Phylogenetic analysis
The almost complete 16S rRNA gene sequence of strain XHP0099T was 1,346 bp in length, and was deposited into the GenBank database under the accession number ON329670. The results of the 16S rRNA gene sequence analysis on the EzBioCloud server revealed that strain XHP0099T belongs to the genus Paracoccus in the family Rhodobacteraceae and showed the highest sequence identity with “P. siganidrum” M26 (98.2%), followed by P. alkanivorans 4-2T (97.6%) and P. alkenifer DSM 11593T (97.4%). The NJ and ML trees (Fig. S1 and S2) showed that strain XHP0099T formed a clade with “P. siganidrum” M26 and P. alcaliphilus TCM 7364T. However, strain XHP0099T clustered with P. alkanivorans 4-2T, P. onubensis 1011MAR3C25T, P. saliphilus YIM 90738T, according to the clades of the MP phylogenetic tree (Fig. S3). It is noticeable that the position of strain XHP0099T on all three trees were not well supported (bootstraps <70%). In addition, a maximum-likelihood tree (Fig. 1) based on the 120 ubiquitous single-copy protein-coding genes showed that strain XHP0099T clustered with P. salsus EGI L200073T with high confidence (bootstrap value 0.999), while P. onubensis 1011MAR3C25T and P. alkanivorans 4-2T as well as “P. siganidrum” M26 and P. alcaliphilus DSM 8512T formed two more deeply branching clades. Above all, phylogenetic trees exhibited similar topologies that strain XHP0099T clustered within a clade that accommodated the genus Paracoccus. These results strongly suggested that strain XHP0099T was a member of the genus Paracoccus.
Genomic analysis
The whole genome shotgun data of strain XHP0099T have been deposited in the NCBI GenBank database under the accession number JAHKNG000000000. The draft genome of strain XHP0099T was 4,179,628 bp in length with 155 contigs. The lowest and highest genomic DNA G+C content among species belonging to the genus Paracoccus are 58.7% and 71.0%, respectively. (Lee et al. 2011; Ohara et al. 1990). The G+C content of the genomic DNA of strain XHP0099T was 66.0%, therefore within the range for recognized Paracoccus species. Of the 4,158 predicted genes, 3,997 were protein-coding genes, 109 were pseudogenes and 52 were RNA genes (one 5S rRNA, one 16S rRNA, one 23S rRNA, 46 tRNAs and 3 ncRNAs). Results from eggNOG server (Huerta-Cepas et al. 2017) suggested that there were 384, 2,227 and 3,732 genes were assigned to Gene Ontology (GO) (Carbon et al. 2019), Kyoto Encyclopedia of Genes and Genomes (KEGG) (https://www.kegg.jp/) (Kanehisa et al. 2016) and Cluster of Orthologous Groups (COG) databases (Galperin et al. 2015), respectively.
The ANI values between strain XHP0099T with “P. siganidrum” M26, P. alkanivorans 4-2T and P. saliphilus DSM 18447T were 81.4%, 80.1% and 78.3%, respectively, which were all below 95-96% based classification at the species level (Chun et al. 2018). The dDDH values comparing the genome sequences of strain XHP0099T with “P. siganidrum” M26, P. alkanivorans 4-2T and P. saliphilus DSM 18447T were 24.1%, 23.0% and 21.2%, respectively (Table 1), which lower than the 70% threshold value (Winter et al. 1987) as the proposed and generally accepted species boundaries for the identification of a novel species. Otherwise, the AAI values between strain XHP0099T and “P. siganidrum” M26, P. alkanivorans 4-2T and P. saliphilus DSM 18447T were 75.2%, 74.5% and 74.9%, respectively, which were also below the threshold value of the species boundary (95%) (Rodriguez-R and Konstantinidis 2014). Based on the analysis of phylogeny, ANI, dDDH and AAI, strain XHP0099T should represent a novel species in the genus Paracoccus.
Further analysis of the KEGG reconstruction showed that strain XHP0099 had 28 genes involved in flagellar assemblies (ko02040), of which 10 genes were involved in coding flagellar hook-basal body and flagellar basal body complex protein, including KNW02_RS13770 (flgA), KNW02_RS13800 (flgB), etc. However, cells of strain XHP0099T were non-motile. There were 18 genes involved in nitrogen metabolism (ko00910) and 18 genes involved in sulfur metabolism (ko00920). Further analysis of the occurrence of methylotrophy genes and other closely related type strains within the genus Paracoccus showed that strain XHP0099T had a cluster consisting of genes for glutathione-dependent formaldehyde oxidation (gfa, flhA, fghA) and the XoxF-type methanol dehydrogenase and Calvin cycle gene cluster type 1 (Fig. 2, Table S2). In addition, a set of genes, KNW02_RS15430 (impL), KNW02_RS15425 (impK), KNW02_RS15390 (hcp), KNW02_RS08305 and KNW02_RS15440 (vgrG), KNW02_RS15415 (vasD) and KNW02_RS15480 (vasG) were identified to be involved in type VI secret system (T6SS) assembly (Coulthurst 2019), but the genes coding the regulatory proteins PpkA, Fha1, and PppA were not found.
TwoCRISPR-Cas systems, type II-C1 and incomplete type I-E, were identified in the genome of strain XHP0099T, according to previous classification standard (Makarova et al. 2020). The type II-C1 CRISPR-Cas system comprised one Cas cluster with three Cas genes (cas1/KNW02_RS20015, cas2/KNW02_RS20010, and cas9/KNW02_RS20020) and a CRISPR array of 6 spacers (all were 30 bp in length). The incomplete type I-E system comprised three Cas genes (cas1/KNW02_RS19740, cas2/KNW02_RS19735, and cas6/KNW02_RS19745) and a CRISPR array of 14 spacers (all were 32 bp in length).
Physiology and biochemical analysis
The phenotypic characteristics of strain XHP0099T were consistent with those members of the genus Paracoccus. Cells were Gram-stain-negative, non-motile coccoid and 0.5-0.6 μm in diameter (Fig. S4). Besides, colonies on R2A were observed to be circular, convex, smooth and white in colour. Growth occurred at 20–37°C (optimum, 28°C), pH 5.0–9.0 (optimum, pH 7.0–8.0), and with 0–7.0% NaCl (optimum, 2.0–3.0%). Strain XHP0099T was found to grow well on NA plates, R2A agar plates, TSA plates and 2216E plates, but couldn’t grow well on BHI medium and LB agar plates. In the test about the utilization of methanol, no growth of strain XHP0099T was observed in methylotrophic substrates no matter whether methanol was supplemented or not according to the result of the turbidity of the growing broth culture (Fig. S5). Subsequently, colonies of strain XHP0099T were recovered on R2A agar plates from the methylotrophic substrates after the experiment, which suggested that strain XHP0099T was inhibited in the basic medium. In view of its complete gene clusters for C1 substrates oxidation and Calvin cycle (Fig. 2, Table S2), strain XHP0099T should be regarded as a potential methylotrophic bacterium (Czarnecki and Bartosik 2019).
In BiOLOG GEN III microtest system, acetic acid, acetoacetic acid, citric acid, D-arabitol, D-aspartic acid, D-fructose, D-malic acid, D-maltose, D-mannitol, D-mannose, D-salicin, D-sorbitol, D-trehalose, D-turanose, formic acid, glycero, L-alanine, L-aspartic acid, L-glutamic acid, L-histidine, L-lactic acid, L-malic acid, L-pyroglutamic, L-rhamnose, L-serine, methyl pyruvate, myo-inositol, propionic acid, p-hydroxy-phenylacetic acid, quinic acid, sucrose, Tween 40, α-D-glucose, α-keto-glutaric acid, α-hydroxy-butyric acid, β-hydroxy-D, L butyric acid and γ-amino-butryric acid were utilized as sole carbon sources. The strain was also found to be resistant to fusidic acid, niaproof 4, tetrazolium violet, but sensitive to aztreonam, guanidine HCl, lincomycin, lithium chloride, nalidixic acid, potassium tellurite, rifamycin SV, sodium butyrate and 1% sodium lactate using BiOLOG GEN III MicroPlateTM. Other physiological characteristics was shown in Table 2 and Table S3.
Chemotaxomic characterization
The predominant isoprenoid quinone of strain XHP0099T was ubiquinone Q-10, which was characteristic to members of the genus Paracoccus. The major cellular fatty acids (>10%) were summed feature 8 (C18:1 ω7c/C18:1 ω6c), and C18:0. Fatty acid compositions of strain XHP0099T and other closely related type strains within the genus Paracoccusare shown in Table S1. The polar lipid profiles included diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylcholine (PC) and phosphatidylethanolamine (PE), which is consistent with the characteristics of those closely related strains “P. siganidrum” M26, followed by P. alkanivorans 4-2T and P. saliphilus DSM 18447T. In addition, an unknown aminolipid (AL) and one unidentified polar lipid (L) were also detected.Two-dimensional thin-layer chromatogram of polar lipid profiles of strains XHP0099T,“P. siganidrum” DSM 26381 and P. alkanivorans CGMCC 1.13669T are shown in Fig. S6.
Taxonomic Conclusion
In conclusion, strain XHP0099T can be considered to represent a distinct lineage within the genus Paracoccus based on its low 16S rRNA gene sequence identity (<98.65 %) with members of the genus Paracoccus. Futhermore, phylogenetic tree analysis showed that strain XHP0099T clustered within in the genus Paracoccus. The ANI and dDDH values between strain XHP0099T and closely related species were all below the boundaries for species definition. On the basis of phylogenetic, phenotypic, biochemical and chemotaxonomic characteristics, strain XHP0099T was identified as the type strain of a novel species within the genus Paracoccus, for which the name Paracoccus marinaquae sp. nov. is proposed. Differential characteristics of strain XHP0099T from closely related type species were listed in Table 2.
Description of Paracoccus marinaquae sp. nov.
Paracoccus marinaquae (ma.rin.a’quae. L. masc. adj. marinus, of the sea; L. fem. n. aqua, water; N.L. gen. fem. n. marinaquae, of seawater)
Cells are Gram-stain-negative, non-motile, coccoid and 0.5-0.6 μm in diameter. Colonies on R2A agar (supplemented with 2.5% sea salt) are circular, convex, smooth and white in color after cultivation for 2 days at 28°C. Growth occurred at 20–37°C (optimum, 28°C), pH 5.0–9.0 (optimum, pH 7.0–8.0), and with 0–7.0% NaCl (optimum, 2.0–3.0%). Cells are positive for oxidase and catalase tests. In the API 20NE test, strain XHP0099T was positive for hydrolysis of esculin, reduction of nitrate and 4-nitrophenyl β-D-galactopyranoside. and positive for cystine arylamidase, lipase (C14), N-acetyl-β-glucosaminidase, trypsin, valine arylamidase, α-chymotrypsin, α-fucosidase, α-galactosidase, α-mannosidase, β-galactosidase, β-glucosidase and β-glucuronidase in the API ZYM tests. The respiratory quinone is Q-10. The polar lipids are diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylcholine (PC), aminolipid (AL) and an unidentified polar lipid (L). The major cellular fatty acids are summed feature 8 (C18:1 ω7c/C18:1 ω6c), and C18:0. The G+C content of the genomic DNA is 66.0%.
The type strain XHP0099T (=JCM 34661T=GDMCC 1.2414T=MCCC 1K05846T), was isolated from a sample of seawater collected at the Yellow Sea, China.
The 16S rRNA gene sequence has been deposited at GenBank/EMBL/DDBJ accession number ON329670, while the genome sequence has been deposited at GenBank/EMBL/DDBJ under accession number JAHKNG000000000.