Rhodohalobacter sulfatireducens sp. nov., isolated from a marine solar saltern

A novel Gram-stain-negative, oxidase-positive, catalase-positive, non-motile, facultatively anaerobic, rod-shaped bacterium, designated WB101T, was isolated from a marine solar saltern located in Wendeng, PR China. Strain WB101T shared a high level of 16S rRNA gene sequence similarity with Rhodohalobacter barkolensis 15182T (93.5%), R. halophilus JZ3C29T (93.2%), and ‘R. mucosus’ 8A47T (92.1%). Strain WB101T formed a species-level branch within the genus Rhodohalobacter in both phylogenetic and phylogenomic topologies. The DNA G + C content was 42.0%. Strain WB101T was found to have menaquinone-7 as the only respiratory quinone. The dominant cellular fatty acid (≥ 10%) was iso-C15:0. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylcholine. Characterisation based on phylogenetic, physiological, and biochemical properties indicated that strain WB101T represents a novel species of the genus Rhodohalobacter, and the name Rhodohalobacter sulfatireducens sp. nov. is proposed. The type strain is WB101T (= KCTC 92204T = MCCC 1H00518T).


Introduction
The genus Rhodohalobacter, belonging to the family Balneolaceae (Xia et al. 2016), was first established by Xia et al. (2017). At the time of writing, there were two valid species: R. halophilus (Xia et al. 2017) and R. barkolensis (Han et al. 2018), and one un-validated species: 'R. mucosus' (Wang Communicated by Erko Stackebrandt. The GenBank accession number for the 16S rRNA gene sequence of Rhodohalobacter sulfatireducens WB101 T is OM301685, and the draft genome has been deposited at DDBJ/ENA/GenBank under the accession JAKLWS000000000. 1 3 457 Page 2 of 10 et al. 2021), according to the List of Prokaryotic names with Standing in Nomenclature (LPSN) (https:// lpsn. dsmz. de). All these strains were isolated from saline environments, including salterns and saline lakes.
Microorganisms can metabolise sulphur in both oxidised and reduced states (Carbonero et al. 2012). In assimilatory sulphate reduction (ASR), sulphate is reduced to sulphide, which then participates in the synthesis of organic compounds, such as sulphur amino acids. Compared to dissimilatory sulphate reduction, ASR is a more favourable metabolic pathway for bacteria because no toxic sulphide is produced during this process.
In this study, we proposed a novel species with the basis of the results of polyphasic taxonomy, named Rhodohalobacter sulfatireducens sp. nov. Genome of the novel strain encodes a complete ASR pathway. This study increases the number of members of the genus Rhodohalobacter and enriches the research directions of studies on assimilatory sulphate-reducing bacteria.

Isolation and maintenance conditions
Coastal sediment was used as a bacterial isolation source, and was sampled from a marine solar saltern in Wendeng, PR China (122° 1′ 45″ E, 36° 59′ 38″ N) in November 2020. The sample was serially diluted 10 -4 times with sterilised seawater, and 0.1 mL aliquots of each dilution were spread onto marine agar 2216 (MA; BD). The plates were incubated at 33 °C for 2 weeks. Finally, tiny, reddish, convex, circular, and regular-edged colonies were isolated, purified, and subsequently designated as strain WB101 T . For long-term storage, the strain was stored at -80 °C in sterile 15% (v/v) glycerol supplemented with 5.0% (w/v) NaCl. 'R. mucosus' 8A47 T , R. halophilus JZ3C29 T (both isolated and studied in our laboratory), and R. barkolensis MCCC 1K03442 T (obtained from the Marine Culture Collection of China, MCCC) were collected as related type strains.

Phylogenetic and phylogenomic analyses
The 16S rRNA gene sequence was amplified by PCR using universal primers 27F and 1492R (Liu et al. 2014). After electrophoresis, the purified PCR product was recovered using an agarose gel DNA extraction kit (TaKaRa), ligated into the pMD19-T vector (TaKaRa), and cloned according to the manufacturer's instructions. Sequencing was performed by Tsingke Biotechnology Co., Ltd. (Qingdao, PR China) using universal primers M13F and M13R. The 16S rRNA gene sequence annotated from the genome sequences was submitted to GenBank. Similar sequences were identified using BLAST (https:// www. ncbi. nlm. nih. gov). The EzBioCloud identification service (https:// eztax on-e. ezbio cloud. net) (Kim et al. 2012) was used to determine similarity values amongst the sequences. Genomic DNA was extracted and purified using a bacterial genomic DNA extraction kit (TaKaRa) following the manufacturer's instructions. The draft genome was sequenced by Novogene Biotechnology Co., PR China using the Illumina HiSeq platform (Illumina, San Diego, CA, USA). Raw sequencing reads were assembled using ABySS v. 2.0.2 (https:// www. bcgsc. ca/ resou rces/ softw are/ abyss) (Simpson et al. 2009).
Phylogenetic trees were constructed using MEGA 7 (Kumar et al. 2016) with the neighbour-joining (NJ) (Saitou and Nei 1987) method based on the complete 16S rRNA gene retrieved from the genome sequences. Maximum-parsimony (MP) (Fitch 1971) and maximum-likelihood (ML) (Felsenstein 1981) methods were used to confirm the phylogenetic status of strain WB101 T . Evolutionary distances were calculated using the Kimura two-parameter model (Kimura 1980). Bootstrap analysis with 1,000 replications was used to evaluate the tree topologies.

Morphological, physiological, and biochemical analyses
The morphological and physiological features of strain WB101 T were examined after incubation at 37 °C on MA for three days. A scanning electron microscope (Nova NanoSEM 450; FEI) was used to observe cell morphology. A late exponential phase culture (three days, according to the growth the curves in Supplementary Fig. S1) in marine broth 2216 (MB; BD) was collected and fixed with 2.5% glutaraldehyde solution preserved at 4 °C. Gradient dehydration was then performed with 50%, 70%, 80%, 90%, and 100% ethanol before observation. Gram staining was performed using a Gram stain kit (Hopebio), according to the manufacturer's instructions. Motility was examined using a 3.0% (w/v) NaCl solution according to the hanging-drop method (Bowman 2000). Gliding was assessed using MB supplemented with 0.3% (w/v) agar, according to the method described by Bernardet et al. (2002). Oxidase activity was tested using an oxidase reagent kit (bioMérieux), according to the manufacturer's instructions. Catalase activity was determined by visible bubble production in 3% (v/v) H 2 O 2 solution.
The effects of different growth temperatures on MA were tested at 4, 15, 20, 25, 28, 30, 33, 35, 37, 40, 42, and 45 °C. Growth at different NaCl concentrations was assessed using a modified MA (1 g yeast extract L −1 , 5 g peptone L −1 , 0.1 g ferric citrate L −1 , and 18 g agar L −1 ), prepared with artificial seawater (0.32% MgSO 4 , 0.12% CaCl 2 , 0.07% KCl, and 0.02% NaHCO 3 , all w/v). The NaCl concentrations were adjusted from 0 to 13.0% (w/v) at 1.0% intervals. To test the effect of pH on growth, MB was adjusted to different pH levels with additional buffers (Sangon): MES (pH 5.5 and 6.0), PIPES (pH 6.5 and 7.0), HEPES (pH 7.5 and 8.0), Tricine (pH 8.5), and CAPSO (pH 9.0 and 9.5) at a concentration of 20 mM. The effects of temperature, NaCl tolerance, and pH on growth were recorded every 12 h in a 96-h-incubation (determined by growth curve) featuring visible colonies or optical density at 600 nm wavelength (OD 600 ). Anaerobic growth was determined at 37 °C for 3 weeks on modified MA and modified MA with the addition of 0.1% (w/v) KNO 3 in an anaerobic culture bag (Hopebio) with an oxygen indicator (Hopebio) and an AnaeroPack (MGC). Nitrate reduction ability was evaluated following the method described by Cowan and Steel (1974). H 2 S production was tested according to lead acetate papers (Wang et al. 2012) and producing black precipitates in triplesugar iron agar (TSI; BD). The hydrolysis of DNA, starch, casein, alginate, carboxymethyl cellulose (CMC), and esters, including Tweens 20, 40, 60, and 80, was investigated according to the methods of Smibert and Krieg (1994). Susceptibility to antibiotics was assessed as described by the Clinical and Laboratory Standards Institute (CLSI) (2012) using MA at 37 °C. The sizes of the inhibition zones generated by different drug-sensitive papers were measured until visible bacterial lawns were observed.
Other physiological and biochemical characteristics were tested using API 20NE, API ZYM, and API 50CH (bioMérieux). The oxidising potential of strain WB101 T for various carbon sources was assessed using the Biolog GEN III. All API and Biolog tests were performed with three related type strains, according to the manufacturer's instructions, except that the NaCl concentration was adjusted to 5.0%, 3.0% and 10.0% (w/v), respectively, which were their optimal salinities.

Chemotaxonomic analyses
To determine the respiratory isoprenoid quinones and polar lipid composition, cells of strain WB101 T and related type strains were cultured in MB at 37 °C until the bacterial communities reached the late exponential growth stage (according to the growth curve). Respiratory isoprenoid quinones were extracted from 300 mg of freeze-dried cell material and separated into different classes using thin-layer chromatography (TLC) on silica gel. The effective components were removed from the plate by relying on the spots and analysed further using reverse-phase high-performance liquid chromatography (HPLC) according to the methods described by Kroppenstedt (1982). Polar lipids were extracted from 100 mg of fresh cell material and separated via TLC on silica gel plates (8 × 8 cm, no. 5554; Merck) according to the methods described by Tindall et al. (2007). Total lipid material was detected using molybdatophosphoric acid solution, and specific functional groups were tested using additional staining reagents (Sigma-Aldrich) on three other separate TLC plates, which included molybdenum blue solution (phosphates), α-naphthol sulfuric solution (carbohydrates), and ninhydrin solution (amines).
To determine the fatty acid composition, cells of strain WB101 T , and related type strains were cultured on MA at 37 °C until the bacterial communities reached the late exponential growth stage (according to the four-quadrant streak method) (Sasser 1990). Fatty acids were extracted from 40 mg of fresh cell material, saponified, methylated, and extracted using the standard protocol of the Sherlock Microbial Identification System (MIDI) equipped with an Agilent model 6890 N gas chromatograph. Fatty acids with percentages were determined and calculated using MIS standard software with the TSBA40 database (Buyer 2002).

Phylogenetic and phylogenomic analyses
The only complete 16S rRNA gene sequence (1,532 bp) extracted from the draft genome shared 99.9% similarity with nearly complete sequences (1,505 bp) obtained by conventional Sanger sequencing, which confirms their authenticity. Strain WB101 T exhibited the highest similarity to R. barkolensis 15182 T (93.5%), followed by R. halophilus JZ3C29 T (93.2%), 'R. mucosus' 8A47 T (92.1%), and other valid species of the family Balneolaceae (on the edge of or lower than 90.0%). The phylogenetic tree based on the NJ algorithm demonstrated that strain WB101 T was distinct from R. halophilus JZ3C29 T , R. barkolensis 15182 T , and 'R. mucosus' 8A47 T within the genus Rhodohalobacter at a high confidence level (Fig. 1), which was also emphasised by additional MP and ML phylogenetic trees.
The draft genome of strain WB101 T was 5,104,032 bp in size, and the DNA G + C content was 42.0%. A total of 138 contigs were assembled from the raw sequences, and the N50 value and coverage depth were 123,641 and 275.0 × , respectively. The detailed contents of the tRNAs coded by strain WB101 T were reverse-complemented into codons and are shown in Supplementary Table S1. Strain WB101 T had a significantly larger genome than members of the genus Rhodohalobacter, although they all shared a similar number of tRNAs and G + C content. Detailed genomic information on strain WB101 T and related type strains is listed in Table 1. The OrthoANIu, dDDH, AAI, POCP, and TETRA values between strains WB101 T and 'R. mucosus' 8A47 T , R. barkolensis 15182 T , and R. halophilus JZ3C29 T are shown in Table 2. The OrthoANIu values between each of these strains were far lower than 95.0-96.0%, the threshold for identifying potential novel species (Richter and Rosselló-Móra 2009). Moreover, the ANIb and ANIm values were below 90.0%. All dDDH values were below the threshold  (Li et al. 2010). The TETRA values were lower than 0.99, which is the threshold for novel species (Richter and Rosselló-Móra 2009). For the taxonomic boundaries for genera, all AAI and POCP values between strain WB101 T and each of the three related type strains were over 60.0% and 50.0%, respectively, which were argued as genus boundaries (Rodriguez-R and Konstandtinidis 2014; Qin et al. 2014). Additionally, the same taxonomic status shown by the phylogenetic trees was also demonstrated by the phylogenomic tree based on the GTDB (Fig. 2).

Genomic analyses
No known secondary metabolite clusters were identified with high similarity according to the antiSMASH. Compared to other genus Rhodohalobacter members, strain WB101 T uniquely has a non-ribosomal peptide synthetase cluster (NRPS), suggesting that an unknown peptide synthesis pathway is encoded by its genome. Unclassified hserlactone, terpene, and T3PKS were encoded by strain WB101 T and three related type strains. OrthoVenn2 analysis clustered 7211 protein entries into 1789 clusters shared by strain WB101 T and the three related type strains. Figure 3 shows the overlapping cluster relationships. Strain WB101 T contained 442 unique protein entries, clustered into 151 clusters. Furthermore, 103 entries of these unique proteins were annotated by KEGG and were mainly responsible for carbohydrate metabolism, glycan biosynthesis and metabolism, and unclassified metabolism.
Based on the RAST programme, the galactosylceramide and sulphatide metabolism pathways encoded by the strain WB101 T genome were more abundant than those of the three related type strains. The results of analyses by KEGG Orthology and Links Annotation (KOALA) indicated that the genome contained enzymes involved in ASR. The sulphate reduction occurs within cells of strain WB101 T with adenylylsulphate kinase (cysC), sulphate adenylyltransferase subunit 1 (cysN), sulphate adenylyltransferase subunit 2 (cysD), phosphoadenosine phosphosulphate reductase (cysH), and sulphite reductase (sir). As an intermediate during this process, 3′-phosphoadenosine-5′-phosphosulphate (PAPS) is reduced to sulphite. Then, sulphide is transformed to l-cysteine by O-acetyl serine-(thiol)-lyase (Schiff 1979). The sulphate/thiosulphate transport system encoded by the cysPUWA operon, an ATP-binding cassette (ABC) type transporter, mediates high-affinity sulphate and thiosulphate uptake (Kushkevych et al. 2020). Thus, the lack of cysPUWA operon suggests that strain WB101 T might not utilise extracellular sulphates. According to the genomic data from GenBank, the assimilatory sulphate reduction pathways were incomplete in 'R. mucosus' 8A47 T and R. barkolensis 15182 T (both lacking cysH and sir) and were even absent in R. halophilus JZ3C29 T (lacking cysC, cysN, cysD, cysH and sir). Conversion amongst polysulphides can be achieved by the sulfhydrogenase subunits gamma, beta, alpha, and delta (hydG, hydB, hydA, and hydD) (Ma et al. 2000). Dissimilatory or assimilatory nitrate reduction was incomplete within the genus Rhodohalobacter, as confirmed by the experimental results. Ribokinase (rbsK) allows cells of strain WB101 T to transform d-ribose 5-phosphate to d-ribose. Phosphatidylcholine (lecithin) can be synthesised using cdp-diacylglycerol and phosphatidylcholine synthase (pcs). In summary, compared to three related type strains, strain WB101 T had more numerous metabolic pathways, which explains why it had a relatively larger genome and demonstrates its potential application value.
According to API 20NE, strain WB101 T and the three related type strains were negative for nitrate reduction and indole production. Compared to R. barkolensis MCCC 1K03442 T and R. halophilus JZ3C29 T , strain WB101 T had a stronger positive effect on the assimilation of capric acid. Positive reactions for β-galactosidase and N-acetylβ-glucosaminidase differentiated strain WB101 T from the three related type strains, and negative reactions for lipase (C14) and α-fucosidase were consistent in the genus Rhodohalobacter. Strain WB101 T oxidised d-glucuronic acid and glucuronamide more strongly than the other three related type strains.
The integrated morphological, physiological, and biochemical characteristics of strain WB101 T are provided in the species description. The details that distinguish strain WB101 T from the related type strains are summarised in Table 3. Additional Supplementary Table S2 lists the traits of strain WB101 T and the generic traits of neighbouring genera of the family Balneolaceae.

Chemotaxonomic analyses
The sole respiratory quinone of strain WB101 T was menaquinone-7 (MK-7), which was consistent with species of the genus Rhodohalobacter. Diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), and phosphatidylcholine (PC) were the major polar lipids detected in strain WB101 T . Additionally, minor amounts of an unidentified phospholipid (PL1), three glycolipids (GL1, GL2, and GL3), and four unidentified lipids (L1, L2, L3, and L4) were present. Compared to the three related type strains, cells of strain WB101 T could synthesise lecithin, which was also identified in genomic analyses. The absence of aminolipids (AL1, AL2, and AL3) could differentiate strain WB101 T from 'R. mucosus' 8A47 T and R. barkolensis MCCC 1K03442 T . Further details of the polar lipid content of strain WB101 T and the three related type strains are shown in Supplementary Fig. S3. High iso-C 15:0 content was detected in strain WB101 T (53.8%), and in other members of the genus Rhodohalobacter, which was considered the major fatty acid (≥ 10.0%). As minor fatty acids (≥ 1.0% and ≤ 10.0%), C 12:0 and C 16:0 appeared in the cells of the four strains. Detailed discrepancies between strain WB101 T and related type strains are listed in Supplementary Table S3.

Conclusion
Combined with the results of the genotypic, phenotypic, and chemotaxonomic analyses, the similarities and differences between strain WB101 T and other related taxa were explicitly demonstrated. Based on the phylogenetic and phylogenomic tree topologies, we concluded that strain WB101 T belongs to the genus Rhodohalobacter but differs from 'R. mucosus', R. barkolensis, and R. halophilus at a novel species level. Therefore, Rhodohalobacter sulfatireducens sp. nov. was proposed, with strain WB101 T as the type strain.
The type strain WB101 T (= KCTC 92204 T = MCCC 1H00518 T ) was isolated from a marine solar saltern in Weihai, PR China.
The GenBank accession numbers are OM301685 for the 16S rRNA gene and JAKLWS000000000 for the draft genome.