Paracoccus marinaquae sp. nov., isolated from coastal water of the Yellow Sea

A Gram-stain-negative, non-motile and coccoid bacterial strain, designated XHP0099T, was isolated from the coastal water of the Yellow Sea, China. Growth occurred at 20–37 ℃ (optimum, 28 ℃), pH 5.0–9.0 (optimum, pH 7.0–8.0), and with 0–7.0% NaCl (optimum, 2.0–3.0%). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain XHP0099T was related to members of the genus Paracoccus and shared the highest sequence similarity with “P. siganidrum” M26 (98.2%), followed by P. alkanivorans 4-2 T (97.6%) and P. alkenifer DSM 11593 T (97.4%). The average nucleotide identity, amino acid identity, and digital DNA–DNA hybridization values of strain XHP0099T against related members in the genus Paracoccus were below the cut-off points proposed for the delineation of a novel species. The major cellular fatty acids (> 10%) were summed feature 8 (C18:1ω7c/C18:1ω6c), and C18:0. The major isoprenoid quinone was Q-10 and the polar lipids contained diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylcholine (PC), aminolipid (AL) and unidentified polar lipids (L). The G + C content of the genomic DNA of strain XHP0099T was 66.0%. Genomic analysis suggested that strain XHP0099T harbored gene clusters for formaldehyde and the XoxF-type methanol oxidation and type 1 Calvin cycle, which could confer the methylotrophy pathway. Based on the phenotypic, phylogenetic, biochemical and chemotaxonomic analysis, strain XHP0099T represents a novel species of the genus Paracoccus, for which the name Paracoccus marinaquae sp. nov. is proposed. The type strain is XHP0099T (= JCM 34661 T = GDMCC 1.2414 T = MCCC 1K05846T).


Introduction
The genus Paracoccus within the family Rhodobacteraceae of the class Alphaproteobacteria was first proposed by Davis et al. (1969) and the genus description was significantly amended several times (Katayama et al. 1995;Liu et al. 2008;Ludwig et al. 1993). At the time of writing, there are 78 species with validly published names in the genus Paracoccus according to the List of Prokaryotic names with Standing in Nomenclature (LPSN) (http:// www. bacte rio. net/, accessed on Sept. 13th 2022) (Parte et al. 2020). Bacteria of the genus Paracoccus were isolated from other various habitats, such as air (Xue et al. 2017), soil (Sun et al. Communicated by Erko Stackebrandt. Hua-Peng Xue and Zi-Yue Fu have contributed equally to this work. 2015), sediment (Zhang et al. 2019), seawater (Lyu et al. 2021) and sediment of freshwater (Wang et al. 2009). They were also isolated from some plants (Rai et al. 2020), animal species (Lee et al. 2019) and some extreme environments, such as hot spring (Ye et al. 2020), plateau permafrost (Zhu et al. 2013) and even oil-contaminated water (Zhang et al. 2020b). Numerous representatives of the genus Paracoccus can grow chemolithoautotrophically, many Paracoccus spp. are methylotrophs and can utilize methanol (MeOH) and methylamine (MA) as sole carbon and energy sources, e.g. P. denitrificans and closely related P. versutus and P. kondratievae (Kelly et al. 2006). Some strains with C1 compounds are able to metabolize dimethylamine (DMA), trimethylamine (TMA) and N,N-dimethylformamide (DMF), etc. (Sanjeevkumar, et al. 2013). The diversity of methylotrophy in the genus Paracoccus at the biochemical and genetic levels was described by Czarnecki (Czarnecki and Bartosik 2019), including an examination of the origin and evolution of C1 metabolism in this group of bacteria. In addition, P. denitrificans (Robertson and Kuenen 1984) is one of the earliest reported aerobic denitrifying bacteria and most species of the genus Paracoccus are positive for the reduction of nitrate (Chen et al. 2020;Liu et al. 2013). Interestingly, there is a butachlor-degrading strain Paracoccus sp. FLY-8 T (Zhang et al. 2011) that can degrade and utilize chloroacetamide herbicides as carbon sources for growth, which has an important potential use in situ bioremediation of chloroacetamide herbicides and their metabolite-contaminated environment.
Hereby, we report a bacterium of the genus Paracoccus, designated XHP0099 T which was isolated from a surface water sample of the Yellow Sea, northern China.

Isolation and culturing conditions
Strain XHP0099 T , was isolated from a Yellow Sea surface seawater sample, northern China (34°46′40″N, 119°27′37″E). For strain isolation, 1.0 mL of seawater sample was diluted in 9.0 mL sterile distilled water solution containing 2.5% (w/v) sea salt (BOSSKAS, Shanghai, China), and mixed by vortexing. A 10 -2 dilution of the sample was performed in the same solution of sea salt and 0.1 mL was spread on R2A agar plates (supplemented with 2.5% sea salt unless otherwise mentioned). The plate was incubated at 28 ℃ for 7 days, and individual colonies were selected and purified on fresh R2A agar plates. The strain was routinely cultured on R2A plates at 28 ℃ and stored as aqueous glycerol suspensions (20%, v/v) at -80 ℃. "P. siganidrum" DSM 26,381, P. alkanivorans CGMCC 1.13669 T and P. saliphilus DSM 18447 T were used as reference strains for morphological, physiological, biochemical and chemotaxonomic analyses under the same experimental conditions as strain XHP0099 T .

Phylogenetic analysis
Genomic DNA was extracted from strain XHP0099 T using the Ezup Column Bacteria Genomic DNA Purification Kit (Sangon Biotech, China) following the manufacturer's instructions. PCR amplification of the 16S rRNA gene fragment was conducted with the bacterial-specific primers 27F and 1492R as described previously (Weisburg et al. 1991). Identification of phylogenetic neighbors and calculation of 16S rRNA gene sequence identities were performed using EzBioCloud server (https:// www. ezbio cloud. net/). The phylogenetic trees were constructed with neighbor-joining (NJ) (Saitou and Nei 1987), maximum-likelihood (ML) (Felsenstein 1981) and maximum-parsimony (MP) (Fitch 1971) methods using the MEGA X software package (Kumar et al. 2018) after alignments. Evolutionary distance matrices of the phylogenetic trees were calculated through Kimura's two-parameter model (Kimura 1980) with bootstrap analysis based on 1,000 replicates.

Genome sequence analysis
Whole-genome sequencing of strain XHP0099 T was performed using a paired-end sequencing method with the Hiseq X platform (Illumina) at Magigene Company, Guangzhou, China. Sequence assembly was performed with SPAdes v3.13.0. The ANI between the genomes of different strains were calculated using an ANI Calculator tool (https:// www. ezbio cloud. net/). The dDDH values between the genomes of the strains were performed with the Genometo-Genome Distance Calculator (GGDC) (https:// ggdc. dsmz. de). Formula 2 was used for the dDDH analysis. AAI value was calculated on web server (http:// enve-omics. ce. gatech. edu/ aai/). The completed genome sequences of strain XHP0099 T were submitted to the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (https:// www. ncbi. nlm. nih. gov/ genome/ annot ation_ prok/) to annotate genomes, including prediction of protein-coding genes, as well as other functional genome units such as structural RNAs, tRNAs, small RNAs, pseudogenes, direct and inverted repeats, insertion sequences, transposons and other mobile elements. The results of the genome annotation were submitted to eggNOG online server (https:// eggnog-mapper. embl. de/) for further analysis. The CRISPRCasFinder (https:// crisp rcas. i2bc. paris-saclay. fr/ Crisp rCasF inder/ Index) was used to conduct the identification of repeats and spacers in the CRISPR-Cas systems of the genomes. The DNA G + C content was calculated from the draft genome sequence. The 120 ubiquitous single-copy protein-coding genes (Parks et al. 2018) were built as profile hidden Markov models (HMMs) and were used to construct a robust phylogeny using EasyCGTree version 4.0 (https:// github. com/ zdf19 87/ EasyC GTree4) under Windows operation system (OS), of which the algorithm was originally introduced previously (Zhang et al. 2020a). EasyCGTree version 4.0 integrates HMMER (http:// www. hmmer. org/) for gene calling, MUSCLE for sequence alignment, trimAl for alignment trimming, and FastTree for phylogeny construction.

Morphology, physiology, and biochemical analysis
Brain Heart Infusion (BHI) medium, Luria Bertani (LB) agar plates, nutrient agar (NA) plates, Reasoner's 2A (R2A) agar plates, Tryptone Soya Agar (TSA) plates and 2216E plates (Solarbio, China) were used for checking optimal growth conditions of strain XHP0099 T . Expect for the 2216E medium, the other four were all supplemented with 2.5% (w/v) sea salt. Gram staining was performed according to the manufacturer's instructions in the Gram Staining kit (G1060, Solarbio, China). The temperature, pH, and salinity tolerance of the strains were tested on R2A agar plates. The range of growth temperatures and pH tolerance were set to 4, 10, 20, 28, 37, 42 ℃ and pH 4.0-10.0 (at intervals of 1.0 unit) on R2A agar, respectively. Salt tolerance test was performed at different NaCl concentrations (0, 0.5, 1.0, 2.0, 3.0, 5.0, 7.0, 10.0 and 12.0%, w/v). The motility of cells was visualized after growing at 28 ℃ for 3 days in a test tube containing R2A medium (0.5% ager, w/v). Oxidase activity was determined using 1% (v/v) p-tetramethyl phenylenediamine as the substrate. Catalase activity was assessed by observing bubble production after 3% hydrogen peroxide was added to a sample of XHP0099 T cells. The utilization of methanol by strains was explored by analyzing the growth curves with the medium introduced previously (Kelly et al. 2006). The basic medium was supplemented with methanol (30 mM), yeast extract (0.1 g⋅L −1 ), and 2.5% (w/v) sea salt, and medium without methanol was used as a control. Growth curves were performed through measuring McFarland turbidity of the four strains in different methylotrophic substrates. BiOLOG GEN III microtest system (BiOLOG, USA), API 20NE, and API ZYM system (bioMérieux, France) were used to study additional carbon utilization and characterizations of chemosensitivity, following the manufacturer's instructions.

Chemotaxonomic characterization
The biomass used for the analysis of cellular fatty acids, polar lipids, and quinones, were obtained from cultures grown on R2A agar for 2 days at 28 ℃ with vibration (180 rpm). The cellular fatty acids were extracted, methylated and analyzed according to the instructions provided by MIDI (Sherlock Microbial Identification System) (Sasser 1990). Polar lipids were extracted and identified using twodimensional thin-layer chromatography as the previously described method (Athayle et al. 1984). Respiratory quinone was purified and analyzed using high-performance liquid chromatography (HPLC) as described previously (Hiraishi et al. 1996).

Phylogenetic analysis
The almost complete 16S rRNA gene sequence of strain XHP0099 T was 1346 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 XHP0099 T 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 4-2 T (97.6%) and P. alkenifer DSM 11593 T (97.4%). The NJ and ML trees ( Fig. S1 and S2) showed that strain XHP0099 T formed a clade with "P. siganidrum" M26 and P. alcaliphilus TCM 7364 T . However, strain XHP0099 T clustered with P. alkanivorans 4-2 T , P. onubensis 1011MAR3C25 T , P. saliphilus YIM 90738 T , according to the clades of the MP phylogenetic tree (Fig. S3). It is noticeable that the position of strain XHP0099 T 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 proteincoding genes showed that strain XHP0099 T clustered with P. salsus EGI L200073 T with high confidence (bootstrap value 0.999), while P. onubensis 1011MAR3C25 T and P. alkanivorans 4-2 T as well as "P. siganidrum" M26 and P. alcaliphilus DSM 8512 T formed two more deeply branching clades.

Genomic analysis
The whole genome shotgun data of strain XHP0099 T have been deposited in the NCBI GenBank database under the accession number JAHKNG000000000. The draft genome of strain XHP0099 T was 4,179,628 bp in length with 155 contigs. The G + C content of the genomic DNA of strain XHP0099 T was 66.0%, therefore within the range for recognized Paracoccus species (58.7-71.0%) (Lee et al. 2011;Ohara et al. 1990). 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 XHP0099 T with "P. siganidrum" M26, P. alkanivorans 4-2 T and P. saliphilus DSM 18447 T were 81.4%, 80.1% and 78.3%, respectively, which were all below 95-96% based classification at the species level (Richter and Rosselló-Móra 2009). The dDDH values comparing the genome sequences of strain XHP0099 T with "P. siganidrum" M26, P. alkanivorans 4-2 T and P. saliphilus DSM 18447 T were 24.1%, 23.0% and 21.2%, respectively (Table 1), which lower than the 70% threshold value (Wayne et al. 1987) as the proposed and generally accepted species boundaries for the identification of a novel species. The AAI values between strain XHP0099 T and "P. siganidrum" M26, P. alkanivorans 4-2 T and P. saliphilus DSM 18447 T were 75.2%, 74.5% and 74.9%, respectively, which were also below the threshold value of the species boundary (95%) Fig. 1 A maximum-likelihood tree based on the 120 ubiquitous single-copy protein-coding genes showing the phylogenetic relationship of XHP0099 T in the genus Paracoccus. Bootstrap values based on 1000 replicates are showed at the branch points nodes. RefSeq assembly accession number or GenBank assembly accession number is indicated in the bracket. Hyphomonas polymorpha PS728 T is used as out group. Bar, 0.2 substitutions per nucleotide position (Rodriguez-R and Konstantinidis 2014). Based on the analysis of phylogeny, ANI, dDDH and AAI, strain XHP0099 T should represent a novel species in the genus Paracoccus. Further analysis of the KEGG reconstruction showed that strain XHP0099 T 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 XHP0099 T were observed to be 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 the genome of strain XHP0099 T harbors 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) was 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.
Two CRISPR-Cas systems, type II-C1 and incomplete type I-E, were identified in the genome of strain XHP0099 T , 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 XHP0099 T 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 ℃ (optimum, 28 ℃), pH 5.0-9.0 (optimum, pH 7.0-8.0), and with 0-7.0% NaCl (optimum, 2.0-3.0%). Strain XHP0099 T 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 XHP0099 T 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, the methylotrophic substrates with strain XHP0099 T were spread on R2A agar plates after the experiment, and growth of was observed. This result suggested that strain XHP0099 T 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 XHP0099 T should be regarded as a potential methylotrophic bacterium (Czarnecki and Bartosik 2019).
The strain was 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 MicroPlate™. Other physiological characteristics was shown in Table 2  and Table S3.

Chemotaxomic characterization
The predominant isoprenoid quinone of strain XHP0099 T was ubiquinone Q-10, which was characteristic to members of the genus Paracoccus. The major cellular fatty acids (> 10%) were summed feature 8 (C 18:1 ω7c/C 18:1 ω6c), and C 18:0 . Fatty acid compositions of strain XHP0099 T and other closely related type strains within the genus Paracoccus are 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 4-2 T and P. saliphilus DSM 18447 T . 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 XHP0099 T , "P. siganidrum" DSM 26,381 and P. alkanivorans CGMCC 1.13669 T are shown in Fig. S6.
The type strain XHP0099 T (= JCM 34661 T = GDMCC 1.2414 T = MCCC 1K05846 T ), was isolated from a sample of seawater collected at the Yellow Sea, China. The GenBank/ EMBL/DDBJ accession numbers are ON329670 (16S rRNA gene) and JAHKNG000000000 (genome),respectively.