Draft-genome sequence of Shewanella algae strain C6G3

Shewanella algae strain C6G3, isolated from the 2 uppermost centimeters of muddy sediment of Arcachon Bay (SW Atlantic French coast, sampled in October 2007) has the capability to use a large panel of terminal electron acceptors under anaerobic condition, such as nitrate, nitrite and metal-oxide, and presents a great metabolic versatility. Here, we present the non-contiguous draft-genome sequence of Shewanella algae C6G3, which consists of a 4,879,425 bp. The chromosome contains 5792 predicted genes. In total, the genome consists of 24 rRNA genes, 86 tRNA genes and 5660 genes assigned as protein-coding genes.


Introduction
The genus Shewanella comprises several Gram-negative species which are widely distributed in marine and freshwater environments. Shewanella algae (formerly classified as S. putrefaciens) has been frequently isolated from marine water samples and spoiling fish [1][2][3]. They are capable of reducing trimethylamine N-oxide (TMAO) to trimethylamine and producing hydrogen sulfide, both of which are main components of the fishy odor present during low temperature storage. They were also isolated from human feces, skin and other clinical samples [4,5]. The collected strains were heterogeneous with G + C values ranging from 43 % to 55 %. However, there were differences between environmental and clinical isolates. Most of the strains isolated from human clinical specimens and identified as S. putrefaciens showed betahemolysis on sheep blood agar whereas environmental strains were nonhemolytic [6,7]. During a screening study of heterotrophic bacteria from the sediment of Arcachon Bay [8], a large set of isolates was obtained from different sampling sites and years (2). Among the 24 isolates, 15 strains belong to Shewanella genus and were able to reduce Mn(III/IV) and/or nitrate. The genus seems to play an important role in the turnover of organic matter coupled to anaerobic respiration electron acceptors, such as Fe(III), Mn(III/IV) and NO 2/3 . Here we report on further taxonomic and physiological studies on strain Shewanella algae strain C6G3 and present its main genomic features.

Chemotaxonomic data
The fatty acid analysis was performed on two strains of S. algae (C6G3, ATCC 51192 T ) and on S. oneidensis MR-1 T (Additional file 1: Table S2). At the end of aerobic culture, fatty acids were extracted from cell pellet by alkaline hydrolysis and analyzed using chromatography-electron ionization mass spectrometry (GC-EIMS) following the protocol described in Zabeti et al., [13]. The overall fatty acid pattern of S. algae C6G3 is rather common for the genus Shewanella. The major ones were C16:1ω7 (35.2 %), C16:0 (34.6 %) (generally reported between 16-55 % and 5-31 %, respectively [14]) and 3OH-C12:0 (7.7 %). Interestingly, the C15:0 br is much lower in strain C6G3 (2. The proposed Shewanella species have been chosen from the List of Prokaryotic names with Standing in Nomenclature (type strain and sequence accession number) for their ability to use nitrate, nitrite and / or metal oxides. Species whose genome has been sequenced are marked with star (*). Escherichia coli [44] was used as out-group respectively). S. algae C6G3 presents also a relatively higher percentage of short-chain fatty acids (shorter than C15, 16.0 %) than both S. oneidensis MR-1 T and S. algae ATCC 51192 T (8.5 %).

Genome sequencing information
Genome project history S. algae C6G3 was isolated from intertidal marine sediment on the basis of its ability to use large range of electron acceptors particularly nitrate, nitrite and metal-oxides [8]. The genome of S. algae C6G3 is the second to be reported from that species, the other one being S. algae ACDC [15]. The genome project of S. algae C6G3 is deposited in the Genome On Line Database [16]. A summary of the project and information on compliance with MIGS version 2.0 [17] are shown ( Table 2).

Growth conditions and DNA isolation
S. algae C6G3 was grown aerobically at 30°C under stirring condition on artificial sea water [18] amended with lactate (3 g/L), yeast extract (1 g/L) and tryptone (5 g/L). DNA was extracted from cells collected in exponential growth phase using the protocol of Marteinsson et al. [19]. DNA concentration and purity were checking on biophotometer® (Eppendorf ) before sequencing.

Genome sequencing and assembly
The genome sequencing of S. algae C6G3 was generated at the Molecular Research LP MR DNA Laboratory (USA). De novo whole-genome shotgun sequencing was performed using the Ion Torrent PGM (Life Technologies [20]) sequencing platform. This produced 1,444,981 reads with an average length of 200 bp for a total number of sequenced bases of 288,996,200 representing a sequencing Evidence codes -IDA: Inferred from Direct Assay; TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [55]. You will find the table of associated MIGS Record in additional file (Additional file 2) depth of 50-fold. The assembly of S. algae C6G3 genome was generated at MR DNA Laboratory using the SeqMan NGen® software assembler (DNASTAR). The final assembly identified 43 contigs generating a genome size of 4,9 Mb.

Genome annotation
Genome annotation was performed on two platforms: on RAST [21] and on IMG/ER [22] (DOE Joint Genome Institute [23]). The tRNAscan-SE tool [24] (RAST and IMG/ER) was used to find tRNA genes, whereas ribosomal RNAs were detected using RMAmmer [25] (IMG/ER) and tool "search_for_rnas" (developed by Niels Larsen (available by the author), RAST). Open Reading Frames (ORFs) were predicted using GLIM-MER2 [26] in RAST and using GeneMark (v.2.6.r) [27] and GenePRIMP [28] as a part of the DOE-JGI genome annotation pipeline. Gene prediction analyses and functional annotations were performed in RAST with a series of BLAST against FIG hands-curated subsystems [29]. They were also analyzed with FIGfams collection databases, and through comparative approaches with Integrated Microbial Genome -Expert Review platform (RPS-BLAST, BLAST, BLASTp, Hmmsearch (HMMER)) against non-redundant databases including COGs, Pfam [30], TIGR-fam [31], KEEG [32], IMG. The total is based on either the size of the genome in base pairs or the total number of protein coding genes in the annotated genome

Not in COGs
The total is based on the total number of protein coding genes in the genome Additional functional annotations were performed within the SEED framework (RAST) [33] and the IMG/ER (DOE-JGI) platform.

Genome properties
The assembly of non-contiguous finished draft genome consists of 43 contigs representing overall 4,879,425 pb. The DNA G + C content was 53.08 %. Using RAST and IMG/ER, 5770 and 5795 genes were respectively predicted. Among them 4149 and 5660 protein-coding genes and 108 and 135 RNAs were identified by RAST and IMG/ER, respectively. The properties and the statistics of the genome (IMG/ER data) are summarized in Table 3 and Fig. 3. Putative COG functions were assigned for 39.28 % of the protein-coding genes. The distribution of genes into COGs functional categories is presented in Table 4.

Nitrate reduction
Nitrate respiration involves two distinct enzyme systems: the NapAB localized in the periplasm and the membranebound nitrate reductase NarGHI enzyme localized on the cytoplasmic face of the cytoplasmic membrane. The sole nitrate reductase of S. algae C6G3 is NapAB (Fig. 4). As most of the Shewanella species, S. algae C6G3 genome encodes the two NAP isoforms, each comprising three catalytic subunits: NapA where nitrate reduction takes place, a di-haem cytochrome NapB and a maturation chaperone NapD. The two isoforms present different membrane-intrinsic subunits [34] named NAP-α (NapE-DABC) and NAP-β (NapDAGHB). NAP-β (NapDAGBH) possesses NapGH, an iron-sulfur cluster ferredoxins instead of NapC. The functional differences between these systems may be explained by differential regulation in the composition of the available quinol pool. S. oneidensis MR-1 T encodes only Nap-β isoform in which NapC is lacking (Fig. 4). This membrane-anchored tetrahem c-Cyt mediates electron transport from the quinol pool to NapB. The function of NapC in NAP-β in S. oneidensis MR-1 T may be met by CymA, an homologue of periplasmic tetrahem c-Cyt of the NapC/NirT family, which is also found in S. algae C6G3 genome.

Dissimilative reduction of nitrite into ammonium
Nitrite can be reduced to ammonium (NH 4 ) by a periplasmic nitrite reduction system (NRF) [35]. As NAP Colored proteins are annotated from KEGG and putatively functional systems, there are two types of NRF: NrfABCD and NrfAH types. NrfA is the terminal reductase while NrfBCD/NrfH are responsible for electron transfer from menaquinol pool to NrfA. Shewanella strains are known for encoding NrfABCD system only. Genome annotations of S. algae C6G3 and S. oneidensis MR-1 T identify nrfA in both cases but nrfBCD were found in S. algae C6G3 only (Fig. 5). As previously described for NAP system, S. oneidensis MR-1 T genome lacks genes encoding for the specific compounds that deliver electrons to the terminal reductase (nrfBCD). Indeed, nrfB is lacking and nrfCD are present but proposed to be pseudogene because of truncation [36]. Gao et al., [37] suggested that the tetraheme c-Cyt CymA, a cytoplasmic membrane electron transport protein, is likely to be the functional replacement of both NapC and NrfBCD/ NrfH allowing to NrfAH-like system to be efficient in S. oneidensis MR-1 T .

Metal oxide reduction
S. algae C6G3 can utilize extracellular mineral metal oxides of Fe(III) and Mn(III/IV) as respiratory electron acceptors (unpublished data). Inspection of its genome confirmed the presence of genes involved in pathway (i.e. metal-reducing or MTR pathway) for transferring electrons from the inner membrane through the periplasm and across the outer membrane where metal oxides are reduced [38]. In S. algae C6G3 genome, genes that encode MtrCBA and OmcA are located in the same region, which also includes mtrD (an mtrA homologue), mtrE (an mtrB homologue) and mtrF (an mtrC homologue) (Fig. 6). Table 5 shows the BLASTP for amino acid sequences of MTR pathway in S. algae C6G3 versus those in metal-reducing S. oneidensis MR-1 T .
The numbers of genes found in the MTR clusters of the analyzed Shewanella strains varies from four, such as omcA1-mtrC-mtrA-mtrB in Shewanella frigidimarina [11], to nine, such as mtrD-mtrE-mtrF-omcA1-undB-omcA1-mtrC-mtrA-mtrB in Shewanella halifaxensis [39,40]. CymA identified as the entry point for electrons into the MTR pathway [41] is not located in the MTR gene cluster in S. algae C6G3 as described in S. oneidensis MR-1 T . Furthermore, S. algae C6G3 has an additional protein encoding for decahem c-Cyt of the OmcA/MtrC family. The role of this cytochrome is not defined. On RAST platform, this ORF has been annotated as mtrH in S. algae C6G3 and S. halifaxensis HAW-EB4 T .

Conclusion
The Shewanella genus comprises a diverse group of facultative anaerobes. Their ability to couple the oxidation of various carbon sources to the reduction of a broad range of terminal electron acceptors imparts a respiratory flexibility that allows colonization of varied and changeable marine and freshwater environments [39,42,43]. The occurrence of the two different NAP operons, NRF, and that of CymA in S. algae C6G3 accords with the renowned anaerobic respiratory flexibility of Shewanella. S. algae C6G3 is also capable of using solid Fe(III) and Mn(III/IV) as terminal electron acceptors. Reduction of these particulates occurs at the cell surface and is catalyzed by multihaem cytochromes whose properties are beginning to emerge.

Additional files
Additional file 1: Table S1. Presentation of positives carbon sources (Biolog GN2 microplateTM) & electron acceptors for S. algae strain C6G3 and ATCC 51192T and S. oneidensis MR-1T (differences are distinct in bold type). Table S2. Main fatty acids composition (90.6 %) of S. algae C6G3 and percentage of this fatty acids in S. algae ATCC 51192T (73.6 % of total pattern) and S. oneidensis MR-1T (92.5 % of total pattern).