dataesr/scientific-paragraphs-categorization huggingface dataset:
Together with the results of RNA-Seq experiments, these data demonstrate that the motility defect of the fliZ mutant results from a global decrease in the expression of flagellar genes, resulting in the production of low amounts of the flagellin monomer. The Expression of FliZ-Dependent Hemolysin and Class II-III Flagellar Genes Is Heterogeneous at the Single-Cell Level Microscopic observations performed during insect infection have revealed that the expression of the fliC and xaxAB genes in Xenorhabdus is heterogeneous differing between individual cells  [23]. We investigated the heterogeneity of fliC, xaxAB and xhlBA gene expression during bacterial growth in vitro, by measuring the expression of these genes in the wild type strain carrying the P fliCgfp[AAV], P xaxAB -gfp[AAV] or P xhlBA -gfp[AAV] fusion by flow cytometry.
RNA-Seq Analysis Transcriptomic high-throughput sequencing data were analyzed with a bioinformatic pipeline implemented within the Microscope platform  [56]. The pipeline currently used is a ''Master'' shell script that launches the various parts of the analysis (i. e. a collection of Shell/Perl/R scripts) and checks that all tasks are completed without error. We first assessed RNA-Seq data quality by including options, such as read-trimming or the use of merging/split paired-end reads. We then mapped reads onto the contigs of the X. nematophila F1 genome sequence (accession number: CAVM000000000) with the SSAHA2 package  [57] , which combines the SSAHA searching algorithm (sequence information encoded in a perfect hash function) for identifying regions of high similarity, and the cross-match sequence alignment program, which aligns these regions, using a banded Smith-Waterman-Gotoh algorithm.
An alignment score covering at least half of the read is required for a hit to be retained. We minimized the false positive discovery rate, by using SAMtools (v. 0. 1. 8) to extract reliable alignments from SAM-formatted files. The number of reads matching each genomic object harbored by the reference genome was then calculated with the Bioconductor-Genomic Features package. If reads matched several genomic objects, the count number was weighted so as to keep the total number of reads constant. Finally, the Bioconductor-DESeq package  [58]  was used with default parameters for the analysis of raw count data and to determine whether expression levels differed between conditions. The complete dataset from this study has been deposited in the GEO database under accession no. GSE47365.
RT-qPCR Analysis RT-qPCR was performed in two steps. First, the cDNA was synthesized from 1 mg of total RNA from each replicate used for RNA-Seq (0. 2 mg of total RNA was used for sorted cells), with Super Script II Reverse Transcriptase from Invitrogen and random hexamers (100 ng. ml 21 ) from Applied Biosystems. We then carried out qPCR in triplicate with the LightCycler 480 SYBR Green I Master kit from Roche Diagnostics, with 1 ml of cDNA synthesis mixture (diluted 1:100 or 1:20) and 1 mM of specific primers for the genes studied (Table  S3 ). The enzyme was activated by heating for 10 minutes at 95uC. All qPCRs were performed in three technical replicates, with 45 cycles of 95uC for 5 s, 60uC for 5 s and 72uC for 10 s, and were monitored with the Light Cycler 480 system (Roche). Melting curves were analyzed for each reaction and each curve contained a single peak.
The recA gene was used as the reference housekeeping gene and 16S, mre or ampD was used as an internal control. The data for each sample are expressed relative to the expression level of recA, as follows  [59] : ratio~E target À Á Dcp target control{sample ð Þ E ref ð Þ Dcp ref control{sample ð Þ. The relative expression ratio for a target gene was calculated on the basis of its real-time PCR efficiency (E) and the crossing point (CP) difference (D) between a sample and the control (DCP control -sample ). Crossing points (CP) for each sample and each gene were calculated with LightCycler480 (Roche) software, using second-derivative maximum analysis and the CP medians of the technical replicates for each biological sample used. All relative quantifications were assessed with REST software 2009, using the pairwise fixed randomization test with 2,000 permutations, with PCR efficiencies calculated with serial dilutions of a mixture of cDNAs  [59].
Quantification of fliC Expression in Bacterial Populations Wild type, fliA and fliZ strains carrying either P fliC -gfp[AAV] or P fliC -gfp[AAV] -P tet -fliZ constructs were cultured in black-sided, clear-bottomed 96-well plates (Greiner). For each well, 20 ml of a 1/50 dilution of an overnight culture was added to 180 ml of LB supplemented with kanamycin, and 200 ng. ml 21 of aTc when required. Then, the plates were incubated, with shaking on an orbital shaker, at 28uC, in an Infinite M200 microplate reader (Tecan). Absorbance at 600 nm and GFP fluorescence intensity, with excitation at 48564. 5 nm and emission at 520610 nm, were measured after 15 hours of growth. Specific fluorescence was obtained by dividing fluorescence units by the absorbance value. As a control, we checked that addition of aTc did not affect the expression of P fliC -gfp[AAV] fusion in the wild type strain (data not shown).
Acknowledgments We thank  Nadege Ginibre  and  Christine Laroui  for technical assistance and  Sylvie Page  `s for assistance with the pathology analysis. Image acquisition and analysis were performed on the workstations of the Montpellier RIO Imaging facility of the IGMM. We also thank  Christophe Duperray  and  Myriam Boyer  from the  Montpellier RIO Imaging  facility of the IRB and IGMM institutes for technical assistance with cell sorting manipulations and  Philippe Clair  from the  Montpellier GenomiX facility  for expert technical assistance with real-time PCR.
Classification and features 16S rRNA gene analysis A representative genomic 16S rRNA gene sequence of L. arenae DSM 19593 T was compared using NCBI BLAST  [4, 5]  under default settings (e. g. , considering only the high-scoring segment pairs (HSPs) from the best 250 hits) with the most recent release of the Greengenes database  [6]  and the relative frequencies of taxa and keywords (reduced to their stem  [7] ) were determined, weighted by BLAST scores. The most frequently occurring genera were Jannaschia (38. 1%), Thalassobacter (15. 4%), Octadecabacter (11. 7%), Roseovarius (10. 7%) and Roseobacter (10. 2%) (28 hits in total). Regarding the three hits to sequences from other members of the genus, the average identity within HSPs was 96. 0%, whereas the average coverage by HSPs was 98. 7%.
Among all other species, the one yielding the highest score was 'Octadecabacter orientus' (DQ167247), which corresponded to an identity of 99. 2% and an HSP coverage of 99. 6%. (Note that the Greengenes database uses the INSDC (= EMBL/NCBI/DDBJ) annotation, which is not an authoritative source for nomenclature or classification). The highestscoring environmental sequence was FJ664800 (Greengenes short name 'Quantitative dynamics cells plankton-fed microbial fuel cell clone plankton D11'), which showed an identity of 97. 0% and an HSP coverage of 99. 6%. The most frequently occurring keywords within the labels of all environmental samples which yielded hits were 'lake' (9. 9%), 'tin' (9. 8%), 'xiaochaidan' (9. 4%), 'microbi' (2. 6%) and 'sea' (2. 5%) (222 hits in total). Environmental samples which yielded hits of a higher score than the highest scoring species were not found.
The substrate utilization and resistance patterns of L. arenae DSM 19593 T were also determined for this study using Generation-III microplates in an OmniLog phenotyping device (BIOLOG Inc. , Hayward, CA, USA). The microplates were inoculated at 28°C with a cell suspension at a cell density of 95-96% turbidity and dye IF-A. Further additives were vitamin, micronutrient and sea salt solutions. The exported measurement data were further analyzed with the opm package for R  [26, 27] , using its functionality for statistically estimating parameters from the respiration curves such as the maximum height, and automatically translating these values into negative, ambiguous, and positive reactions. The strain was studied in two independent biological replicates, and reactions with a different behavior between the two repetitions, if any, were regarded as ambiguous.
Genome project history The strain was first chosen for genome sequencing in the Genomic Encyclopedia of Bacteria and Archaea (GEBA) phase I project according the GEBA criteria  [31, 32] , but then sequenced as part of the "Ecology, Physiology and Molecular Biology of the Roseobacter clade project: Towards a Systems Biology Understanding of a Globally Important Clade of Marine Bacteria" funded by the German Research Council (DFG). Project information is stored in the Genomes OnLine Database  [33]. The Whole Genome Shotgun (WGS) sequence is deposited in Genbank and the Integrated Microbial Genomes database (IMG)  [34]. A summary of the project information is shown in Table  2.
(The version deposited at Genbank contains two scaffolds less, which did not meet the requirements for the minimal contig length. The additional fragments 'thalar_Contig12. 1' and 'thalar_Contig18_1. 4' can be found in the IMG database). The combined sequences provided a 195× coverage of the genome. Table  1. Classification and general features of L. arenae DSM 19593 T according to the MIGS recommendations  [16]  published by the Genome Standards Consortium  [17].
Genome annotation Genes were identified using Prodigal  [42]  as part of the JGI genome annotation pipeline  [43]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt, TIGR-Fam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Identification of RNA genes were carried out by using HMMER 3. 0rc1  [44]  (rRNAs) and tRNAscan-SE 1. 23  [45]  (tRNAs). Other non-coding genes were predicted using INFER-NAL 1. 0. 2 [46]. Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes -Expert Review (IMG-ER) platform  [34]. CRISPR elements were detected using CRT  [47]  and PILER-CR  [48].
Recent discoveries of associated bacteria in two families (Nucinellidae and Montacutidae) suggest further species and habitats should be explored  (Oliver and Taylor, 2012; Oliver et al. , 2013). Tight evolutionary relationships exist between metazoans and their associated symbionts in various habitats, with several studies providing evidence that deep-sea vent and seep bivalve species originated from shallow-water ancestors  (Craddock et al. , 1995; Little and Vrijenhoek, 2003; Lorion et al. , 2010; Williams et al. , 2004). Examining the intra-and interspecific diversity of chemosynthetic symbioses in bivalves, within the context of functional, ecological, biogeographical and evolutionary trends in symbiotic systems, will provide greater insight into this adaptation to reducing habitats. The Atlantic Ocean opened relatively recently (∼ 180 Mya), after fragmentation of Pangaea.
, 2007; Génio et al. , 2008; Olu et al. , 2010; Olu-LeRoy et al. , 2007b). A check list of species in which symbiosis has been documented is also provided (Table  1 ). Table  1. Summary of chemosymbiotic bivalve species for which data are available regarding symbiosis in the Atlantic, Gulf of Mexico, and Mediterranean Sea. Numbers in parentheses indicate the number of species for which symbiosis is documented using electron microscopy and molecular data, possibly including in situ hybridization of symbionts (bold). Synonymized taxa, according to the World Register of Marine Species (WORMS, www. marinespecies. org), are included as many former names were in use when symbioses were initially documented, and thus appear in the tree (Fig. 2 ). Species within genus "Pliocardia" were recently ascribed to undetermined genus  (Audzijonyte et al.
Possible settlement is also reported on wood block colonization experiments deployed in the western Mediterranean canyons at 500 m deep (personal observation, N. Le Bris, personal communication, 2012). Interestingly, molecular and microscopic data suggest the absence of methane-oxidizing symbionts. Furthermore, specimens from the Meknès and Gorringe sites do not seem to have sulfur-oxidizers either. Identified bacteria either belong to the "Gammaproteobacteria G" group, present in I. modiolaeformis, or to a clade unrelated to any known symbiont (clone G-4), or to the Bacteroidetes. Overall, symbioses in the I. macdonaldi/I. modiolaeformis clade appear unexpectedly variable. This could be linked with the wide range of habitats colonized, which include alfalfa and wood colonization devices, natural wood falls, carbonates close to reduced sediment, and siboglinid tubes.
In the Gulf of Mexico, vesicomyids are represented by Pliocardia ponderosa (formerly Calyptogena ponderosa) and Pliocardia cordata (formerly Vesicomya cordata) (Table 1). They both display closely related sulfur-oxidizing symbionts (Fig. 2 )  (Brooks et al. , 1987; Distel et al. , 1994; Stewart et al. , 2009b)  and an abundance of hemoglobin  (Scott and Fisher, 1995). Species from seeps in the Gulf of Guinea have recently received attention. These include Elenaconcha guiness, Isorropodon bigoti and Calyptogena valdiviae. E. guiness was found to display a very typical vesicomyid symbiosis, as documented in many other species around the world  (Duperron et al. , 2012). This species displays a single symbiont 16S rRNA phylotype, with high similarity to other vesicomyid-associated symbiont sequences available in Genbank, and sulfur metabolism is supported by the presence of the gene encoding APS (adenylyl-sulfate) reductase and by carbon stable isotope values in the range of those reported for seep vesicomyids  (Olu et al.
, 2012). Finding closely related symbionts in closely related species is somewhat expected, although instances of non-parental acquisition are reported  (Stewart et al. , 2008 (Stewart et al. , , 2009b;; Decker et al. , 2013). The homogeneity of symbiont populations within a single species was also questioned recently by the finding of distinct bacterial 16S rRNA lineages in distinct specimens of Calyptogena valdiviae (Fig. 2 ). Overall, new data about symbioses in the Atlantic Vesicomyidae further support the existence of a certain level of symbiont heterogeneity within species, and of environmental acquisition of non-parental symbiont strains in co-occurring host species, which can potentially lead to symbiont co-occurrence, displacement, or genetic recombination among symbionts. These phenomena might be of great significance for the evolution of vesicomyid symbioses.
Solemyidae All documented Solemyidae live in obligate symbiosis with sulfur-oxidizing Gammaproteobacteria. They make U-or Yshaped burrows that allow access to both the bottom seawater, rich in oxygen, and sediment pore-waters that contain reduced sulfur  (Fisher, 1990; Stewart and Cavanaugh, 2006). Due to their infaunal lifestyle, deep-sea Solemyidae are rarely sampled in great numbers, which limits the possibility to investigate them in detail. Most data were obtained from coastal species. Although a weak suspensionfeeding capability is retained, dependency upon symbiotic nutrition is maximal, as shown by the drastic reduction (Solemya velum), or even absence (Solemya reidi) of the digestive system and reduction in labial palps. Authors estimated that more than 97 % of the carbon is derived from symbionts  (Conway et al.
For example, carbon fixation in symbionts of Solemya velum is carried out by a Type IA RubisCO, while a Type II RubisCO is present in Acharax sp. Guiness  (Duperron et al. , 2012; Robinson and Cavanaugh, 1995; Schwedock et al. , 2004). Other differences could exist besides the type of RubisCO. Additional 16S rRNA sequences were recovered besides that of gammaproteobacterial symbionts in Solemya elarraichensis and Acharax gadirae branching within the Epsilonproteobacteria, Betaproteobacteria, Chlamydiae, Firmicutes, and Actinobacteria. Their status as symbionts or pathogens was not resolved, but at least some might have been contaminants. In the Sea of Oman, a family related to Solemyidae, the Nucinellidae, was recently shown to display chemosynthetic sulfur-oxidizing bacteria in their gills based on detailed electron microscopy approaches, but without supporting molecular data  (Oliver and Taylor, 2012).
, 2003a) , which live in reduced sediment near seagrass beds, also have identical symbionts to those of Codakia orbicularis. The symbiont from L. pectinata not only diverges from those of other lucinids, but the host tissue also displays a high concentration of haemoglobin, which suggests that the symbiosis might function in a different way  (Frenkiel et al. , 1996). Another species, L. nassula, occurs in seagrass beds near Florida and has a very similar symbiont sequence (Durand and  Gros, 1996)  Additionally, symbiosis was characterized based on gill tissue ultrastructure or molecular evidence in a variety of other coastal reduced sediment or seagrass bed species including Myrtea spinifera, L. floridana, L. borealis, and Loripes lucinalis (synonymized with Loripes lacteus, for which symbiont 16S sequences are in GENBANK but unpublished), with estimates that the latter could be responsible for up to 16 % of the primary production observed in seagrass bed habitats in a lagoon in upper Corsica  (Dando et al.
Colors relate to the family to which the host belongs; sequences in bold are from species present in the area of focus for the review. For clarity, only host species name is mentioned without mention of "symbiont". Properly identified bivalve-associated 16S rRNA sequences above 1200 bp were used; short sequences available for Bathymodiolus tangaroa, Acharax gadirae and Idas macdonaldi were not included. Tree was reconstructed using a maximum likelihood algorithm from 1129 nucleotide positions. Based on Bayesian information criterion, a Tamura 3-parameter with Gamma-distribution of rates (T92 + G) was employed. Node support values (> 60 % shown) are based on 500 bootstrap replicates using the same methodology. Analyses were run using MEGA 5. 0  (Tamura et al. , 2011). Although some names have recently taken on new synonyms (see Table  1 ), we used names included in the original GENBANK records.
Much is believed to depend upon the seasonality of reproduction and whether larval dispersal takes place in cold deep waters or in warmer surface waters (Fig. 4a ,  Tyler et al. , 2007). Currently, however, such information is scarce for chemosymbiotic bivalves. Vesicomyidae, Solemyidae and Lucinidae bivalves generally have large oocytes (from 100 µm up to 600 µm diameter) rich in yolk (Table  1 , Fig. 3c ), although a few exceptions occur (Le  Pennec and Beninger, 2000). Most Vesicomyidae seem to be continuous spawners, but some also display periodicity like Calyptogena pacifica and Calyptogena kilmeri from bathyal depths in Monterey Bay  (Lisin et al. , 1997). The large oocyte size in several species indicates that the veliger larva is probably lecithotrophic. To date, no data are available regarding their dispersal capacities  (Lisin et al.
Conclusions Chemosymbiotic bivalves are diverse. Four of the five welldocumented families are associated with sulfur-oxidizing Gammaproteobacteria. Members of the family Mytilidae (clade Bathymodiolinae) are characterized by a broader diversity of associated bacteria. Approximately 200 species belonging to these families (considering only subfamily Bathymodiolinae for Mytilidae) are documented from the North Atlantic, Gulf of Mexico and Mediterranean. Data regarding symbiosis are available for 26. 5 % of these species. Despite many similarities, important differences exist between bivalve families with regards to host behavior, reproductive biology and larval dispersal capabilities. Important differences also exist among their symbioses, with regards to the mode of acquisition, the establishment and the functioning of their bacterial partners.
Acknowledgements. Authors thank the  EU programs Mediflux, HERMES, HERMIONE  for funding, as well as the  ESF/FCT EuroDEEP CHEMECO. We are grateful to other funding resources, the  BIOZAIRE program  (Ifremer),  French ANR DeepOases  (PI:  D. Desbruyères ),  GDR Ecchis ,  Portuguese Fundac  ¸ão para a Ciência e a  Tecnologia (FCT post doctoral fellowship   SFRH/BPD/64154/2009 ). We acknowledge  UPMC ,  CNRS ,  Ifremer  and  U. Aveiro  for additional funding. We also thank crews, pilots and scientists of the cruises Nautinil (chief scientist: J. P. Foucher),  DeepSeeps (C. R. Fisher), Bionil (A. Boetius) ,  Medeco  ( J. Sarrazin  and  C. Pierre ), Biozaire 2 (M. Sibuet),  Guineco (A. Boetius) ,  WACS (K. Olu) ,  TTR14-17  ( M. Ivanov ) and  MSM1/3  ( O. Pfannküche ) during which we collected the material on which this synthesis is based.
8 ml sitting drops using a Cartesian 4000 XL robot. Optimal quality crystals were grown at 298 K using a 1:1 ratio of complex solution and reservoir solution [100 mM MES pH 6. 5, 5-15%(v/v) PEG 3350, 5-15%(v/v) 2-propanol]. Crystals grown after 2-3 d were cryoprotected by adding 30%(v/v) 2-propanol to the mother liquor and were flash-cooled in liquid nitrogen. Diffraction data were collected at 100 K using synchrotron radiation on the PXI-XS06 beamline at SLS, Villigen, Switzerland. Data-processing and scaling were accomplished with XDS  (Kabsch, 2010;   1  and Supplementary Fig. S2 ). The anomalous Patterson showed the presence of two possible sites, and two Ta 6 Br 12 clusters were found using the SHELX package  (Sheldrick, 2008). The search model for molecular replacement was based on a polyalanine backbone of three RVD repeats derived from PDB entry 3v6t  (Deng et al.
Data reduction The obtained detector frames around the two different Bragg peaks of a full set of maps are reconstructed into threedimensional RSMs by translating angular coordinates to reciprocal-space coordinates  (Lazzari, 2002; Kriegner, 2013) , q x , q and q z , as shown in Fig. 4 (a). The average Bragg position is obtained by computing the center of mass (COM) of the Bragg peak, COM ¼ 1=I total ð Þ P i q i I i ; ð1Þ where the sum runs over all reciprocal space points q i with the corresponding scattering intensities I i. This data treatment is inspired by scanning transmission X-ray microscopy with pixelated detectors  (Menzel et al. , 2010)  where the deflection of the beam is used to image objects in transmission with differential phase contrast. In the present work the COM provides a direct measurement of strain and bending in the sample and it can be determined with a much higher precision than the beam divergence.
FEM simulations To verify our results, FEM calculations were conducted with the software package COMSOL Multiphysics 4. 3, using the linear elastics module. The dimensions of the structure, the stiffness tensor entries as well as the densities of Si and Ge served as model input parameters  (Wortman & Evans, 1965). The simulation included a 2. 1 mm Ge layer on top of a Si substrate and an initial strain state with " xx = " yy = 0. 18% and " zz = À0. 12%, which was patterned with the shape of the measured bridge. The orientation was chosen with the bridge direction along [010], as was the case in the real sample. Asymmetric etching effects [Fig. 5(a) ] and cracks [Fig. 5(b) ] found through scanning electron microscopy (SEM) inspection of the sample were incorporated into the FEM model Fig. 5(c)].