Non-contiguous finished genome sequence and description of Sulfurimonas hongkongensis sp. nov., a strictly anaerobic denitrifying, hydrogen- and sulfur-oxidizing chemolithoautotroph isolated from marine sediment

Here, we report a type strain AST-10 representing a novel species Sulfurimonas hongkongensis within Epsilonproteobacteria, which is involved in marine sedimentary sulfur oxidation and denitrification. Strain AST-10T (= DSM 22096T = JCM 18418T) was isolated from the coastal sediment at the Kai Tak Approach Channel connected to Victoria Harbour in Hong Kong. It grew chemolithoautotrophically using thiosulfate, sulfide or hydrogen as the sole electron donor and nitrate as the electron acceptor under anoxic conditions. It was rod-shaped and grew at 15-35°C (optimum at 30°C), pH 6.5-8.5 (optimum at 7.0-7.5), and 10-60 g L-1 NaCl (optimum at 30 g L-1). Genome sequencing and annotation of strain AST-10T showed a 2,302,023 bp genome size, with 34.9% GC content, 2,290 protein-coding genes, and 42 RNA genes, including 3 rRNA genes.


Introduction
The genus Sulfurimonas was formally proposed in 2003, and included only one species, Sulfurimonas autotrophica OK10 T , at that time [1]. Since then, several novel species have been identified, such as Sulfurimonas paralvinellae GO25 T [2], Sulfurimonas denitrificans DSM 1251 T (reclassified, previously known as Thiomicrospira denitrificans) [2], and Sulfurimonas gotlandica GD1 T [3]. Here, we report another novel species, Sulfurimonas hongkongensis AST-10 T , isolated from coastal sediment, and describe its features, together with the genome sequencing and annotation. Currently, all known Sulfurimonas members were isolated from marine sediments except for strain GD1 from deep seawater [4]. The most widely shared feature of Sulfurimonas members is chemolithoautotrophy; strains can grow by oxidizing hydrogen gas, elemental sulfur, hydrogen sulfide, or thiosulfate [1][2][3][4][5][6][7]. In our previous studies, anoxic sulfur-oxidizing bacteria were demonstrated to dominate the nitrate induced marine sedi-ment remediation process [8][9][10]. Phylogenetic analysis based on 16S rRNA genes showed that Epsilonproteobacteria closely related to S. denitrificans constituted the major bacterial population during such remediation of the sediment at Kai Tak Approach Channel, Hong Kong, China. Strain AST-10 T was isolated from the sediment and named Sulfurimonas hongkongensis sp. nov., based on its unique physiological and phylogenetic characteristics.

Classification and features
Sediment was collected 10-50 cm below the seawater/sediment interface at the Kai Tak Approach Channel connected to Victoria Harbor in Hong Kong, China. Sewage and industrial effluent had been discharged there for decades until the installation of a new sewage collection system in the late 1990s. The long lasting sulfate-reducing conditions resulted in a high sulfide concentration in the sediment, where an AVS (Acid-Volatile Sulfide) of 198 μmol g -1 had been measured [8]. The pore water after centrifugation at 4,000 rpm for 15 min had a pH of 7.89 and a salinity of 2.9%.
Enrichments were prepared by adding 20 g of wet sediment (32.0% dry matter) to serum bottles containing 70 mL of sterilized seawater, purged with N2 and incubated for at least 24 h at room temperature. Potassium nitrate (1 g L -1 ) and sodium phosphate, monobasic (0.1 mmol L -1 ), were then added from sterilized stock solutions. The bottles were incubated at 28°C in a water bath for 72 h. The enrichments were plated onto agar plates of DSM113-S medium, a salinity modified version of DM113 medium that is recommended by DSMZ for nitrate-reducing and sulfideoxidizing bacteria. One liter of DSM113-S contained: KH2PO4 (2.0 g), KNO3 (4.0 g), NH4Cl (1.0 g), MgSO4·7H2O (0.8 g), Na2S2O3·5H2O (5.0 g), NaHCO3 (1.0 g), FeSO4·7H2O (2.0 mg), NaCl (25.0 g) and 2 ml of trace element solution SL-4. Solid media contained 1.5% bacterial agar from Difco. All media were sterilized by autoclaving and cooled under N2 atmosphere. Colonies formed on plates were picked and further purified by re-streaking single colonies on agar plates for more than 20 rounds (4-10 d round -1 ). A colony isolated and purified from the above process was defined as strain AST-10 T .
The 16S phylogenetic tree shown in Figure 1 indicated that strain AST-10 T is a member of the genus Sulfurimonas, (Table 1). An online BLAST query in NCBI using the 16S rRNA gene sequence from strain AST-1 T showed a relatively low identity to all currently identified Sulfurimonas species, including S. denitrificans DSM 1251 T (97% identity), S. gotlandica GD1 T (95% identity), S. autotrophica OK10 T (95% identity), and S. paralvinellae GO25 T (94% identity). Using the commonly accepted criterion of a 97% 16S rDNA sequence similarity cutoff for defining species [19,20], strain AST-10 T could accordingly be identified as a novel species within the genus Sulfurimonas.  , 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 [18].
Cell morphology was examined by Scanning Electron Microscopy (SEM). As shown in Figure 2, the cells of AST-10 T were rod-shaped, 0.2-0.4 μm in diameter, and 0.5-1.2 μm in length. On solid medi-um, AST-10 T grew and formed small, white, transparent, round shaped colonies with smooth boundaries.

Physiology
Effects of temperature, pH, and salinity on the growth of strain AST-10 T were investigated, showing that it grew at 15-35°C (optimum at 30°C), pH 6.5-8.5 (optimum at 7.0-7.5), and 10-60 g L -1 NaCl (optimum at 30 g L -1 ). The generation time of strain AST-10 T under optimal conditions was tested as 6.1 h. It was significantly shorter than other species, such as S. paralvinellae GO25 T and S. denitrificans DSM 1251 T . The cell yield of strain AST-10 T was 5.2 g dry weight per mole of S2O3 2-. This value is similar to that of its Epsilonproteobacterial relative S. denitrificans DSM 1251 T (5.72 g), but only about one-half of the Betaproteobacterial Thiobacillus denitrificans (11.6 g). Such difference in growth efficiency might be attributed to the different pathways used for carbon fixation and metabolism.
To determine whether electron acceptors other than NO3 -would sustain the growth of strain AST-10 T , SO4 2-, NO2 -, Fe 3+ , and O2 were separately tested with S2O3 2-as the sole electron donor. No growth was observed using any of these electron acceptors. S2O3 2-, HS -, and H2 can support the growth of strain AST-10 T as electron donors, however, acetate, lactate, malate, formate, pyruvate, glucose, glycerol, and yeast extract cannot. Hence, strain AST-10 T was a chemolithoautotroph, using NO3 -as an electron acceptor and S2O3 2-, HS -, or H2 as an electron donor. The time course of S2O3 2-oxidation and NO3 -reduction during strain AST-10 T growth was monitored. N2 was the dominant denitrification product, no accumulation of N2O and NO2 -was detected, when it was cultivated using DSM113-S at 30°C and pH 7.5. Significant production of insoluble S 0 occurred when it was cultured with an excess amount of S2O3 2-(molar ratio of S2O3 2-/NO3 -> 2). SO4 2-became the dominant oxidation product under excess NO3 -conditions (molar ratio of S2O3 2-/ NO3 -< 0.25). This was quite similar to the well-characterized strain Thiomicrospira CVO [21]. But for S. denitrificans DSM 1251 T , no accumulation of insoluble S 0 was observed even under a high molar ratio of S2O3 2-/NO3 - [5].

Genome sequencing and annotation Genome project history
The strain was selected for genome sequencing on the basis of its 16S rRNA gene-based phylogenetic position within the genus Sulfurimonas (Table 1). It is the first sequenced genome of Sulfurimonas hongkongensis sp. nov. A summary of the genome sequencing project information is shown in Table  2. The genome consists of 28 contigs, which has been deposited at DDBJ/EMBL/GenBank under accession number AUPZ00000000. The version described in the present study is the first version.

Growth conditions and DNA isolation
As described above, the strain was grown in DSM113-S medium under anoxic condition with optimal growth at 30°C, pH7.0-7.5, and NaCl 30 g L -1 . The genomic DNA used for shotgun sequencing was prepared by DSMZ.

Genome sequencing and assembly
The genome shotgun sequencing project was finished by BGI (Beijing Genomics Institute). Briefly, DNA was first mechanically fragmented with an enrichment size of ~500 bp. Then the DNA fragmentation was gel purified and quality checked. The recycled DNA was used for shotgun library construction, which was finally sequenced on an Illumina HiSeq 2000 platform using the pairedend 150 bp sequencing strategy. A total of 6,932,096,700 bp of raw sequence was obtained, which was assembled with CLC Genomics Workbench 6.0.2 using a word size of 40 bp. The draft genome was finally assembled into 28 contigs with a 2,302,023 bp genome size and more than 3,000 fold genome coverage (Table 3).

Genome annotation
The draft genome was annotated by NCBI Prokaryotic Genome Annotation Pipeline (PGAP). Protein-coding genes with function prediction were calculated based on the PGAP result. The COGs (Clusters of Orthologous Groups) functional annotation was conducted by PRSBLAST search against COGs database with an E-value cutoff 1e-10 [22,23]. Pfam domains were annotated using HMMER 3.0 program on Pfam database with an Evalue cutoff 1e-10 [24,25]. SignalP 4.1 Server was employed to analyze proteins with signal peptide [26]. TMHMM Server 2.0 was used to predict transmembrane helices in proteins [27].

Genome properties
The draft genome of Sulfurimonas hongkongensis AST-10 T was assembled into 28 contigs with a total size of 2,302,023 bp and a GC content of 34.9%. 2,332 genes were annotated, 2,290 of which were protein-coding genes. The remaining 42 genes were RNA genes including 3 rRNA genes. A total of 1,146 of the protein-coding genes were assigned putative functions. The remaining 1,144 protein-coding genes were annotated as hypothetical proteins. The AST-10 T genome properties and statis tics are summarized in Tables 2-4 and Figure 3. a 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, b Also includes 54 pseudogenes and 5 other genes.

Conclusion
Description of Sulfurimonas hongkongensis sp. nov.
The type strain AST-10 T = DSM 2096 T = JCM 18418 T , was isolated from coastal sediment at the Kai Tak Approach Channel connected to Victoria Harbour in Hong Kong, China. The GC content of the genome is 34.9%. The genome sequence has been deposited at DDBJ/EMBL/GenBank under accession number AUPZ00000000.