yeast extract; 0.5 g cysteine-HCl; 0.16 g sodium acetate; 1 ml Widdel trace element solution [START_REF] Widdel | Studies on dissimilatory sulfate-reducing bacteria that decompose fatty acids. Isolation of new sulfate-reducing bacteria enriched with acetate from saline environments. Description of Desulfobacter postgatei gen. nov., sp[END_REF]) and 1 ml resazurin 0.1%. When specified, 10 g of elemental sulfur (S 0 ) per litre of medium was added. Before culture inoculation, 0.2 ml of 10% (wt/vol) NaHCO 3 , 0.1 ml of 2% (wt/vol) Na 2 S-9H 2 O and glucose were injected from sterile stock solutions in the culture medium. Mesotoga prima strain MesG1.Ag4.2 T was grown in the same culture medium mentioned above containing fructose (20 mM) instead of glucose in the presence of 25 g l 21 NaCl. Desulfovibrio vulgaris subsp. vulgaris, D. salexigens and D. gibsoniae were grown in the same culture medium as for M. prima strain PhosAc3 with the sugar being replaced by hydrogen (H 2 :CO 2 ; 80:20 vol/vol) as electron donor and elemental sulfur by sodium sulfate (Na 2 SO 4 ) (4 g l 21 ) as electron acceptor. Twenty-five grams per litre NaCl was added to the culture medium for growth of D. salexigens. Methanospirillum hungatei, M. congolense and M. aarhusense, were grown in the same culture medium as for sulfate-reducing bacteria but without sulfate. Co-cultures were performed in the same medium as for culturing Mesotoga species alone but without elemental sulfur that was replaced by sodium sulfate (4 g l 21 ) in the case of co-cultures with sulfate-reducing bacteria.

Cultures and co-cultures were performed under anaerobic conditions in Hungate tubes or in large volume flasks. Cocultures were established by mixing (10% vol/vol each) exponentially growing (i) Mesotoga strain PhosAc3 and either D. vulgaris subsp. vulgaris, D. gibsoniae, M. hungatei, M. congolense or M. aarhusense, (ii) M. infera strain VNs100 T and D. vulgaris subsp. vulgaris, (iii) M. prima strain MesG1.Ag4.2 T and D. salexigens. Fresh medium was inoculated with 10% (vol/vol) of exponentially growing cells and incubated at 378C. Growth was monitored by measurement of the OD at 580 nm of the culture directly in the Hungate tube. Growth curves and metabolites quantification were obtained from at least 2 independent replicates.

Analytical methods

Soluble sulfides were quantified according to the Cord-Ruwish (1985) method by using a Shimadzu UV-160A spectrophotometer (Shimadzu, Kyoto, Japan). Sugars (lactose, glucose), organic acids (acetate, lactate, propionate, butyrate, formate) and ethanol determination was carried out by high performance liquid chromatography (HPLC) as described by [START_REF] Fardeau | Thermotoga hypogea sp. nov., a xylanolytic, thermophilic bacterium from an oil-producing well[END_REF]. For hydrogen quantification, 1 ml of culture headspace sample (gas phase) was injected into a Shimadzu 8A TCD-GC system (Shimadzu, Kyoto, Japan) equipped with a concentric CTR1 column (Alltech, Columbia, MD, USA), connected to a computer running WINILAB III software (Perichrom, Saulx les Chartreux, France) [START_REF] Fardeau | Thermotoga hypogea sp. nov., a xylanolytic, thermophilic bacterium from an oil-producing well[END_REF]. Unless otherwise indicated, analytical measures were performed on duplicate culture tubes or bottles.

Cells counting by microscopy

For preparing the microscope observation chamber, all steps were performed in an anaerobic chamber (Coy Laboratory, Grass Lake, MI, USA). One hundred microlitre of the M. prima strain PhosAc3 and D. vulgaris subsp. vulgaris co-culture were centrifuged for 3 min at 10 000 g. DAPI staining was performed by resuspending the cells pellet in 100 ml of 10 mM Tris-HCl (pH 7.6), 8 mM MgSO 4 and 1 mM KH 2 PO 4 buffer (TPM buffer) containing 5 ng ml 21 of 4 0 ,6-diamidino-2-phenylindole (DAPI). After 20 min of incubation in the dark, cells were washed three times with TPM buffer. The DNA was stained under anaerobic conditions to limit the exposure of the cells to air. The cells were placed between a glass coverslip and a thin layer of LS4D-YE medium supplemented with 10 ng ml 21 of FM4-64V R (Invitrogen) and 1.5% (wt/vol) of Phytagel TM (Sigma-Aldrich) that was previously prepared under anaerobic conditions. The preparation was then transferred in a hermetic microscope observation chamber [START_REF] Fievet | Single-cell analysis of growth and cell division of the anaerobe Desulfovibrio vulgaris Hildenborough[END_REF] and put into a standard temperature-controlled inverted microscope. Pictures were acquired with a Nikon TiE-PFS inverted epifluorescence microscope, 1003 NA1.3 oil PhC objective (Nikon) and Hamamatsu Orca-R2 camera. Image processing was controlled by the NIS-Element software (Nikon).

Hydrogenase activity measurements by spectrophotometry

Pure cultures of M. prima strain PhosAc3 (1 l) were grown in the presence of elemental sulfur until the OD 580nm reached 0.28. Cells were harvested by centrifugation (5000 g for 30 min), rinsed once with 50 ml of 50 mM Tris-HCl, NaCl 200 mM (pH 7.5) buffer and finally resuspended in 500 ml of 100 mM HEPES buffer (pH 8.0). When M. prima strain PhosAc3 was co-cultured with D. vulgaris subsp. vulgaris in separated compartments, cells were prepared as above from 500 ml of culture. For hydrogenase uptake activities, a rubber-stopper sealed cuvette containing 1 ml of 100 mM HEPES buffer (pH 8.0), 75 ml of 100 mM methyl-viologen was bubbled for 5 min with hydrogen. Then, a defined amount of cells suspension (from 10 to 50 ml) and 5 ml of 1/10 Triton X100 diluted in HEPES buffer was added. The OD at 604 nm was recorded over the time. As controls, methyl-viologen reduction was followed in cuvettes without hydrogen bubbling and without addition of cells.

For H 2 production activity, a rubber-stopper sealed cuvette containing 1 ml of 100 mM HEPES buffer (pH 8.0) was bubbled for 5 min with Argon. A defined amount of cells suspension (from 10 to 50 ml) was then added and the cuvette was bubbled with Argon for 2 min longer. Then, 100 ml of dithionite-reduced methyl-viologen (10 mM) and 5 ml of 1/10 Triton X100 diluted in HEPES buffer were added. The decrease of the OD at 604 nm was recorded over the time. The auto-oxidation kinetic of reduced methyl-viologen was measured in cuvette in the absence of cells. H 2 production activity was obtained by subtracting the auto-oxidation activity from the methy-viologen oxidation activity in the presence of cells.

All buffers, reactives and cells suspensions were kept under anaerobic conditions and continuously flushed with Argon.

H 2 production monitoring by Clark-based microsensor

Hydrogen production was also measured on M. prima strain PhosAc3 cells grown on glucose either as mono-culture in the presence of elemental sulfur or as co-culture with D. vulgaris in dialysis membrane-separated compartments as described in Supporting Information Fig. S1. Cells were harvested at the end of the exponential phase and washed three times with an anaerobic 0.1 M Tris-HCl, 0.15 M NaCl (pH 7.5) buffer saturated with N 2 . The measurements were carried out in a 1.9 ml sealed anaerobic cuvette filled with 0.1 M Tris-HCl, 0.15 M NaCl (pH 7.5) buffer equilibrated at 378C and containing cells suspension. After 15 min, 50 mM glucose (final concentration) was added and H 2 production was monitored with a H 2 -MR 500 mm microsensor plugged to a Microsensor Multimeter (Unisense, Aarhus, Denmark). Electrode was calibrated with known amounts of pure H 2 gas saturated in water at 378C (750 mM).

Phylogenetic analyses

Two thousand seven hundred seventy-five complete prokaryotic proteomes were downloaded at the NBCI (http://www. ncbi.nlm.nih.gov/). Only one representative proteome per species was kept for analyses. The 1369 corresponding proteomes (including 14 Thermotogae species, Supporting Information Table S1) were gathered to build a local database. This database was queried with BLASTP (v2.2.26) [START_REF] Altschul | Basic local alignment search tool[END_REF] to identify homologues of the studied proteins. Mesotoga prima strain PhosAc3 or T. maritima strain MSB8 protein sequences were used as seed. The 60 protein sequences displaying the highest scores and E-values lower than 10 23 were retrieved. Homologues from M. prima strain PhosAc3 and M. infera strain VNs100 T were downloaded from the MAGE platform at the genoscope (https://www.genoscope.cns.fr). The retrieved homologues were aligned with MAFFT v7 (option L-INS-I) [START_REF] Katoh | MAFFT multiple sequence alignment software version 7: improvements in performance and usability[END_REF]. The resulting multiple alignments were visually inspected with SEAVIEW v.4.5.4 [START_REF] Gouy | SeaView version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building[END_REF] and trimmed with BMGE v1.1 [START_REF] Criscuolo | BMGE (Block Mapping and Gathering with Entropy): a new software for selection of phylogenetic informative regions from multiple sequence alignments[END_REF].

Maximum likelihood phylogenetic trees were inferred with PHYML v.3.1 [START_REF] Guindon | New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0[END_REF] implemented in the SEA-VIEW program [START_REF] Gouy | SeaView version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building[END_REF] using the 'Le and Gascuel evolutionary model' [START_REF] Le | An improved general amino acid replacement matrix[END_REF] and a gamma distribution with four categories of sites to take into account the heterogeneity of site evolutionary rates. The NNI 1 SPR option was used to explore tree topologies. The robustness of the resulting trees was evaluated using the non-parametric bootstrap procedure implemented in PHYML (100 replicates of the original dataset).

Fig

  Fig. 1. A. Glucose consumption in pure culture of M. prima strain PhosAc3 in either the absence (circle) or presence (triangle) of elemental sulfur (S8) or in co-culture with D. vulgaris (square) over the time. B. Acetate production in either pure culture of M. prima in the absence (circle) or presence (triangle) of elemental sulfur (S8) or in co-culture with D. vulgaris (square) over the time.

Fig. 2 .

 2 Fig. 2. A. Growth curves of M. prima strain PhosAc3 (triangle) and D. vulgaris (circle) in pure culture or in co-culture (square) in 20 mM glucose/ 28 mM sulfate containing medium. B. Growth curves of M. prima strain MesG1.A4.2 T in the presence (triangle) or the absence (circle) of S8 and in coculture with D. salexigens (square) in 20 mM fructose/28 mM sulfate medium

Fig. 4 .

 4 Fig. 4. Hydrogen production by M. prima strain PhosAc3 cells. Hydrogen evolution was measured on Mesotoga whole washed cells grown either in dialysis tubing co-cultured with Desulfovibrio vulgaris subsp. vulgaris in glucose/sulfate medium (black line) or grown in pure culture on glucose in the presence of elemental sulfur (grey line). Experiments were carried out in a respirometry chamber (1.9 ml) in Tris-HCl 0.1M NaCl 0.15M buffer (pH 7.5) at 378C using a specific H 2 microsensor. Addition of cells (1.6 mg total protein for both samples) and glucose (50 mM final concentration) are indicated by arrows.

  2 T genome encodes homologous ferredoxin-dependent [FeFe]-hydrogenase (Theba_0443) as well as Na 1 -NQR Obligate sugar oxidation by Mesotoga spp. 287 V C 2017 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 20, 281-292

Table 1 .

 1 End-products quantification of the single and co-cultures of M. prima PhosAc3 and M. prima MesG1Ag4.2T.

				M. prima			M. prima
		M. prima	M. prima	PhosAc3	M. prima	M. prima	MesG1Ag4.2 T
		PhosAc3	PhosAc3 1 S8	1 D. vulgaris	MesG1Ag4.2 T	MesG1Ag4.2 T 1 S8	1 D. salexigens
	Sugar consumed (mM)	1.50 6 0.92	6.57 6 0.19	12.39 6 0.33	1.00 6 0.23	3.27 6 0.85	6.65 6 1.88
	Acetate produced (mM) 1.67 6 0.21	9.21 6 0.13	27.13 6 0.45	0.70 60.41	8.48 6 1.96	13.64 6 1.75
	H 2 S produced (mM)	1.05 6 0.25 24.40 6 0.30	9.70 6 0.85	1.18 6 0.41	18.03 6 5.16	14.00 6 4.52

K. Fadhlaoui et al. V C 2017 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 20, 281-292

V C 2017 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 20, 281-292

K. Fadhlaoui et al. V C 2017 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 20, 281-292

K. Fadhlaoui et al. V C 2017 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 20, 281-292

Acknowledgements

The authors are grateful to Alison Strutt for revising the English text and to Matthieu Robbino for participating in H 2 production measurements. The authors do not have any conflict of interest to declare.

Supporting information

Additional Supporting Information may be found in the online version of this article at the publisher's web-site: Fig. S1. (A) Compartment-separated co-culture of M. prima strain PhosAc3 and D. vulgaris. D. vulgaris was grown inside a dialysis tube (dialysis membrane Spectra/Por, 12-14K MWCO) that was secluded from the surrounding strain M. prima culture by rubber stoppers. The apparatus was incubated in a very slowly moving (30 r.p.m.) rotary shaker at 378C. (B) Experimental assembly to grow the co-culture in two different vials linked by a tubing. Pseudothermotoga lettingae TMO (YP_001469925), Pseudothermotoga thermarum DSM 5069 (YP_004660443), Petrotoga mobilis SJ95 (YP_001568014), Marinitoga piezophila KA3 (YP_005096257), Kosmotoga olearia TBF 19.5.1 (YP_002940621), Thermotoga naphthophila RKU-10 (YP_003346384), Mesotoga prima MesG1.Ag.4.2 (Theba1348), Mesotoga prima PhosAc3 (MESO PHOS-AC3v1_120293), Mesotoga infera VNs100 (MESINFAv2 2581), Thermotoga naphthophila RKU-10 (YP_003346384), Thermotoga petrophila RKU-1 (YP_001244271), Thermo-toga neapolitana DSM 4359 (YP_002533979), Thermotoga maritima MSB8 (NP_228063), Fervidobacterium pennivorans DSM 9078 (YP_005471998), Fervidobacterium nodosum Rt17-B1 (YP_001411073), Thermosipho melanesiensis BI429 (YP_001306837) and Thermosipho africanus TCF52B (YP_002335587). Table S1. List of the 2775 prokaryotic proteomes used in the phylogeny analyses.