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Hyphomonas sediminis sp. nov., isolated from marine sediment

A Gram-staining-negative, aerobic and pear-shaped bacterial strain, designated WL0036T, was isolated from coastal sediment sample collected in Nantong city, Jiangsu province of China (120° 51′ 13″ E, 32° 6′ 26″ N) in October 2020. Strain WL0036T was found to grow at 20–37 °C (optimum, 28 °C) with 0–9.0% NaCl (optimum, 2.5–4.0%) and displayed alkaliphilic growth with the pH range of pH 6.0–10.0 (optimum, pH 7.0–8.0). The polar lipids profile of strain WL0036T included phosphatidylcholine, phosphatidylethanolamine, glycolipid and an unidentified lipid. The major isoprenoid quinone was determined to be Q-11 and the major fatty acids were C16:0, 11-methyl-C18:1ω7c, and summed features 8 (C18:1ω6c and/or C18:1ω7c). The G + C content of genomic DNA was 61.8%. Phylogenetic trees constructed based on 16S rRNA gene sequence and bac120 gene set (a collection of 120 single-copy protein sequences prevalent in bacteria) indicted that strain WL0036T clustered with strains Hyphomonas neptunium ATCC 15444T and H. polymorpha PS728T. The average nucleotide identities between strain WL0036T and strains H. neptunium ATCC 15444T and H. polymorpha PS728T were 80.7% and 81.2%, respectively. Strain WL0036T showed 22.8% and 23.2% of digital DNA-DNA hybridization identities with H. neptunium ATCC 15444T and H. polymorpha PS728T, respectively. As inferred from the phenotypic and genotypic characteristics and the phylogenetic trees, strain WL0036T ought to be recognized as a novel species in genus Hyphomonas, for which the name Hyphomonas sediminis sp. nov. is proposed. The type strain is WL0036T (= MCCC 1K05843T = JCM 34658T = GDMCC 1.2413T).


Introduction
The genus Hyphomonas was originally proposed by Pongratz 1957. The cells of the genus Hyphomonas are Gram-staining-negative, aerobic, heterotrophic and nonsporeforming. The daughter cells are oval to pear-shaped, smaller than the body of the mother cell, moving through polar lateral flagella. Hyphae rarely branches during normal growth (Pongratz 1957). At the time of writing, the genus Hyphomonas includes 12 species, of which 11 have been validly published according to the List of Prokaryotic names with Standing in Nomenclature (LPSN) server (https:// lpsn. dsmz. de/ genus/ hypho monas, accessed on March 30, 2022) (Parte 2018): H. polymorpha (Moore et al. 1984); H. neptunium (Moore et al. 1984); H. oceanitis (Weiner et al. 1985); H. jannaschiana (Weiner et al. 1985); H. adhaerensand (Weiner et al. 2000); H. johnsonii (Weiner et al. 2000); H. atlanticus (Li et al. 2014c); H. beringensis (Li et al. 2014a); H. pacifica (Li et al. 2016), H. rosenbergii (Weiner et al. 2000) and H. chukchiensis (Li et al. 2014a). Nevertheless, H. rosenbergii was recommended to be place on the reject list because the 16S rRNA gene sequence of H. rosenbergii ATCC 43869 T shared a similarity of 92.8% with the primitive sequence (Lai et al. 2015;Li et al. 2014b). Members of the genus Hyphomonas are primarily spread in the marine environment, which include pelagic areas, offshore areas and deep-sea thermal springs.
To investigate the application potential of the bacteria from offshore areas of Jiangsu province, China, a novel bacterium, designated WL0036 T , was isolated from a marine sediment sample of Nantong city. In the present study, we conducted a polyphasic taxonomic study to clarify the taxonomic position of strain WL0036 T .

Materials and methods
Isolation and culture condition Strain WL0036 T was isolated from a marine sediment sample collected in Nantong city, Jiangsu province of China (120° 51′ 13″ E, 32° 6′ 26″ N) in October 2020. Prior to isolation, the sample was stored in 4 °C refrigerator. For isolation, 2 g sample was diluted in sterile physiological saline (per litre of water: 9.0 g sodium chloride, Sangon Biotech, China) (18 mL), and then incubated in a constant temperature shaker incubator at 28 °C for 1 h. The suspension was diluted to 10 -3 and 100 µL were spread on plates of the marine agar medium 2216E (Hopebio, China, per litre: 5.0 g peptone, 1.0 g yeast extract, 15.0 g agar, 0.01 g ferric phosphate, 25 g sea salt, 0.5 g NaOH).

Genome sequencing and annotation
For 16S rRNA gene amplification by PCR, genomic DNA was extracted by using the Ezup column bacterial genomic DNA purification kit (Sangon Biotech, China). The crude extract was used as DNA template for PCR as described previously (Weisburg et al. 1991) with 27F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1492R (5′-AAG GAG GTG ATC CAGCC-3′) primers. The almost full-length 16S rRNA gene sequence was compiled using Seq-Man software (DNASTAR) and ulphate through the EzbioCloud (https:// www. ezbio cloud. net/) server (Yoon et al. 2017) to retrieve sequences of the most closely related types strains. Whole-genome shotgun sequencing of strain WL0036 T was performed using paired-end sequencing method with Hiseq X platform (Illumina) at Magigene Company, Guangzhou, China. The whole genome was assembled using SPAdes version 3.10.1 (Nurk et al. 2013) in UGENE software package (Okonechnikov et al. 2012). Following the proposed minimal standards for the use of genome data for the taxonomy of prokaryote (Goris et al. 2007;Richter and Rosselló-Móra 2009), the average nucleotide identify (ANI) and the digital DNA-DNA hybridization (dDDH) were used to compare strain WL0036 T and related type species genome sequences. The ANI values was calculated by ANI calculator using the OrthoANIu algorithm on the EzBioCloud website (https:// www. ezbio cloud. net/) (Yoon et al. 2017) and the dDDH values was calculated using the Genome-to-Genome Distance Calculator (http:// ggdc. dsmz. de/ ggdc. php/) (Meier-Kolthoff et al. 2013). Genome annotation was conducted by the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (Tatusova et al. 2016), ulpha online server (https:// eggnog-mapper. Embl. de/) (Huerta-Cepas et al. 2017) and KEGG online server (https:// www. kegg. jp/ blast koala/) (Kanehisa et al. 2016), The results of genetic analysis for drug resistance was obtained through the Comprehensive Antibiotic Resistance Database (CARD) website (https:// card. mcmas ter. ca/) (Alcock et al. 2020). The G+C content of genomic DNA was calculated based on the EzBioCloud server from the draft genome sequence.

Phylogenetic analysis
Phylogenetic trees based on 16S rRNA gene sequence were reconstructed by the ulphate-joining (NJ) (Saitou and Nei 1987), minimum-evolution (ME) (Pardi et al. 2010), and maximum-parsimony (MP) (Fitch 1971) methods using MEGA X software (Kumar et al. 2018) after multiple sequence alignments. The evolutionary distance matrix of the phylogenetic tree was commonly calculated using Kimura's two-parameter model (Kimura 1980). Confidence values for tree branches were determined by bootstrap analysis based on 1000 resamples (Felsenstein 1985). Genome-based phylogenies of supermatrix and supertree approaches from protein sequences of the bac120 gene set (a collection of 120 single-copy protein sequences prevalent in bacteria) were constructed by using Easy-CGTree version 3.0 (https:// github. com/ zdf19 87/ EasyC GTree) as described previously (Zhang et al. 2020). The genome sequences of the strains of interest were downloaded from the NCBI genome database (https:// www. ncbi. nlm. nih. gov/ genome/) and detailed information is listed in Suppl. Table S1.
Morphology, physiology, and biochemical analysis A Gram Stain kit (Solarbio, China) was used for testing the Gram reaction. The cell morphology of the bacterium was observed by transmission electron microscopy (JEM, 2100) using cells grown in TSA (Difco) medium supplemented with 2.5% sea salt for 48 h at 28 °C. Motility was determined by observing growth of cells in test tubes containing TSB medium with 0.3% agar after 3 days of incubation at 28 °C. Anaerobic growth was tested in an anaerobic chamber (Oxoid, UK) with the atmosphere generation system (AnaeroGen, Oxoid, UK) for 10 days on TSA medium plates which supplemented with 2.5% sea salt. To determine the development conditions of the strain WL0036 T , growth at various temperatures (4, 10, 20, 28, 37, 45, and 50 °C) was tested in TSA medium supplemented with 2.5% sea salt. Adjusting pH values (pH 4.0-12.0, with 1.0 increments), using NaOH solution (1.0%, w/v) and HCl solution (1.0%, w/v) to adjust pH in the tryptic soy broth (TSB, per litre: 15.0 g tryptone (Sangon Biotech, China), 5.0 g soy papain digest (Sangon Biotech, China), 25.0 g sea salt) medium. To investigate the tolerance to NaCl, growth at various NaCl concentrations (0, 1.5, 2.5, 4.0, 7.0, 9.0, 11.0 and 13.0%) was investigated in TSA medium (per litre: 15.0 g tryptone, 5.0 g soy papain digest).
Catalase activity was detected by observing whether bubbles were produced using 3.0% hydrogen peroxide. Oxidase activity was determined with the use of 1.0% tetramethyl-β-phenylenediamine. Additional biochemical tests were performed using the BIOLOG GEN III microtest system (Biolog, USA), API 20NE and API ZYM systems (bioMérieux, France) in addition to adjusting the salinity to 2.5% with sea salt when culturing strain WL0036 T according to the manufacturer's instructions.

Chemotaxonomic characterization
The strain WL0036 T and the two reference strains H. neptunium ATCC 15444 T and H. polymorpha PS728 T were incubated in TSB medium supplemented with 2.5% sea salt at 28 °C for three days, and then the biomass was collected for fatty acid characterization. Cellular fatty acids was methylated and ulphate using the Sherlock Microbial Identification System (MIDI) according to a previously described method and the manufacturer's instructions (Sasser 1990). The polar lipid profile and isoprenoid quinones were determined using cells grown on TSB medium containing 2.5% sea salt at 28 °C for 3 days. Two-dimensional thin-layer chromatography was performed following previously demonstrated methods to detect polar lipids in cells (Collins and Jones 1980;Minnikin et al. 1984). Phosphomolybdic acids were used for determination of all polar lipids, ninhydrin for phospholipids containing amino or glucosamine, and anisaldehyde and α-naphthol were used for glycolipids. Isoprenoid quinones were extracted (Collins et al. 1977) and ulphate using reversed phase HPLC as described previously (Tamaoka 1986).

Phylogenetic analysis
The complete 16S rRNA gene sequence of strain WL0036 T was 1292 bp (accession number, OL605964) a length of 1072 bp was used to construct the 16SrRNA evolutionary tree of the strain. The results from the EzBioCloud server suggested that the 16S rRNA gene sequence of strain WL0036 T exhibited the highest similarities to those of H. polymorpha PS728 T (98.3%) and H. neptunium ATCC 15444 T (97.8%). By using the maximum-parsimony method, the phylogenetic tree ( Fig. 1) denoted that strain WL0036 T clustered with H. polymorpha PS728 T , H. rosenbergii VP-6 T and H. neptunium ATCC 15444 T , and the clade of these four taxa was closely related to the rest members of the genus Hyphomonas. Meanwhile, the phylogenetic position of strain WL0036 T was well supported (bootstrap value > 70.0%) in the ulphate-joining tree, and it agreed with the trees of ulphate-joining method (Suppl. Fig. S1) and the minimum-evolution method (Suppl. Fig. S2) with high confidence (bootstrap values > 70).
In order to further explore the relationships among strain WL0036 T and related species within the genus Hyphomonas, the bac120 tree constructed following the method of the supermatrix (Fig. 2) demonstrated that strain WL0036 T formed a clade and clustered with the reference strains H. polymorpha PS728 T and H. neptunium ATCC 15444 T . Meanwhile, the phylogenetic tree based on 119 universal protein coding genes of the bac120 set among the strains of interest (Suppl. Fig. S3) showed that strain WL0036 T also formed a clade with H. polymorpha PS728 T and H. neptunium ATCC 15444 T , which was supported Fig. 1 Maximum-parsimony phylogenetic tree based on 16S rRNA gene sequence showing the relationship between strain WL0036 T and its closest relatives. Bootstrap values (expressed as percentages of 1000 replications) of above 70% are shown at the branch nodes. The black dots indicate branches that were also recovered using the minimum-evolution and neighbor-joining methods. Species with no valid publication are marked with double-quote. The Gen-Bank/EMBL/DDBJ accession numbers are indicated in the brackets at the end of the tip labels. Escherichia coli JCM 1649 T is used as out group. The Figure is from this study by 113 of the 119 genes (95.0%) used in supertree method. This result was corresponding to that of the supermatrix method and those based on 16S rRNA gene sequence. Consequently, these findings support that strain WL0036 T was a member of genus Hyphomonas.

Genomic features
The draft genome of strain WL0036 T was comprised of 4 contigs with a size of 3498010 bp. The G+C content of genomic DNA was 61.8%. Automated annotation by NCBI PGAP recognized 3302 protein-coding sequences, 6 rRNA genes, 41 tRNA genes and 3 RNA genes of other type. The 16S rRNA gene sequence (locus tag K1X12_16485) on the genome was identical to the Sanger sequence.
The ANI values between strain WL0036 T and strains H. neptunium ATCC 15444 T and H. polymorpha PS728 T were 80.7% and 81.2%, respectively (Table 1), all of which the ANI values were below threshold for species boundaries (95.0-96.0%) (Richter and Rosselló-Móra, 2009 (Goris et al. 2007). These data illustrated that strain WL0036 T represents a novel species.

Phenotypic characteristics
The cells of strain WL0036 T were Gram-stainingnegative, aerobic, motile and pear-shaped with a single polar flagellum. Slight yellow, round and smooth colonies were observed on TSA medium with 2.5% sea salt for 3 days at 28 °C. More results on phenotypic characteristics were listed in Table 2 and Suppl. Table S2.
The type strain, WL0036 T (= MCCC 1K05843 T = JCM 34658 T = GDMCC 1.2413 T ), the DNA G+C content of genomic DNA is 61.8%. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and the draft genome sequence of strain WL0036 T are OL605964 and JAIEZP000000000, respectively.
Author contributions DFZ and WJL designed research and project outline. LW and DFZ performed isolation, deposition and polyphasic taxonomy. DFZ, LW and WH performed genome analysis. LW, WJL AHZ and DFZ drafted the manuscript. AHZ, WH, ZYG, JKH and CL revised the manuscript. All authors read and approved the final manuscript.
Funding This research was supported by the National Natural Science Foundation of China (No. 31900001), the China Postdoctoral Science Foundation (2020M671312) and the Fundamental Research Funds for the Central Universities (B210202140).
Data availability All of the data supporting the conclusions of this article are included within the article and its additional files. The genome datasets and the 16S rRNA gene sequence of Hyphomonas sediminis WL0036 T generated during the current study are available in the GenBank/EMBL/DDBJ repository under accession number JAIEZP000000000 and OL605964. Other datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. Other genome sequence data detailed information was listed in Suppl. Table S1.

Competing interests
The authors declare no competing interests.
Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.