Pseudodesulfovibrio alkaliphilus, sp. nov., an alkaliphilic sulfate-reducing bacterium isolated from a terrestrial mud volcano

The diversity of anaerobic microorganisms in terrestrial mud volcanoes is largely unexplored. Here we report the isolation of a novel sulfate-reducing alkaliphilic bacterium (strain F-1T) from a terrestrial mud volcano located at the Taman peninsula, Russia. Cells of strain F-1T were Gram-negative motile vibrios with a single polar flagellum; 2.0–4.0 µm in length and 0.5 µm in diameter. The temperature range for growth was 6–37 °C, with an optimum at 24 °C. The pH range for growth was 7.0–10.5, with an optimum at pH 9.5. Strain F-1T utilized lactate, pyruvate, and molecular hydrogen as electron donors and sulfate, sulfite, thiosulfate, elemental sulfur, fumarate or arsenate as electron acceptors. In the presence of sulfate, the end products of lactate oxidation were acetate, H2S and CO2. Lactate and pyruvate could also be fermented. The major product of lactate fermentation was acetate. The main cellular fatty acids were anteiso-C15:0, C16:0, C18:0, and iso-C17:1ω8. Phylogenetic analysis revealed that strain F-1T was most closely related to Pseudodesulfovibrio aespoeensis (98.05% similarity). The total size of the genome of the novel isolate was 3.23 Mb and the genomic DNA G + C content was 61.93 mol%. The genome contained all genes essential for dissimilatory sulfate reduction. We propose to assign strain F-1T to the genus Pseudodesulfovibrio, as a new species, Pseudodesulfovibrio alkaliphilus sp. nov. The type strain is F-1T (= KCTC 15918T = VKM B-3405T).


Introduction
Dissimilatory sulfate-reducing bacteria are widespread in nature and play a significant role in the global cycling of carbon and sulfur (Rabus et al. 2015). Majority of the cultivated sulfate-reducers belong to the phylum Desulfobacterota among which the class Desulfovibrionia is one of the largest (Waite et al. 2020). The first strain of Desulfovibrio was isolated by Beijerink in 1895 and since then more than 100 species of Desulfovibrio have been described (Parte et al. 2020-https://lpsn.dsmz.de/genus/ pseudodesulfovibrio). In 2016 four species of Desulfovibrio were reclassified into the new genus-Pseudodesulfovibrio, mainly according to 16S rRNA gene phylogeny (Cao et al. 2016). In 2020, Desulfovibrio species were subdivided into 13 genera based on the analysis of 120 conserved single-copy marker genes (Waite et al. 2020).
In this study, we report the isolation of an alkaliphilic sulfate-reducing strain F-1 T from a terrestrial mud volcano and describe its physiological, metabolic and genomic properties. Our data suggest that strain F-1 T belongs to the genus Pseudodesulfovibrio, but differs from other species of this genus. Thus we propose to assign strain F-1 T to a new species, Pseudodesulfovibrio alkaliphilus sp. nov.

Materials and methods
Origin of the strain Strain F-1 T was isolated from a sample of mud collected from the active gryphon of terrestrial mud volcano Gnilaya Gora, Taman Peninsula, Krasnodarsky Krai, Russia. Coordinates of the sampling point were 45.251°N, 37.436°E. Samples were collected in May 2017, from the upper 20 cm of mud, pH 8.5, temperature 21°C, 15.7 mM Cl -, 5.3 mM SO 4 2-. Samples were taken anaerobically in plastic tightly stoppered bottles and transported to the laboratory.

Media and cultivation
Strain F-1 T was isolated in pure culture after successive cultivations, using anaerobically prepared, bicarbonate-buffered liquid medium of the following composition (per liter distilled water): 0.33 g KH 2-PO 4 , 0.33 g NH 4 Cl, 0.33 g KCl, 0.33 g CaCl 2 Á6H 2 O, 2.00 g NaHCO 3 , 0.33 g MgCl 2 Á6H 2 O, 10.00 g NaCl, 0.63 g Na 2 SÁ9H 2 O, 0.001 g resazurin, 1 mL of a vitamin solution (Wolin et al. 1963) and 1 mL of a trace element solution (Slobodkin et al. 2012). The medium was prepared by boiling and cooling it under N 2 flow, and then the reducing agent (0.66 g Na 2-SÁ9H 2 O) was added. The medium was dispensed in 10 mL portions into 17 mL Hungate tubes and autoclaved at 121°C for 60 min; the headspace was filled with N 2 . The pH of the sterile medium was 9.0. Magnesium sulfate (14 mM) and sodium lactate (10 mM) were added from the sterile stock solutions before the inoculation of the sample.

Phenotypic characterization
Growth experiments were performed in triplicate. For morphological, physiological, and metabolic characterization, strain F-1 T was cultivated in the same media used for isolation unless noticed otherwise. The effects of temperature, pH and salinity on growth were examined in the reduced medium with magnesium sulfate and sodium lactate. The range of NaCl concentrations for growth was evaluated at 0%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 5%, 6%, 7% (w/v) NaCl concentrations. The range of pH for growth was determined at 6.0-12.0 with 0.5 intervals and the range of temperature from 4 to 50°C with 5°C intervals. The cell-wall structure was examined using the Gram method (Beveridge et al. 2014). The growth of bacteria was determined by direct counting of the cells in the aliquot of a liquid culture with a phase contrast microscope (Zeiss Primo Star) and a counting chamber. Transmission electron microscopy was performed with a model JEM-100 electron microscope (JEOL) as described previously (Bonch-Osmolovskaya et al. 1990). Soluble substrates for growth were added from sterile anaerobic stock solutions before inoculation. Elemental sulfur was added in each Hungate tube with liquid medium. Medium with poorly crystalline Fe(III) oxide (ferrihydrite) was prepared as described previously (Slobodkin et al. 1999). Determination of gaseous products of metabolism was performed by Gas Cromatography equipped with a HayeSep N 80/100 mesh column at 40°C and flow rates of 20 ml min -1 (argon was used as a carrier gas). Sulfide was measured colorimetrically with dimethyl-pphenylenediamine (Trüper and Schlegel 1964). The ability of the strain to grow aerobically was tested in 50 ml bottles sealed with a rubber stopper and aluminum screw cap containing 10 ml aerobically prepared medium (100% air in the gas phase). For checking microaerobic growth, various amounts of air were injected in the headspace of bottles containing anaerobically prepared non-reduced medium.

Chemotaxonomic characterization
For chemotaxonomic analyses strain F-1 T was grown in the same media used for isolation; the cells were harvested in the late exponential phase of growth (48 h) collected by centrifugation, and freeze-dried. Cellular fatty acids were converted to methyl esters by the direct methylation with HCl/MeOH, extracted, and analyzed with GC-MS as described elsewhere (Slobodkina et al. 2020).
16S rRNA gene analysis and genome sequencing, assembly, annotation and comparison DNA for the 16S rRNA gene and complete genome sequencing was obtained using the FastDNA Spin Kit (MP Bio) following the manufacturer's protocol. The 16S rRNA gene was amplified using universal primers for bacteria 27F, 357F, 530F, 1114F, 342R, 519R and 1492R (Weisburg et al. 1991). Sequencing of PCR products was carried out using the Sanger method. The 16S rRNA gene sequence of the isolate was compared with other sequences in GenBank (Benson et al. 1999) using the BLAST program (Altschul et al. 1990) and by means of the EzBio-Cloud server (Yoon et al. 2017; http://www.ezbio cloud.net) to identify it closest relatives. Alignment with a representative set of related 16S rRNA gene sequences was carried out using the ClustalW program implemented in the phylogenetic analysis package MEGA version 7.0 (Kumar et al. 2016). Bootstrap consensus trees were inferred from 1000 replicates (Felsenstein 1985) using the maximum-likelihood method based on the Tamura-Nei model, as well as the neighbor-joining and the minimum evolution methods (Rzhetsky and Nei 1992;Hazkani-Covo and Graur 2007) provided by MEGA version 7.0.
The GenBank/EMBL accession number of 16S rRNA gene sequence of the strain F-1 T is MN601397.
The genome of strain F-1 T was sequenced using MiSeq system (Illumina, San Diego, California, USA. Whole-genome sequence allowed us to specify the taxonomic position of strain F-1 T using two methods: Average Nucleotide Identity (ANI) provided by EzBioCloud ANI calculator (https://www. ezbiocloud.net/tools/ani) (Yoon et al. 2017) and the genome-to-genome distance method (GGDC) with the GGDC 2.0 BLAST ? model provided by Genome-to-Genome Distance Calculator (http://ggdc.dsmz.de) (Meier-Kolthoff et al. 2013). Gene search and annotation were performed using the RAST server (Brettin et al. 2015). SEED viewer was used for the assignment of the predicted genes to subsystem categories (Overbeek et al. 2014). Additionally, the Integrated Microbial Genomes non-redundant database, Pfam, KEGG and COG databases were used for genome analysis.
The Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accessionWODC00000000.

Enrichment and isolation
For the initial enrichment, the mud sample was inoculated (10% w/v) into sterile anaerobic liquid medium with lactate and sulfate as the growth substrate. After 2 days of incubation at 30°C, microbial growth was observed expressing in media turbidity. After three subsequent transfers and following serial tenfold dilutions in the same medium, only one morphological type was observed in the highest positive dilution (10 -9 ). Attempts to obtain separate colonies either anaerobically in agar blocks or aerobically on the surface of the medium with 1.5% of agar were unsuccessful. The purity of strain F-1 T was assessed by routine microscopic examination and confirmed by results of 16S rRNA gene and complete genome sequencing.

Phenotypic and chemotaxonomic characteristics
Mid-exponential-phase cells of strain F-1 T grown on sulfate and lactate were motile vibrios with a single polar flagellum, 2.0-4.0 lm in length and 0.5 lm in diameter (Fig. 1a). Cells stained Gram-negative in both the exponential and the stationary growth phases. The formation of endospores was not observed in the cultures grown under optimal or suboptimal conditions. Ultrathin sections of the strain F-1 T revealed a Gram-stain-negative cell wall type with an outer membrane (Fig. 1b). No intracellular membranes were observed.
The temperature range for growth of strain F-1 T was 6-37°C, with an optimum at 24°C. No growth was detected at 4°C or below and 42°C or above after incubation for a month. The pH range for growth was 7.0-10.5, with an optimum at pH 9.5. No growth was observed at pH values 6.5 or bellow or 11.0 or above. Growth of strain F-1 T was observed at NaCl concentrations from 0.3 to 3.0% (w/v) with an optimum at 0.5-1.0%, no growth was evident at 3.5% (w/v) NaCl or above. The doubling time on lactate/SO 4 2under optimal growth conditions was 1.47 h. Addition of yeast extract (0.1 g/l) did not stimulate growth.

Phylogeny
The 16S rRNA gene sequences of strain F-1 T obtained by amplification with universal bacterial primers and retrieved from whole-genome data were identical. A comparison of 1541 nucleotides of 16S rRNA gene sequences of strain F-1 T with those available in GenBank (Benson et al. 1999) and EzBioCloud (Yoon et al. 2017) databases showed that the novel isolate belongs to the genus Pseudodesulfovibrio and had the highest sequence similarity to Pseudodesulfovibrio aespoeensis DSM 10631 T (98.05%) and Pseudodesulfovibrio indicus J2 T (96.00%). The 16S rRNA gene phylogenetic tree reconstruction revealed that the strain F-1 T constituted a monophyletic branch clearly separated from the most closely related species (Fig. 2).
Pairwise ANI value of the genome of the strain F-1 T and the genome of the closest relative organism, P. aespoeensis (DSM 10631 T ) was 82.07%. The in silico DDH value predicted between strain F-1 T and P. aespoeensis (DSM 10631 T ) by the recommended formula 2, was 24.50%. Both these values are much lower than the threshold for prokaryotic species delineation proposed to be 95-96% (ANI) and 70% (DDH) (Meier-Kolthoff et al. 2013, Rodriguez-R and Konstantinidis 2016).

Genome analysis
The draft genome assembly of strain F-1 T has a total length of 3,227,153 bp and N50 value of 302,886 bp within 29 contigs and the genomic DNA G ? C content was 61.93 mol%. The genome of F-1 T was predicted to contain 3061 protein-coding sequences and 54 RNA genes. A total of 1914 coding sequences were assigned to non hypothetical and 1147 to hypothetical proteins. Most of the annotated genes were responsible for the synthesis of amino acids and derivatives (167), protein metabolism (155), cofactors, vitamins, prosthetic groups and pigment formation (86) (Supplementary Table S2 and Figure S2).
The genome of strain F-1 T contains a full set of genes required for dissimilatory sulfate reduction (Pereira et al. 2011) including sulfate adenylyltransferase, manganese-dependent inorganic pyrophosphatase, APS reductase subunits AprA and AprB, the subunits of dissimilatory sulfite reductase DsrABCD, and electron transfer complexes DsrMKJOP and QmoABC (hereinafter see references in Supplementary Table S3).
The genome of strain F-1 T possessed all genes for glycolysis via the Embden-Meierhoff-Parnas pathway. Surprisingly, the reductive pentose phosphate pathway in the genome of strain F-1 T was absent, although ribulose biphosphate carboxylase, key enzyme of rPP, was present in the proteomes of several Pseudodesulfovibrio strains (Bell et al. 2018).
Strain F-1 T is capable of utilizing molecular hydrogen as an energy source. The genome of strain F-1 T encodes two subunits of periplasmic HynAB hydrogenase which has a bifunctional activity and is required either for the uptake of molecular hydrogen or for H 2 release during fermentation of organic substances. The [Ni-Fe] hydrogenase maturation system HypABCDEF is encoded in the genome of strain F-1 T .
The genome of strain F-1 T contains two copies of arsenate reductase gene, arsC. The presence of arsC is the common feature through the genus Pseudodesulfovibrio. However, there are no published data on the ability of the members of Pseudodesulfovibrio to grow with arsenate as an electron acceptor.
In contrast to the canonical fumarate reductase/succinate dehydrogenase consisting of four subunits (frdABCD), the genome of strain F-1 T contains genes only for three subunits (frdABC), as it was previously reported for Desulfovibrio vulgaris and Desulfovibrio desulfuricans (Zaunmüller et al. 2006). The genome of strain F-1 T contains genes of the nitrogenase complex nifDHK, which is required for N 2 fixation. Two components the iron protein and the molybdenum-iron protein, as well as two genes of P-II family nitrogen regulators are present.

Discussion
Strain F-1 T represents an alkaliphilic, mesophilic, sulfate-reducing bacterium isolated from a terrestrial mud volcano where it could participate in sulfur and carbon cycling. Mud fluids of Gnilaya Gora volcano contain up to 5 mM of SO 4 2-, providing an electron acceptor for the energy metabolism of the new isolate.The ranges of pH, temperature and salinity for growth of strain F-1 T are consistent with the environmental parameters of its habitat, suggesting an indigenous origin of the strain. Phylogenetic analysis based on 16S rRNA gene revealed that strain F-1 T belongs to the genus Pseudodesulfovibrio, where it forms a separate lineage of the species rank. ANI and in silico DDH data also support the assignment of strain F-1 T to a new species. It is the first representative of the genus Pseudodesulfovibrio isolated from a surface terrestrial environment whereas all known species of the genus were recovered from marinerelated or subsurface habitats.
As all members of Pseudodesulfovibrio strain F-1 T is anaerobic mesophilic sulfate-reducing vibrio, but it differs in temperature, pH and salinity ranges and optima for growth and in the electron donors and acceptors utilized (Table 1). The most notable distinction is the growth pH. All Pseudodesulfovibrio species described so far, are neutrophilic bacteria optimally growing at pH around 7.0. Strain F-1 T has the pH optimum at 9.5 and is unable to grow bellow 7.0; thus, it could be considered as obligate alkaliphile.
Metabolic potential encoded in the genome of strain F-1 T is consistent with the phenotypic data. The central carbon metabolism is based on Embden-Meierhoff-Parnas pathway. Sulfate respiration is ensured by the canonical set of genes for dissimilatory sulfate reduction. The reduction of elemental sulfur, fumarate and arsenate is provided by the respective reductases encoded in genome. The presence of gene cluster encoding all enzymes of nitrogenase complex indicates the ability of strain F-1 T to fix N 2 . Therefore, based on phylogenetic position, phenotypic and physiological properties of strain F-1 T we propose to assign it to the genus Pseudodesulfovibrio as a new species, P. alkaliphilus.
Cells are motile vibrios 2.0-4.0 lm in length and 0.5 lm in diameter with a polar flagellum. Growth is observed at NaCl concentrations from 0.3 to 3% (w/v) (optimum 0.5-1%, w/v), in pH range 7.0-10.5 (optimum 9.5), and at temperatures between 6 and 37°C (optimum 24°C). Grows with sulfate as an electron acceptor and lactate, fumarate, D-glucose, D-cellobiose or molecular hydrogen as electron donors. In the presence of sulfate the end products of lactate oxidation are acetate, propionate, H 2 S and CO 2. Sulfite, thiosulfate, elemental sulfur, fumarate or arsenate are used as electron acceptors for growth with lactate as an electron donor, but nitrate, nitrite, selenate or ferrihydrite are not utilized. Pyruvate, malate, formate, acetate, butyrate, ethanol, propanol, and arabinose are not used as electron donors with sulfate as an electron acceptor. In the absence of sulfate lactate and pyruvate are fermented and support growth. The major product of lactate fermentation is acetate. Fumarate, D-glucose and D-cellobiose are not fermented. Capable of weak but sustainable autotrophic growth with sulfate, sulfite, thiosulfate, elemental sulfur, fumarate or arsenate as electron acceptors and molecular hydrogen as an electron donor. Not able to grow by disproportionation of sulfite, thiosulfate and elemental sulfur. The predominant fatty acids are anteiso-C 15:0 , C 16:0 , C 18:0 , and iso-C 17:1x8 .
The genome of the type strain is characterized by a size of 3.23 Mb and a G ? C content of 61.93 mol%. The type strain F-1 T (= KCTC 15918 T = VKM B-3405 T ) was isolated from a terrestrial mud volcano in the Taman peninsula, Russia.