Introduction

The family Rhodobacteraceae is an abundant bacteria group with diverse ecology, metabolism and phenology (Garrity et al. 2005). The number of the genera and species in this family is growing rapidly. For example, 15 novel genera (Park et al. 2018a, b; Feng et al. 2018a, b; Wang et al. 2018; Yu et al. 2018; Klotz et al. 2018; Hu et al. 2018; Zhang et al. 2018; Wirth and Whitman 2018) have been described in 2018, including 6 reclassified novel genera (Cognatishimia, Cognatiyoonia, Flavimaricola, Limimaricola, Pseudaestuariivita and Yoonia) based on phylogenomic analyses (Wirth and Whitman 2018). At the time of writing, the family Rhodobacteraceae contains at least 160 genera encompassing nearly 592 species (List of Prokaryotic Names with Standing in Nomenclature; www.bacterio.net).

Denitrification, an important branch of nitrogen cycle, is a microbial process in which nitrate and nitrite are stepwisely reduced to gaseous forms of nitrogen. As human activities such as the high input of nitrogen-based fertilizers have nearly doubled the nitrogen input to terrestrial and marine ecosystems and consequently lead to water eutrophication, microbial denitrification is getting more attention (Kuypers et al. 2018). Some microbes showing good denitification capacity including Pseudomonas sp. (Sun et al. 2015), Alcaligenes sp. (Ozeki et al. 2001) and Microvirgula sp. (Patureau et al. 2000) have been applied to remove the excess nitrogen from wastewater.

In this study, two bacteria strains showing denitrification capacity were isolated from sediment samples of the Yellow Sea, China. Based on the polyphasic taxonomy study, strain CY05T was proposed to represent a species of a novel genus in the family Rhodobacteraceae. In addition, we explored the existence of genes related to denitrification by analyzing their genomes.

Materials and methods

Samples and cultivation

Two surface marine sediment samples were collected at a water depth of 36 m in the Yellow Sea during a cruise as described previously (Zhang et al. 2017) in 2014. Samples were stored in sterilized plastic bags (250 mL) and transported to the laboratory at 15 ºC. Bacterial strains were isolation using the standard dilution plating technique on TYM agar at 15ºC as described previously (Zhang et al. 2018). Strain CY05T was isolated from sample collected at the site H02 (36° N, 121° E) in November, and H18S-6 was isolated from sample collected at the site H18 (35° N, 121° E) in May, respectively. The temperature and salinity of the seawater at site H02 and H18 measured by CTD system was 18.2ºC and 3.1%, 10.1ºC and 3.1%, respectively. In addition, the following strains: Pelagicola marinus DSW4-44T (obtained from Korean Collection for Type Cultures), Pelagicola litoralis DSM 18290T, Phaeobacter porticola DSM 103148T, Sulfitobacter pseudonitzschiae MCCC 1A00686T, Sedimentitalea nanhaiensis MCCC 1A04178T and Pelagimonas varians DSM 23678T (obtained from Marine Culture Collection of China) were characterized alongside for comparative purposes. Unless otherwise stated, these strains were routinely cultured in marine broth 2216 (MB; Difco) or on marine agar 2216 (MA; MB with 1.5% agar) at 20ºC except for Pelagimonas varians DSM 23678T cultured in corresponding medium supplemented with 0.25 mg/L nicotinic acid.

Phylogenetic analysis

The 16S rRNA gene was amplified by PCR with the universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) (Weisburg et al. 1991). PCR products were purified using the GeneJET gel extraction kit (Thermo). The purified PCR products were ligated into pMD 19-T vector (Tsingke) and sequenced using an automated DNA sequencer (model 3730xl; Applied Biosystems) at Tsingke Biological Technology Co. Ltd (Qingdao, China). The nearly complete 16S rRNA gene sequences were compared to those of species validly published through the EzBioCloud server [http://www.ezbiocloud.net/, (Yoon et al. 2017)]. Phylogenetic trees were constructed with MEGA 6 (Tamura et al. 2013) using maximum-likelihood (Felsenstein 1981), neighbour-joining (Saitou and Nei 1987) and maximum-parsimony (Fitch 1971) methods. The topologies of the phylogenetic trees were evaluated by bootstrap analyses (1000 replications).

Genomic DNA sequencing and analyses

Genomic DNA of strains CY05T and H18S-6 were extracted using a bacterial genomic DNA isolation kit (BioTeke) following the manufacturer’s instructions except that sterile ddH2O was used for DNA elution. Genome sequencing was performed at BGI-Shenzhen (China) using the HiSeq 4000 sequencer system (Illumina). The DNA G + C % content of the two strains were calculated directly from the draft genome sequences.

The obtained draft genomes were annotated using the NCBI Prokaryotic Genome Annotation Pipeline (Tatusova et al. 2016) and RAST v2.0 (Rapid Annotation using Subsystem Technology) (Brettin et al. 2015).

Phylogenomic tree reconstruction and genome comparison

Phylogenomic tree reconstruction was conducted based on an up-to-date bacterial core gene set (UBCG) consisting of 92 single-copy core genes (Na et al. 2018). The alignment file of the UBCG was generated using a JAVA program (Na et al. 2018) from the genome sequences and was used as input for the PhyML 3.0 server (www.atgc-montpellier.fr/phyml/) with smart model selection (Guindon et al. 2010; Lefort et al. 2017). Robustness estimation of the nodes is conducted using bootstrapping with 100 replicates. Genome-based similarity indexes including in silico DNA-DNA hybridization (isDDH), average nucleotide identities (ANI), average amino acid identities (AAI) and the percentage of conserved proteins (POCP) were also calculated. The isDDH values were estimated with the Genome-to-Genome Distance Calculator using the recommended formula 2. ANI values including ANIm and ANIb were calculated in JspeciesWS. AAI values were determined by the website-based AAI calculator (Rodriguez-R and Konstantinidis 2016). POCP values were calculated according to Qin et al. (2014).

Morphological, physiological and biochemical analysis

The colony morphologies of the strains were observed after incubation on MA at 20 ºC for 7 days. Cellular morphologies and flagellum were observed by transmission electron microscope (HITACHI HT7700) with cells from exponentially growing cultures. Flagellum was also checked by the flagella staining method (Qingdao Hope Bio-Technology Co. Ltd., China). Gram staining was carried out using the standard Gram procedure (Murray et al. 1994). Growth at temperatures (5, 10, 15, 20, 25, 30, 37 and 42 °C) was measured in MB. Growth with different NaCl concentrations (0, 0.5% and 1.0%–10.0%, at intervals of 1.0% units; w/v) was determined in NP broth (Zhang et al. 2017) at 20 °C. Growth tests for pH range [5.0–10.0, at intervals of 0.5 pH units, buffered with MES (pH 5.0–6.0, 50 mM), MOPS (pH 6.5–7.0, 50 mM), Tris (pH 7.5–8.5, 50 mM) and CHES (pH 9.0–10.0, 50 mM)] were assessed in NP broth with 2.0% NaCl. Oxidase activity was tested by using commercial oxidase test strips (Tianhe Microorganism Reagent Co.) according to the manufacturer’s instructions. Catalase activity was determined by bubble production in 3% (v/v) H2O2. Hydrolysis of starch, casein, Tween 40, Tween 60 and Tween 80 were examined by using MA as the basal medium and incubation at 20 °C. Growth under anaerobic condition was determined in MB supplemented with potassium nitrate (0.1%, w/v), cysteine hydrochloride (0.05%, w/v) and sodium sulfide (0.05%, w/v) in Hungate tubes filled with oxygen-free N2 at 20ºC for two weeks. The utilization of carbohydrates (0.5%, w/v) as sole carbon and energy sources was determined in basal medium (Farmer et al. 2005) at 20 °C for 14 days. Growth was scored as negative when it was less than, or equal to, that in the negative control (lacking any carbon source). Nitrate reduction, denitrification and nitrite reduction were tested in MB medium supplemented with KNO3 (0.1%, w/v) and NaNO2 (0.01%, w/v) respectively, and contained a small inverted vial. The result were examined as (Dong and Cai 2001) described. MB medium contained a small inverted vial served as control. Susceptibility to antibiotics was examined by the disc-diffusion method on MA after spreading cell suspensions (0.5 McFarland). The discs (Oxoid) contained the following antibiotics: amoxycillin (10 μg), ampicillin (10 μg), bacitracin (10 U), carbenicillin (100 μg), cefotaxime (30 μg), cefoxitin (30 μg), chloramphenicol (30 μg), nitrofurantoin (300 μg), penicillin G (10 U) and rifampicin (5 μg). Antibiotic susceptibility was assessed by measuring diameters of the inhibition zone after 5 days at 20 °C. An inhibition zone diameter more than 10 mm (including the 6 mm diameter of the filter paper) was considered sensitive to antimicrobial agent (Wu et al. 2015). More biochemical properties were determined by using API ZYM and API 20NE strips (bioMerieux) according to the manufacturer’s instructions with sterilized artificial seawater (ASW) (Tindall et al. 2007) as the cell suspension solution and AUX medium supplemented with 2.0% (w/v) NaCl. The results were read after incubation at 20 °C for 24 h, 24 and 48 h for API ZYM and API 20 NE kits, respectively.

Chemotaxonomic analysis

For the analysis of cellular fatty acids, cells were collected from the third quadrant of the quadrant streaked plate (at exponential phase) after incubated on MA medium supplemented with 0.25 mg/L nicotinic acid at 20ºC. Fatty acid methyl esters were analysed by an Agilent 6850 N gas chromatograph and identified using the Sherlock Microbial Identification System (TSBA, version 6.1, MIDI) at Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, P. R. China. For the analysis of polar lipids and quinone, cells of them were grown to early stationary phase in MB at 20 °C. Polar lipids were extracted according to (Komagata and Suzuki 1988) and analysed using two-dimensional TLC on silica gel 60 F254 plates (Merck) with appropriate spraying reagents including ethanolic molybdophosphoric acid (total lipids), ninhydrin (aminolipids) and molybdenum blue (phospholipids). The respiratory quinone of strains CY05T and H18S-6 were extracted, separated and analysed as described by (Lin et al. 2015).

Results and discussion

Phylogenetic analysis

Nearly full-length 16S rRNA gene sequence of CY05T (1425 bp, MN790778) and H18S-6 (1425 bp, MN808553) were obtained. Strains CY05T and H18S-6 showed the highest 16S rRNA gene sequence similarities to Pelagicola marinus DSW4-44T (100% and 99.93%) and less than 96.6% sequence similarities with the closely related type strains of the family Rhodobacteraceae in the class Alphaproteobacteria. The 16S rRNA gene sequence similarity between CY05T and H18S-6 was 99.93%. There was also relatively similar sequence similarities of strains CY05T and H18S-6 to closely related species Pelagicola litoralis DSM 18290T (96.54% and 96.47%, respectively), Phaeobacter porticola DSM 103148T (96.32% and 96.32%, respectively), Sulfitobacter pseudonitzschiae MCCC 1A00686T (96.17% and 96.10%, respectively), Sedimentitalea nanhaiensis MCCC 1A04178T (95.96% and 95.89%, respectively) and Pelagimonas varians DSM 23678T (95.75% and 95.68%, respectively). In the maximum-likelihood tree (Fig. 1), strains CY05T, H18S-6 and Pelagicola marinus DSW4-44T formed a monophyletic clade within the family Rhodobacteraceae with 100% bootstrap support. This stable topology was also supported by the maximum-parsimony (Fig. S1) and neighbour-joining trees (Fig. S2). On the basis of their stable monophyletic grouping, strains CY05T, H18S-6 and Pelagicola marinus DSW4-44T should be placed in a new genus of the family Rhodobacteraceae.

Fig. 1
figure 1

Maximum-likelihood phylogenetic tree based on 16S rRNA gene sequences of strains CY05T, H18S-6 (in bold) and members of closely related taxa. Hyphomonas polymorpha PS728T was used as an outgroup. Bootstrap values (> 70%) based on 1000 replicates are shown at branch points. Bar, 0.02 substitutions per nucleotide position

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Phylogenomic tree reconstruction and genome comparison

Previous studies had shown that 16S rRNA gene sequences lack the resolution for a proper phylogenetic reconstruction inside the Rosebacter group (Luo and Moran 2014; Wirth and Whitman 2018) and genome sequence data was strongly recommended to use for the taxonomy of these bacteria. Therefore, a multigene, genome-based tree using the UBCG tool was constructed to complement the 16S rRNA gene-based trees. In accordance with the 16S rRNA trees, the genome-based tree (Fig. 2) also showed that strains CY05T, H18S-6 and Pelagicola marinus DSW4-44T formed a distinct phylogenetic clade (100% bootstrap value support) within the family Rhodobacteriaceae conforming that strains CY05T, H18S-6 and DSW4-44T should be considered as representing a novel genus of the family Rhodobacteraceae. Further, genome-based similarity indexes including isDDH, ANI, AAI and POCP which were useful for the taxonomy of prokaryotes were calculated and depicted in Table 1. ANI and isDDH percentages were genomic nucleic acid-level comparisons widely used for species delineation. As shown in Table 1, ANIb percentages between strains CY05T and H18S-6, Pelagicola marinus DSW4-44T, Pelagicola litoralis DSM 18290T, Phaeobacter porticola DSM 103148T, Sulfitobacter pseudonitzschiae MCCC 1A00686T, Sedimentitalea nanhaiensis MCCC 1A04178T and Pelagimonas varians DSM 23678T were 94.0%, 94.1%, 69.7%, 72.2%, 69.5%, 70.6% and 68.7%, respectively, and the isDDH values between them were 56.5%, 59.8%, 20.0%, 19.5%, 19.6%, 17.7% and 19.7%, respectively. They were all below the threshold value for ANI (95%–96%) and isDDH (70%) to discriminate bacterial species (Chun et al. 2018). ANI and isDDH percentages indicated that strains CY05T represents a novel genomic species in the family Rhodobacteraceae. The isDDH and ANIb values were 76.7% and 96.8% after comparing strains H18S-6 and Pelagicola marinus DSW4-44T. Considering the recommended threshold value for species discrimination (isDDH < 70% and ANI < 95%–96%), it is clear that strains H18S-6 and Pelagicola marinus DSW4-44T belong to the same species based on the high values of isDDH and ANIb values between them. AAI and POCP percentages were genomic amino acid-level comparisons mainly used for genus delineation. Previously studies showed that the AAI percentages worked perfectly for delimiting genera in the Roseobacter group (Wirth and Whitman 2018) by a gradient of AAI percentages defined by two values: a minimum value (80%) below which species should be separated into different genera. Otherwise, the suggested genus boundary of 50% POCP was found to be too conservative and consequently it was inappropriate for delimiting genera in the Roseobacter group (Wirth and Whitman 2018) and many other bacterial genera (Aliyu et al. 2016; Li et al. 2017). As shown in Table 1, AAI percentages among CY05T, H18S-6 and Pelagicola marinus DSW4-44T were 95.8–97.9%, which were significantly higher than the threshold value for AAI (> 80%) to discriminate bacterial genera (Wirth and Whitman 2018). This result indicated that they represented a genus-level taxon in agreement with the result of the 16S rRNA gene phylogeny, phylogenomic tree and the genome comparison, including ANI and isDDH values. For the POCP percentages, our results confirmed that 50% POCP alone could not be applied to Roseobacter group for genus-level circumscription because all of the calculated POCP percentages were greater than this value. All together, the genome sequence data based on ANI, isDDH and AAI values indicated that strains CY05T, H18S-6 and Pelagicola marinus DSW4-44T represent a novel genus of the family Rhodobacteraceae.

Fig. 2
figure 2

UBCG maximum-likelihood tree. Tree was generated using PhyML server 3.0 with smart model selection. The numbers at the nodes indicate the bootstrap support after 100 replicates. Hyphomonas polymorpha PS728T was used as an outgroup. Bar, 0.1 substitutions per position

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Table 1 ANIb, ANIm, isDDH, AAI and POCP percentages of strains CY05T and H18S-6 with the closely related type species. Strains: (1) CY05T; (2) H18S-6; (3) DSW4-44T; (4) Pelagicola litoralis DSM 18290T; (5) Phaeobacter porticola DSM 103148T; (6) Sulfitobacter pseudonitzschiae MCCC 1A00686T; (7) Sedimentitalea nanhaiensis MCCC 1A04178T; (8) Pelagimonas varians DSM 23678T
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Genomic DNA sequencing and analyses

The assembled draft genome of strain CY05T was 4,856,535 bp in length, with 40 contigs and a N50 value of 277,157 bp. According to annotation by PGAP, A total of 4660 genes were predicted, with 4557 protein-coding genes, 53 RNA genes and 50 pseudogenes. The assembled draft genome of strain H18S-6 was 5,037,133 bp in length, with 292 contigs and a N50 value of 264,929 bp. According to annotation by PGAP, A total of 4934 genes were predicted, with 4808 protein-coding genes, 69 RNA genes and 57 pseudogenes. The calculated genomic DNA G + C content of strains CY05T and H18S-6 were 54.5 and 54.2%, similar to that of Pelagicola marinus DSW4-44T (54.3%), but relatively lower than those of other type strains in related genera (Table 2).

Table 2 Differential characteristics of strains CY05T, H18S-6 and the type strains of its phylogenetically closest relatives in the family Rhodobacteraceae. Strains: (1) CY05T; (2) H18S-6; (3) Pelagicola marinus DSW4-44T; (4) Pelagicola litoralis DSM 18290T; (5) Phaeobacter porticola DSM 103148T; (6) Sulfitobacter pseudonitzschiae MCCC 1A00686T; (7) Sedimentitalea nanhaiensis MCCC 1A04178T; (8) Pelagimonas varians DSM 23678T. Symbols: + Positive; – Negative; w Weakly positive
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In accordance with the phenotypic results that strains CY05T and H18S-6 could reduce nitrate to gaseous forms of nitrogen, the genomes of strains CY05T and H18S-6 contained a complete set of genes encoding the enzymes involved in denitrification, including nitratereductase, nitrite reductase, nitric oxide reductase and nitrous-oxide reductase (Table. S1). Although the related genes of denitrification of the two isolates are located in different positions far away from each other, suggesting that these genes may not function simultaneously, strains CY05T and H18S-6 were positive for nitrate and nitrite reduction and denitrification based on the experimental results. There may be other ways to compensate and the specific reasons need to be further explored. Furthermore, based on the genome annotation and gene sequence comparison, the genome of strain H18S-6 also contained an assimilatory pathway by which this strain could reduce nitrate to ammonia for growth (Table. S2). However, whether or not these genes could function is not clear.

Morphological, physiological and biochemical analysis

Both strains were Gram negative, aerobic, rod-shaped and non-flagellated. Growth of the two strains was found to occur between 4 and 30 °C (optimum 20 °C). They were able to reduce nitrate and nitrite to gaseous forms of nitrogen. They were sensitive to amoxycillin (10 μg), ampicillin (10 μg), bacitracin (10 U), carbenicillin (100 μg), cefotaxime (30 μg), cefoxitin (30 μg), chloramphenicol (30 μg), nitrofurantoin (300 μg), penicillin G (10 U) and rifampicin (5 μg). There were many differences between the two strains. Cells of strains CY05T and H18S-6 were (0.7–1.8) µm and (0.8–1.3) µm in width, and (1.6–3.9) µm and (1.9–4.5) µm in length, respectively (Fig. S3). Growth occurs in 1.0%–5.0% NaCl (optimum, 2.0%–3.0%) for strain CY05T and 2.0–5.0% (optimum, 3.0%) for strain H18S-6. The pH for growth was pH 6.5–9.0 (optimum, 7.5–8.0) for CY05T and pH 7.0–9.0 (optimum, 7.5) for H18S-6. The strain H18S-6 also distinguished from strain CY05T by the ability to utilise D-cellobiose and inositol as single carbon resource for growth and the presence of α-galactosidase activities. The strains CY05T differed from those of phylogenetically related taxa by the ability of nitrate reduction and denitrification, flagellum, the assimilation of carbohydrates, the tolerance to sodium chloride and temperatures. (Table 2 and S3). Further data on the morphological, physiological and biochemical characteristics of the isolates were mentioned in Table 2 and the species description.

Chemotaxonomic analysis

The chemotaxonomic results also supported the results of the phylogenetic analysis. Cellular fatty acid comparison of CY05T, H18S-6 and the type strains of closely related species were shown in Table 3. The predominant cellular fatty acids of strains CY05T and H18S-6 were summed feature 8 (C18:1 ω7c and/or C18:1 ω6c) (> 80%), which are characteristics of the family Rhodobacteraceae. But the proportions of some fatty acids were distinguishable. C16:0 were the significant amounts in strains Pelagicola litoralis DSM 18290T, Phaeobacter porticola DSM 103148T, Sulfitobacter pseudonitzschiae MCCC 1A00686T and Pelagimonas varians DSM 23678T, but were detected at lower levels in strains CY05T, H18S-6, Pelagicola marinus DSW4-44T and Sedimentitalea nanhaiensis MCCC 1A04178T. Strain Sedimentitalea nanhaiensis MCCC 1A04178T contained a higher amount of C12:0 3OH, but it was not detected in CY05T, H18S-6, Pelagicola marinus DSW4-44T (Table 3). The distinctiveness of our isolates was also evident in the polar lipid profiles. The polar lipids of strain CY05T included phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, one unidentified aminolipid, three unidentified phospholipids, one unidentified lipid and one unidentified amino phospholipid. The polar lipids of strain H18S-6 included phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, three unidentified phospholipids, two unidentified aminolipids and one unidentified lipid. Unidentified amino phospholipid was not detected in other strains except for strains CY05T and Pelagimonas varians DSM 23678T. However, more unidentified lipids were detected in Pelagimonas varians DSM 23678T than in strain CY05T. Two unidentified aminolipids were only detected in strain H18S-6 (Fig. S4). The major respiratory quinone detected in strains CY05T and H18S-6 was ubiquinone-10, typical in members of the family Rhodobacteraceae (Park and Yoon 2013).

Table 3 Fatty acid compositions (%) of strains CY05T, H18S-6 and the type strains of some phylogenetically related species
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In conclusion, strain CY05T is not closely affiliated with any closely related genera by polyphasic characteristic differences, such as the ability of nitrate reduction, nitrite reduction and denitrification, the tolerance to sodium chloride and temperatures (Table 2). Especially significant differences between the content of polar lipids, fatty acid profiles and DNA G + C contents as described above. Furthermore, the phylogenetic position based on the 16S rRNA gene sequences and the genome sequence also reflected this relationship. And strain CY05T could also be distinguished from H18S-6 to be recognized as separate species, such as culture conditions, the ability to utilise D-cellobiose and inositolthe, content of polar lipids and fatty acid profiles (Table 2).

Based on the polyphasic data presented in this study, strain CY05T should be considered to represent a novel species of a novel genus within the family Rhodobacteraceae, for which the name Zongyanglinia huanghaiensis gen. nov. sp. nov. is proposed. Moreover, the genome sequence data based on ANI, isDDH and AAI values indicated that strains H18S-6 and Pelagicola marinus DSW4-44T belong to the same species. Combined with the above mentioned polyphasic data, such as the phylogenetic trees and the genome sequence data based on ANI, isDDH and AAI values, strain DSW4-44T should be transferred to this new genus Zongyanglinia as Zongyanglinia marinus comb. nov., the type strain is DSW4-44T (= KCTC 62762T = KCCM 43261T = JCM 33637T).

Description of Zongyanglinia gen. nov.

Zongyanglinia (Zong.yang.li’ni.a. N.L. fem. N. Zongyanglinia named to honour Chinses microbiologist Zong-Yang Lin).

Cells are Gram-stain negative. The major respiratory quinone is ubiquinone-10 (Q-10). The main polar lipids included phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, unidentified phospholipid, unidentified aminolipid and lipid. The predominant cellular fatty acids are summed feature 8 (C18:1 ω7c and/or C18:1 ω6c). The DNA G + C content of the genomic DNA are 54.2%–54.3%. Phylogenetically, the genus Zongyanglinia is affiliated to the family Rhodobacteraceae of the class Alphaproteobacteria. The type species is Zongyanglinia huanghaiensis CY05T (= MCCC 1K04409T = KCTC 62200T).

Description of Zongyanglinia huanghaiensis sp. nov.

Zongyanglinia huanghaiensis (hu.ang.hai.en’sis. N.L. masc. adj. huanghaiensis pertaining to Huanghai, the Chinese name for the Yellow Sea, the geographical origin of the type strain).

Strain exhibits the following properties in addition to those given in the genus description. Cells are aerobic, rods or ovoid-shaped rods and approximately 0.7 μm –1.8 μm in width and 1.6 μm –3.9 μm in length. Colonies are circular with regular edges, opaque, slightly convex and approximately 0.2 mm–1.1 mm in diameter on MA agar after 7 days incubation at 20 °C. Growth occurs at 5 °C–30 °C (optimum, 20 °C) and in pH range 6.5–9 (optimum, 7.5–8.0). Grows with 1.0%–5.0% NaCl (optimum, 2.0%–3.0%), does not grow without NaCl. Positive for catalase, oxidase, nitrate and nitrite reduction and denitrification, but could not hydrolysis of casein, starch, Tween 40, Tween 60 and Tween 80. The following substances are utilised as single carbon resource for growth: D-glucose, sucrose, inulin, D-raffinose pentahydrate, D-galactose, D-mannose, D-maltose monohydrate and xylan (weakly), but not D-cellobiose, inositol, D-xylose, xylitol, D-fructose, D-mannitol, sorbitolum, L-sorbose, L-arabinose and D-trehalose. In the API ZYM strips, positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase and naphthol-AS-B1-phosphophydrolase, but negative for lipase (C14), cystine arylamidase, trypsin, α-chymotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase activities. In API 20NE tests, positive for nitrate reduction, but negative for indole production, acid production from glucose, arginine dihydrolase, urease, β-glucosidase, gelatinase and beta-galactosidase, or assimilattion of glucose, arabinose, mannose, mannitol, N-acetyl-glucosamine, maltose, gluconate, caprate, adipate, malate, citrate and phenyl-acetate.

The type strain is CY05T (= MCCC 1K04409T = KCTC 62200T), isolated from sediment of the Yellow Sea. The G + C content of the genome is 54.2%, its approximate genome size is 4.86 Mbp. The GenBank accession numbers for the 16S rRNA gene sequence and the draft genome sequence of the type strain are MN790778 and WQLV00000000, respectively.

Description of Zongyanglinia marinus comb. nov.

Zongyanglinia marinus (ma.ri′nus. L. masc. adj. marinus of the sea, marine).

Basonym: Pelagicola marinus Choi et al. 2019.

The description is the same as that given by Choi et al. (2019) with the following modification. Positive for nitrite reduction and denitrification, could grow at 5ºC, weakly utilize inulin as single carbon resource for growth. The type strain is DSW4-44T (= KCTC 62762T = KCCM 43261T = JCM 33637T).