High-quality draft genome sequence of Sedimenticola selenatireducens strain AK4OH1T, a gammaproteobacterium isolated from estuarine sediment

Sedimenticola selenatireducens strain AK4OH1T (= DSM 17993T = ATCC BAA-1233T) is a microaerophilic bacterium isolated from sediment from the Arthur Kill intertidal strait between New Jersey and Staten Island, NY. S. selenatireducens is Gram-negative and belongs to the Gammaproteobacteria. Strain AK4OH1T was the first representative of its genus to be isolated for its unique coupling of the oxidation of aromatic acids to the respiration of selenate. It is a versatile heterotroph and can use a variety of carbon compounds, but can also grow lithoautotrophically under hypoxic and anaerobic conditions. The draft genome comprises 4,588,530 bp and 4276 predicted protein-coding genes including genes for the anaerobic degradation of 4-hydroxybenzoate and benzoate. Here we report the main features of the genome of S. selenatireducens strain AK4OH1T.


Introduction
Selenium (Se) is an intriguing element in that microbes actively metabolize it through reduction, oxidation, methylation and demethylation reactions, using some of these to conserve energy. Of particular interest is the process of dissimilatory Se reduction, where the Se oxyanion, selenate [Se(VI)], is sequentially reduced to selenite [Se(IV)] and further to insoluble elemental Se(0). The ability to respire selenate/selenite is comparatively rare, nonetheless, is found in phylogenetically diverse anaerobes [1]. SeRB display a tremendous phylogenetic diversity, and yet the metabolic function seems to be conserved (or alternatively horizontally dispersed) in these unrelated groups. Furthermore, the physiologies of the known selenate-respiring bacteria appear to vary greatly. For example, they are able to couple growth to a wide range of electron acceptors such as arsenate, [2,3] cobalt oxide (Co(III)) [4], and tellurite [5] to name a few. SeRB have been isolated from a variety of different locations. A few examples are: in California in the San Joaquin Valley [6], from estuarine sediment in NJ [7], from a glass manufacturing plant in Japan [8], and from the dead sea [9].
Sedimenticola selenatireducens type strain AK4OH1 T (= DSM 17993 T = ATCC BA-1233 T ) is a member of the Gammaproteobacteria isolated from estuarine sediment for its unique ability to couple the oxidation of aromatic acids to selenate respiration. The genus Sedimenticola currently includes seven cultivated strains of which two species have been named and described: S. selenatireducens strain AK4OH1 T , the type strain of the type species for this genus [10], S. selenatireducens strain CUZ [11], S. thiotaurini strain SIP-G1 [12], Sedimenticola sp. strain Ke4OH1 [7], and Sedimenticola sp. strain NSS [11].
Here we summarize the physiological features of Sedimenticola selenatireducens AK4OH1 T and provide a description of its genome.

Organism information
Classification and features S. selenatireducens strain AK4OH1 T was isolated from estuarine sediment in the New York-New Jersey harbor estuary (40°586′N, 74°207′E) [10]. The position of strain AK4OH1 T relative to its phylogenetic neighbors is shown in Fig. 1. S. selenatireducens strain CUZ [11] is the closest relative to strain AK4OH1 T with a 16S rRNA gene similarity of 100 %, yet interestingly, it has not been found to respire selenate. In addition to these two, there are five other cultivated strains of the genus Sedimenticola: S. thiotaurini strain SIP-G1 T [12], Sedimenticola sp. strain NSS [11], and Sedimenticola sp. strain Ke4OH1 [7]. The isolate TT-Z (accession number AM292414) [13] groups among the Sedimenticola strains ( Fig. 1) suggesting that it is part of the Sedimenticola genus. The isolate IR (accession number AF521582) groups closely with strain AK4OH1 T and strain CUZ, and its position in the phylogenetic tree suggests that it Fig. 1 Phylogenetic analysis highlighting the position of Sedimenticola selenatireducens strain AK4OH1 T relative to its closest neighbors based on the 16S rRNA gene. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model [29]. The tree with the highest log likelihood (-3985.1130) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained by applying the Neighbor-Joining method to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 15 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 1276 positions in the final dataset. Evolutionary analyses were conducted in MEGA6 [30]. The strains and their corresponding GenBank accession numbers for 16S rRNA genes are listed in parentheses. The genome accession number and locus tag of strain AK4OH1 T are NZ_ATZE00000000.1 and A3GODRAFT_03746. (T = type strain). Bar: 0.01 substitutions per nucleotide position. C. okenii was used as an outgroup Cells of strain AK4OH1 T are Gram-negative and rodshaped [10] (Fig. 2 and Table 1). The strain can grow heterotrophically or lithoautotrophically under hypoxic and anaerobic conditions [12]. Motility is observed during early to mid-exponential growth on liquid MB2216 medium, but not in late exponential phase, and cell morphology varies depending on growth conditions [10,12].

Genome sequencing information
Genome project history S. selenatireducens strain AK4OH1 T was selected for sequencing in 2011 based on its phylogenetic position [14,15] and is part of the study Genomic Encyclopedia of Type Strains, Phase I: the one thousand microbial a 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 [36] genomes project (KMG-I) [16]. The goal of the KMG-I study was to increase the coverage of sequenced reference microbial genomes [17]. The Quality Draft (QD) assembly and annotation were made available for public access on June 18, 2014. Table 2 presents the project information and its association with MIGS version 2.0 compliance [18]. The NCBI accession number for the Bioproject is PRJNA165429. The genome accession number is ATZE00000000.1 consisting of 41 contigs (ATZE01000001-ATZE01000041) and 37 scaffolds.

Growth conditions and genomic DNA preparation
S. selenatireducens strain AK4OH1 T was grown in mineral salt medium at 28°C with 10 mM Na 2 SeO 4 as electron acceptor and 250 μM 4-hydroxybenzoate as carbon source, as previously described [10]. Genomic DNA was isolated from 0.5 g of cell paste using JetFlex Genomic DNA Purification Kit (GENOMED) as recommended by the manufacturer.

Genome sequencing and assembly
Sequencing was achieved using an Illumina [19] platform using a std paired-end library obtaining 273× fold coverage. The sequencing was done at the DOE Joint Genome Institute. ALLPATHS assembly software [20] was used to obtain 41 final contigs. Quality check and assembly statistics were performed at JGI. The raw sequences were screened against contaminants and 0.1 % of the reads were removed.

Genome annotation
Gene calling was performed using Prodigal 2.5 [21]. The genome sequence was analyzed using the Joint Genome Institute IMG system [22]. Ribosomal RNAs were predicted based upon sequence similarity, using BLAST, against the non-redundant nucleotide database and/or using Infernal and Rfam models. tRNA genes were found using tRNAscan-SE [23]. The predicted CDS were searched using the NCBI non-redundant protein database. The major metabolic pathways and predicted protein set were searched using KEGG, SwissProt, COG, Pfam, and InterPro protein databases implemented in the IMG. Additional gene prediction analysis and manual functional annotation were performed within IMG and using Artemis software (release 13.0, Sanger Institute).

Genome properties
The high quality draft genome sequence consists of 37 scaffolds that account for a total of 4,588,530 bp with a 56.6 % G + C content. In total, 4331 genes were predicted, 4276 of which are protein-coding genes, 55 RNA genes, and no pseudogenes. The majority of the predicted genes (79 %) were assigned a predicted function. The properties and statistics of the genome are summarized in Table 3 and Table 4.

Insights from the genome sequence
The respiratory flexibility of anaerobic prokaryotes allowing them to employ different terminal electron acceptors for respiration enables these organisms to thrive in dynamic redox environments. Among the enzymes that catalyze oxidation-reduction reactions of metals and metalloids are those that are highly conserved and belong to the DMSO reductase family [24]. Key members   The total is based on the total number of protein coding genes in the genome Fig. 3 Phylogenetic analysis highlighting the relation of Sedimenticola selenatireducens strain AK4OH1 T genes to known DMSO reductases by Maximum Likelihood method. The evolutionary history was inferred by using the Maximum Likelihood method based on the JTT matrix-based model [37]. The tree with the highest log likelihood (-17325.9218) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained by applying the Neighbor-Joining method to a matrix of pairwise distances estimated using a JTT model. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 13 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 724 positions in the final dataset. Evolutionary analyses were conducted in MEGA6 [30]. GenBank accession numbers are listed in parentheses. Bar = 0.5 substitutions per nucleotide position of the DMSO family of reductases, which transfer electrons to a variety of substrates that act as terminal electron acceptors for energy generation, are nitrate reductases (Nar, Nap, Nas), arsenate reductase (Arr), selenate reductase (Ser), and chlorate reductase (Clr), among others. S. selenatireducens strain AK4OH1 T can use nitrate, nitrite and selenate as the terminal electron acceptors for anaerobic growth, while using the electron donors acetate, lactate, pyruvate, benzoate, 3-hydroxybenzoate, and 4-hydroxybenzoate [10]. Chlorate and perchlorate can be used as electron acceptors when peptone is used as an energy source [12]. (Micro-)aerobic growth with oxygen as electron-acceptor and peptones as electron-donor is also detected [12] Within the AK4OH1 T genome, there are several likely DMSO reductases. Figure 3 shows the grouping of AK4OH1 T genes with closely matching, known, DMSO reductases. A3GODRAFT_03903 groups closely with the NapA, from Magnetospira sp. QH-2. A3GOD-RAFT_01428 clusters together with the NarG of Escherichia coli K-12 MG1655. Both of these genes are organized in gene clusters similar to known nap and nar operons [25]. BLAST searches of the AK4OH1 T genome using arsenate reductases showed no genes with significant similarity. This agrees with strain AK4OH1's inability to respire arsenate [10]. A3GODRAFT_02603 and A3GODRAFT_03351 from strain AK4OH1 T cluster closely with the chlorate reductase from Diaphorobacter sp. J5-51 and with the selenate reductase from Thauera selenatis. A3GODRAFT_02603, which groups closest with ClrA, resembles the gene organization of a clr operon [26]. While the only well-studied respiratory selenate reductase, serA, is from Thauera selenatis, A3GODRAFT_03351 and its neighboring genes follow the same organization as found with serABDC [27]. Gene A3GODRAFT_04296 clusters together with the perchlorate reductase from Dechloromonas aromatica, and appears to have the same gene organization as a pcr operon [28].

Conclusions
The complete genome of the estuarine bacterium Sedimenticola selenatireducens AK4OH1 T provides a stronger foundation from which to learn more about the process of dissimilatory selenate reduction. As AK4OH1 T was the first organism isolated capable of coupling the respiration of selenate to the oxidation of benzoic acids, its genome also provides a starting point for learning more about this unique capability.