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Pseudodesulfovibrio sediminis sp. nov., a mesophilic and neutrophilic sulfate-reducing bacterium isolated from sediment of a brackish lake

A novel mesophilic and neutrophilic sulfate-reducing bacterium, strain SF6T, was isolated from sediment of a brackish lake in Japan. Cells of strain SF6T were motile and rod-shaped with length of 1.2–2.5 μm and width of 0.6–0.9 μm. Growth was observed at 10–37 °C with an optimum growth temperature of 28 °C. The pH range for growth was 5.8–8.2 with an optimum pH of 7.0. The most predominant fatty acid was anteiso-C15:0. Under sulfate-reducing conditions, strain SF6T utilized lactate, ethanol and glucose as growth substrate. Chemolithoautotrophic growth on H2 was not observed, although H2 was used as electron donor. Fermentative growth occurred on pyruvate. As electron acceptor, sulfate, sulfite, thiosulfate and nitrate supported heterotrophic growth of the strain. The complete genome of strain SF6T is composed of a circular chromosome with length of 3.8 Mbp and G+C content of 54 mol%. Analyses of the 16S rRNA gene and whole genome sequence indicated that strain SF6T belongs to the genus Pseudodesulfovibrio but distinct form all existing species in the genus. On the basis of its genomic and phenotypic properties, strain SF6T (= DSM111931T = NBRC 114895T) is proposed as the type strain of a new species, with name of Pseudodesulfovibrio sediminis sp. nov.


Introduction
The genus Pseudodesulfovibrio encompasses species of Gram-stain-negative sulfate-reducing bacteria with rodshaped motile cells (Galushko and Kuever 2019). The type species is P. indicus (Cao et al. 2016). According to the List of Prokaryotic Names with Standing in Nomenclature (LPSN), there are 10 species with validly published names in this genus, as of the end of February 2022. They include seven species which were originally described as Desulfovibrio species, i.e., D. halophilus (Caumette et al. 1991), D. profundus (Bale et al. 1997), D. aespoeensis (Motamedi and Pedersen 1998), D. tunisiensis (Ben Ali Gam et al. 2009), D. portus (Suzuki et al. 2009), D. piezophilus (Khelaifia et al. 2011), D. senegalensis (Thioye et al. 2017). These species were transferred to the genus Pseudodesulfovibrio in subsequent works (Cao et al. 2016;Galushko and Kuever 2019;Waite et al. 2020). P. hydrargyri (Ranchou-Peyruse et al. 2018) and P. mercurii (Gilmour et al. 2021) were described as novel species of Pseudodesulfovibrio, although their type strains had been classified in the genus Desulfovibrio in the past. 'P. alkaliphilus' (Frolova et al. 2021) and 'P. cashew' (Zheng et al. 2021) were recently proposed, while they have not been included in the validation list yet. It has also been indicated that D. oxyclinae (Krekeler et al. 1997), 'D. dechloracetivorans' (Sun et al. 2000) and 'Desulfovibrio brasiliensis' (Warthmann et al. 2005) should be reclassified into the genus Pseudodesulfovibrio (Galushko and Kuever 2019;Waite et al. 2020). Although D. oxyclinae is validly published name, proposed name for its reclassification, 'P. oxyclinae', has not been validated because its type strain is only available in one culture collection (Waite et al. 2020 renamed, as its type strain is not available in culture collections at present. On the other hand, the type strain of 'D. brasiliensis' is currently available in two culture collections (as DSM 15816 and JCM 12178). It was also indicated that 'Paradesulfovibrio onnuriensis' is the closest relative of P. senegalensis (Kim et al. 2020), and belongs to a lineage in the Pseudodesulfovibrio. Phylogenetic analysis based on the 16S rRNA gene indicated that there are two distinct phylogenetic groups within the genus Pseudodesulfovibrio (Galushko and Kuever 2019). The divergence between the groups (referred to as "cluster 1" and "cluster 2", respectively) is large enough to separate them into different genera. In other words, reclassification of cluster 2 as a separate genus is to be expected (Galushko and Kuever 2019).
In this study, a novel sulfate-reducing bacterium isolated and characterized, as a representative of a new species in the genus Pseudodesulfovibrio.

Enrichment and isolation
The novel isolate, strain SF6 T was isolated from sediment of a brackish lake, Lake Akkeshi in Japan. Water depth of the sampling site (43.05° N 144.89° E) was 1.6 m. At the time of sampling, temperature and of pH of overlying water were 22.3 °C and 8.0, respectively. Throughout this study, a bicarbonate-buffered and sulfide-reduced defined medium was used as basal medium. The basal medium for marine sulfate-reducing bacteria was prepared as described previously (Widdel and Bak 1992), and headspace of culture bottles was filled with N 2 /CO 2 (80:20, v/v). To establish the first enrichment, 0.2 g of the sediment was taken from 5 to 6 cm layer and inoculated into the basal medium supplemented with 5 mM formate. The culture bottle was incubated at 18 °C in the dark. The grown culture was transferred to the same medium three times. The resulting enrichment culture was subjected to agar shake dilution. A black colony was picked up in the same medium and incubated at 18 °C. After growth became visible, grown culture was transferred to the basal medium supplemented with 5 mM lactate, and incubation temperature was changed to 28 °C. Finally, pure culture of strain SF6 T was obtained from the culture grown on lactate, by agar shake dilution. Purity of the resulting culture was confirmed by microscopic observation with a phase-contrast microscope (Axioplan 2; Zeiss) and repeated sequencing of the 16S rRNA gene fragments.

Phylogenetic analysis based on the 16S rRNA gene
Nearly full length of the 16S rRNA gene was amplified by PCR with primer pair of 27F and 1492R (Lane 1991). The PCR product was directly sequenced, and the resulting sequence was subjected to blastn search to identify the closest relatives. Phylogenetic analysis was conducted using MEGA version 11 (Tamura et al. 2021), as described below. The 16S rRNA gene sequence of strain SF6 T was aligned with those of type strains in the genus Pseudodesulfovibrio, using the MUSCLE algorithm. With the resulting alignment, models for genetic distance calculation were evaluated using the model selection tool in MEGA. With the best model giving the lowest Bayesian Information Criterion (BIC) score, genetic distances were calculated by excluding positions with gaps.

Phenotypic characterization
In all experiments for phenotypic characterizations, strain SF6 T was cultured at 28 °C in the basal medium supplemented with 5 mM lactate, unless otherwise specified. Its growth was monitored as turbidity of cultures.
For cellular fatty acid analysis, strain SF6 was grown in the basal medium supplemented with 20 mM lactate. The fatty acid profile was obtained with the Sherlock Microbial Identification System (MIDI) version 6.0 (database; MOORE6).

Genomic characterization
Whole genome sequencing was performed using the platforms of Illumina NextSeq and Nanopore GridION. Short and long reads from the platforms were subjected to hybrid assembly using Unicycler (Ver 0.4.7). The assembled genome sequence was annotated with DFAST (Tanizawa et al. 2018).
As genome relatedness indices between SF6 T and its close relatives, values of average nucleotide identity (ANI) and average amino acid identity (AAI) were calculated using tools provided by Kostas lab (http:// enve-omics. ce. gatech. edu/). The Genome-to-Genome Distance Calculator provided by DSMZ were used to calculate digital DNA-DNA hybridization (dDDH) values, by applying the formula 2 (Meier- Kolthoff et al. 2013).
A genome-based taxonomic classification was carried out with the Genome Taxonomy Database (GTDB) (Parks et al. 2018). Taxonomic position of the strain SF6 T in the GTDB (release 95) was identified using GTDB-Tk (Chaumeil et al. 2020).

Physiological and chemotaxonomic characteristics
The fundamental characteristics of strain SF6 T are summarized in Table 1 and presented in the species description. Cells of strain SF6 T were motile, rod-shaped, 0.6-0.9 μm in width, 1.2-2.5 μm in length. Under the sulfate-reducing conditions, strain SF6 T grew at 10-37 °C with optimum growth at 28 °C, and grew at pH range of 5.8-8.2 with the optimum pH of 7.0. The NaCl range for growth was 0.6-6.5%, with optimum growth at 2.0%.
In the presence of sulfate, lactate, ethanol and glucose supported heterotrophic growth of SF6 T accompanying sulfide production. The molar ratio of generated sulfide to consumed lactate never exceeded 0.8. This upper limit is clearly lower than expected ratio for complete oxidation of lactate (1.5), suggesting incomplete lactate oxidation by strain SF6 T . Chemolithotrophic growth on hydrogen was not observed. Formate and hydrogen were utilized as electron donor, but acetate was required as carbon source for growth. Among the substrate tested, only pyruvate supported fermentative growth of the strain. The pyruvate-dependent growth was also observed in the presence of sulfate, but sulfide was not detected in this case. This means that strain SF6 T grows by fermentation of pyruvate, but does not use it as electron donor for sulfate reduction. This pattern of pyruvate utilization was previously reported in P. alkaliphilus F-1 T (Frolova et al. 2021). In addition to sulfate, sulfite, thiosulfate and nitrate were used as electron acceptor for lactate oxidation.

Genomic features
The complete genome of strain SF6 T was reconstructed by assembling 3,394,816 DNBSEQ reads and 126,221 Grid-ION reads, with coverage of 330-fold. It consists of a single circular chromosome with size of chromosome 3,764,150 bp and G+C content of 54.0% (Table 1). In the genome, 3527 protein-coding sequences, 9 RNA genes and 57 tRNA genes were predicted. Three copies of the 16S rRNA gene had identical sequence. The encoded proteins include those involved in glycolysis via Embden-Meyerhof pathway, membrane transport of monosaccharides, respiratory nitrate reduction to nitrite and nitrogen fixation.
Some genes encoding key enzymes for inorganic carbon fixation by sulfate reducers were not identified in the genome of strain SF6 T . The genome lacks the fhs and acsB genes, encoding and formate-tetrahydrofolate ligase and carbon monoxide dehydrogenase/acetyl-CoA synthase, respectively. These enzymes are key components of the Wood-Ljungdahl pathway. In addition, formate-tetrahydrofolate ligase also plays a critical role in carbon fixation via reductive glycine pathway (Sánchez-Andrea et al. 2020).

Taxonomic assignment
In the blastn analysis of the 16S rRNA gene sequence, high sequence identities were observed between strain SF6 T and type strains of Pseudodesulfovibrio species (Table 1). Among them, P. indicus J2 T showed the highest identity of 97.4%. By constructing phylogenetic tree of the 16S rRNA gene, it was indicated that strain SF6 T belongs to the genus Pseudodesulfovibrio (Fig. 1). The tree also indicated that strain SF6 T is phylogenetically distinct from existing species,  (Caumette et al. 1991); 4, P. profundus 500-1 T (Bale et al. 1997); 5, P. aespoeensis P1B T (Krekeler et al. 1997); 13, 'P. cashew' SRB007 T (Zheng et al. 2021); 14, 'P. alkaliphilus' F-1 T (Frolova et al. 2021). 15, 'Paradesulfovibrio onnuriensis' IOR2 T (Kim et al. 2020); 16, 'P. hontreensis' ME T (Tarasov et al. 2015). Data were retrieved from respective references except for genomic features of some  and belongs to the cluster 1 defined in the previous study (Galushko and Kuever 2019). Some genomic characteristics are consistent with the results of 16S rRNA gene analysis which suggested that strain SF6 T represents a novel species. The G+C content of strain SF6 T is distinct from those of other type strains of Pseudodesulfovibrio species (except for P. profundus), with differences greater than 4% (Table 1). In general, differences between genomic G+C contents of strains from the same species are 1% or smaller (Meier-Kolthoff et al. 2014). The values of ANI, AAI and dDDH between strain SF6 T and the type strains of Pseudodesulfovibrio species are shown in Table 1. All these values are lower than threshold for species delineation. Further, the genome of strain SF6 T was subjected to phylogenomic analysis with the GTDB-tk. By phylogenetic analysis based on 120 conserved proteins (Parks et al. 2018), strain SF6 T was classified as a novel species in the genus Pseudodesulfovibrio.
The creation of new species, suggested by the phylogenetic analyses, is supported by some phenotypic characteristics which differentiate strain SF6 T from other species (Table 1). For the species represented by strain SF6 T , the name Pseudodesulfovibrio sediminis sp. nov. is proposed here.
Cells and rod shaped, 1.2-2.5 μm in length and 0.6-0.9 μm in width. Grows at 10-37 °C with an optimum growth at 28 °C. The pH range for growth is 5.8-8.2, with an optimum pH of 7.0. Grows with 0.6-6.5% NaCl (optimum 2.0%). Predominant fatty acid is anteiso- The type strain SF6 T (= DSM111931 T = NBRC 114895 T ) was isolated from sediment of a brackish lake in Japan.
The GenBank/EMBL/DDBJ accession number for the complete genome of strain SF6 T is AP024485.

Supplementary Information
The online version contains supplementary material available at https:// doi. org/ 10. 1007/ s00203-022-02870-5.  Fig. 1 Phylogenetic position of strain SF6 T within the genus Pseudodesulfovibrio, based on the 16S rRNA gene sequences. The phylogenetic tree was inferred using the maximum likelihood method and Kimura 2-parameter model. A discrete gamma distribution was used to model evolutionary rate differences among sites, allowing some sites to be invariable. All positions containing gaps and missing data were eliminated, leaving a total of 1340 positions in the final dataset. Numbers on nodes represent percentage values of 1000 bootstrap resampling