Introduction

The Roseobacter clade (family Rhodobacteraceae, class Alphaproteobacteria) comprises a large group of bacterial genera (Garrity et al. 2005; Brinkhoff et al. 2008) which have been reported to be wide-spread microorganisms in marine environments, being isolated from seawater, sediments, polar sea ice, microbial mats, seaweeds and animals (Buchan et al. 2005). A recent phylogenomic study of Wirth and Whitman (2018) demonstrated the insufficiency of the 16S rRNA gene sequence analysis for phylogeny of the Roseobacter group in the family Rhodobacteraceae, whereas phylogeny based on a set of house-keeping core gene sequences is more reliable. As a result, applying this approach a number of genera and species of Alphaproteobacteria were reclassified and proposed as novel or recognized taxa (Wirth and Whitman 2018; Hördt et al. 2020). During a survey of microorganisms capable of dwelling shallow sediments of the Sea of Japan the Gram-negative, non-motile bacterium designated KMM 3653T was found and investigated by using phenotypic and molecular methods and results obtained are reported in the present study. Based on the phylogenetic analyses data and distinctive phenotypic characteristics, a novel genus and species Harenicola maris gen. nov., sp. nov. is described to accommodate the strain KMM 3653T (= KCTC 82575T).

Materials and methods

Bacterial strains

Strain KMM 3653T was isolated from a sandy sediment sample collected from the Sea of Japan seashore, Russia, in July 2002 as described in a previous paper (Romanenko et al. 2004). Strain KMM 3653T was grown aerobically on/in Marine agar 2216 (MA) or Marine broth 2216 (MB) (both BD Difco), and stored at − 70 °C in MB supplemented with 30% (v/v) glycerol. The strain KMM 3653T has been deposited in the Collection of Marine Microorganisms (KMM), G. B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, Vladivostok, Russia, and in the Korean Collection for Type Cultures (KCTC), Korea, under a number of KCTC 82575T. The type strains, Nioella nitratireducens KCTC 32417T, Oceanicola litoreus (Vannielia litorea) KCTC 32083T, Pacificibacter aestuarii JCM31805T, and Nioella aestuarii JCM 30752T were purchased from the Korean Collection for Type Cultures (KCTC), Korea, and Japan Collection of Microorganisms (JCM), Japan, respectively, and used in the phenotypic and lipid analyses.

Phenotypic characterization

Gram-staining, oxidase and catalase reactions, and motility (the hanging drop method) were determined as described by Gerhardt et al. (1994). The morphology of cells grown in MB and negatively stained with a 1% phosphotungstic acid on carbon-coated 200 mesh copper grids was examined by electronic transmission microscopy [Libra 120 FE (Carl Zeiss), provided by the Far Eastern Centre of electronic microscopy, Zhirmunsky Institute of Marine Biology, Far Eastern Branch of the Russian Academy of Sciences]. Hydrolysis of starch, casein, gelatin, Tweens 20, 40, 80, DNA, l-tyrosine, chitin, xanthine, hypoxanthine, nitrate reduction (sulfanilic acid/α-naphthylamine test), formation of H2S from thiosulfate, and growth at different salinities (0–12% NaCl), temperatures (4–45 °C) and pH values (4.5–10.5) were carried out using artificial sea water (ASW) as described in previous papers (Romanenko et al. 2011b, 2013). The artificial sea water (ASW) contained (per liter of distilled water): 24 g NaCl, 4.9 g MgCl2, 2.0 g MgSO4, 0.5 g CaCl2, 1.0 g KCl, 0.01 g FeSO4. Acid production from carbohydrates was examined using the oxidation/fermentation medium of Leifson (1963). Biochemical tests were performed using API 20E, API 20NE, API ID32 GN, and API ZYM test kits (BioMérieux, France) as described by the manufacturer except the cultures were suspended in ASW. Antibiotic susceptibility was examined using commercial paper discs (Research Centre of Pharmacotherapy, St. Petersburg) impregnated with the following antibiotics (µg per disc, unless otherwise indicated): ampicillin (10), benzylpenicillin (10 U), vancomycin (30), gentamicin (10), kanamycin (30), carbenicillin (100), chloramphenicol (30), neomycin (30), oxacillin (10), oleandomycin (15), lincomycin (15), ofloxacin (5), rifampicin (5), polymyxin (300 U), streptomycin (30), cephazolin (30), cephalexin (30), erythromycin (15), nalidixic acid (30), and tetracycline (30).

Chemotaxonomic characterization

Strain KMM 3653T and type strains of related genera, N. nitratireducens KCTC 32417T, N. aestuarii JCM 30752T, Oceanicola litoreus (V. litorea) KCTC 32083T, P. aestuarii JCM 31805T, Pacificibacter maritimus KMM 9031T were grown on MA 2216 at 28 °C. Lipids were extracted using the method of Folch et al. (1957). Two-dimensional thin-layer chromatography of polar lipids was carried out on Silica gel 60 F254 (10 × 10 cm, Merck, Germany) using chloroform–methanol–water (65:25:4, v/v) for the first direction, and chloroform–methanol–acetic acid–water (80:12:15:4, v/v) for the second one (Collins and Shah 1984) and spraying with specific reagents (Collins et al. 1980). Respiratory lipoquinones were analyzed by reversed-phase high-performance thin-layer chromatography as described by Mitchell and Fallon (1990). Fatty acid methyl esters (FAMEs) were prepared according to the procedure of the microbial identification system (MIDI) (Sasser 1990). The analysis of FAMEs was performed using the GC-2010 chromatograph (Shimadzu, Kyoto, Japan) equipped with capillary columns (30 m × 0.25 mm I.D.), one coated with Supecowax-10 and the other with SPB-5. Identification of FAMEs was accomplished by equivalent chain length values and comparing the retention times of the samples to those of standards. In addition, FAMEs were analyzed using a GLC-MS Shimadzu GC–MS model QP2020 (column Shimadzu SH-Rtx-5MS, the temperature program from 160 to 250 °C, at a rate of 2 °C/min). Production of bacteriochlorophyll a (Bchl a) was spectrophotometrically tested in methanolic extracts of cells grown on MA and MB in the dark as described by Lafay et al. (1995).

16S rRNA gene sequence and phylogenetic analysis

Genomic DNA of the strain KMM 3653T was extracted using a commercial genomic DNA extraction kit (Fermentas, EU) following the manufacturer’s instruction. The universal bacterial primers 8F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1522R (5’-AAGGAGGTGATCCAGCCGCA-3’) (Edwards et al. 1989) were used for amplification of the 16S rRNA gene. The 16S rRNA gene was PCR-amplified and sequenced as described in a previous paper (Romanenko et al. 2019). The 16S rRNA gene sequence obtained for strain KMM 3653T was compared with those of the closest relatives using the BLAST (http://www.ncbi.nlm.nih.gov/blast/) and EzBioCloud service (Yoon et al. 2017). Phylogenetic analysis was conducted using molecular evolutionary genetics analysis (MEGA X) (Kumar et al. 2018). Phylogenetic trees were constructed by the neighbor-joining and the maximum-likelihood and the distances were calculated according to the Kimura two-parameter model (Kimura 1980). The robustness of phylogenetic trees was estimated by the bootstrap analysis of 1000 replicates.

Whole-genome sequencing and genome-based phylogenetic analysis

The genomic DNA was obtained from the strain KMM 3653T using the high pure PCR template preparation kit (Roche, Basel, Switzerland). The quantity and quality of the genomic DNA were measured using DNA gel electrophoresis and the Qubit 3.0 Fluorometer (Thermo Fisher Scientific, USA). Preparation of the DNA sequencing library was carried out using Nextera DNA Flex kits (Illumina, San Diego, CA, USA) and whole-genome sequencing was performed subsequently using paired-end runs on an Illumina MiSeq platform with a 150 bp read length. The reads were trimmed using Trimmomatic (Bolger et al. 2014) and their quality assessed using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Filtered reads were assembled into contigs with SPAdes version 3.11.1 (Bankevich et al. 2012) and genome metrics were calculated with the help of QUAST (Galaxy version 5.0.2+galaxy0) (Gurevich et al. 2013). The draft genome assembly was annotated using NCBI prokaryotic genome annotation pipeline (PGAP) (Tatusova et al. 2016). Comparisons of the average nucleotide identity (ANI) and in silico DNA–DNA hybridization (dDDH) values of the strain KMM 3653T and its closest neighbors were performed with the JSpecies web server (Richter et al. 2016), and TYGS platform (Meier-Kolthoff and Göker 2019), respectively. The phylogenomic analysis was performed using PhyloPhlAn 3.0 software based on a set of 400 conserved bacterial protein sequences (Asnicar et al. 2020).

Results and discussion

16S rRNA gene sequencing and genome-based phylogenetic analysis

Comparative 16S rRNA gene sequence analysis showed that the novel strain KMM 3653T belongs to the family Rhodobacteraceae (class Alphaproteobacteria) and its closest phylogenetic neighbours were found to be members of the genera Pacificibacter (95.2–94.7% gene sequence similarity) and Nioella (95.1–94.5%), respectively. Different treeing algorithms (neighbor-joining, Fig. 1, and maximum-likelihood, Supplementary Figure S1) revealed that strain KMM 3653T formed a separate branch located between Nioella and Pacificibacter clusters. The low values of 16S rRNA gene sequence similarities with related Alphaproteobacteria demonstrated that strain KMM 3653T can be representative of a novel genus in the family Rhodobacteraceae. The phylogenetic tree based on concatenated 400 protein sequences from whole-genome sequences supported the placement of the novel strain KMM 3653T in the family Rhodobacteraceae as a separate line adjacent to V. litorea KCTC 32083T [former Oceanicola litoreus (Park et al. 2013; Hördt et al. 2020)] (Fig. 2). The ANI and dDDH values between strain KMM 3653T and V. litorea KCTC 32083T, N. nitratireducens KCTC 32417T, P. aquimaris CECT 7680T, and Litoreibacter albidus KMM 3851T were 71.1, 70.3, 71.0, and 69.6%, and values of 18.5% to each of the first three strains and 19.5% to the last, respectively. These values obtained were significantly lower than the ANI and dDDH values of 95–96% and 70%, respectively, which have been accepted for bacterial species discrimination (Chun et al. 2018). The draft genome features of the strain KMM 3653T are summarized in Table S1. The phylogenomic analysis data evidence that the strain KMM 3653T does not belong to any of the recognized genera and could be classified as an individual genus and species of the family Rhodobacteraceae.

Fig. 1
figure 1

Neighbor-joining tree based on 16S rRNA gene sequences available from the GenBank/EMBL/DDBJ databases (accession numbers are given in parentheses) showing relationships of strain KMM 3653T and related taxa. Bootstrap values based on 1000 replications are given as percentages at the branching points and numbers indicate percentages greater than 60%. Bar, 0.01 substitutions per nucleotide position

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Fig. 2
figure 2

Maximum-likelihood tree based on concatenated 400 protein sequences translated from genome sequences showing phylogenetic position of strain KMM 3653T among related members of the family Rhodobacteraceae. Bootstrap values are based on 100 replicates. Bar, 0.05 substitutions per amino acid position

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Morphological, physiological and chemotaxonomic characteristics

Morphological, physiological, biochemical and chemotaxonomic characteristics of strain KMM 3653T are given in Tables 1, 2, Supplementary Figures S2, S3, and in the genus and species descriptions. Strain KMM 3653T was ovoid or rod-shaped bacteria dividing by the budding process and capable of producing extracellular material (Supplementary Figure S2). The novel bacterium KMM 3653T was not able to hydrolyse a number of polymeric substrates (Table 1) and assimilate most carbon sources in API 32GN, API 20E and API 20NE tests (Table S2). Strain KMM 3653T contained ubiquinone Q-10 as the major respiratory quinone and C18:1ω7c as the major fatty acid followed by C16:0 (Table 2). Fatty acid profiles found in all related type strains were similar in a large proportion of C18:1ω7c that is characteristic for the members of the family Rhodobacteraceae although strain KMM 3653T differed in content of some fatty acids, such as C14:1 3-OH, C12:0 3-OH, C16:0 2-OH or C19:0 cyclo (Table 2). The polar lipids of the strain KMM 3653T were found to be phosphatidylcholine (PC), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), an unidentified phospholipid (PL), two unidentified aminolipids (AL) and five unidentified lipids (L) (Fig. S3). The polar lipid profile of the strain KMM 3653T was similar to those of Pacificibacter species (Romanenko 2020) and differed from the other related genera in the absence of PE. As reported members of Vannielia (Park et al. 2013), Litoreibacter (Romanenko et al. 2011a; Kim et al. 2012) and Nioella (Rajasabapathy et al. 2015; Cha et al. 2017; Liu et al. 2017; Yang et al. 2020) contained PE in their lipid profiles (Table 1). In the present study, PE was present in V. litorea KCTC 32083T but was not found in N. nitratireducens KCTC 32417T and N. aestuarii JCM 30752T (Fig. S3). The DNA G+C content of 61.8% was calculated from the genome sequence. The value obtained for the strain KMM 3653T is close to some extent to those of Nioella and Litoreibacter members but significantly differed from those of Pacificibacter and Vannielia (Table 1). The phylogenetic relationships observed on the basis of 16S rRNA gene and whole-genome sequences, and genetic distinctness as revealed by ANI and dDDH analyses were supported by phenotypic differences of the novel isolate KMM 3653T in its growth temperature and salinity ranges, and substrate hydrolysis. Differential phenotypic characteristics are indicated in Table 1. Based on the combined phylogenetic evidence, phenotypic and biochemical characteristics, it is proposed to classify strain KMM 3653T as a novel genus and species, Harenicola maris gen. nov., sp. nov., with the type strain of the type species KMM 3653T (= KCTC 82575T).

Table 1 Differential characteristics of strain KMM 3653T and related genera
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Table 2 Fatty acid composition (%) of strain KMM 3653T and the type strains of related genera of the family Rhodobacteraceae
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Description of Harenicola gen. nov.

Harenicola (Ha.re.ni’co.la. L. fem. n. harena, sand; L. masc./fem. suff. -cola, inhabitant; N.L. masc. n. Harenicola, inhabitant of sand).

Gram-negative, aerobic, oxidase- and catalase positive, ovoid or rod-shaped bacteria enlarged at one pole due to the cell division by budding. Chemoorganoheterotrophic. Sodium ions are essential for growth. The predominant isoprenoid quinone is Q-10. Polar lipids include phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, two unidentified aminolipids, an unidentified phospholipid, and five unidentified lipids. The major fatty acid is C18:1ω7c followed by C16:0. Isolated from the marine environments. On the basis of the 16S rRNA gene sequence analysis, the genus represents a separate branch within the family Rhodobacteraceae of the class Alphaproteobacteria. The type species of the genus is Harenicola maris.

Description of Harenicola maris sp. nov.

Harenicola maris (ma’ris. L. gen. n. maris, of the sea)

In addition to properties given in the genus description the species is characterized as follows:

cells are ovoid or rod-shaped 0.9–1.4 μm in diameter and 1.0–2.7 μm in length. Cells are capable producing of capsular material. Non-motile. Whitish-pigmented, smooth and shiny colonies with the regular edges of 2–3 mm in diameter are produced on MA. Growth occurs at 7–36 °C (optimum, 28–30 °C) and in the presence of 1–5% (w/v) NaCl (optimum, 2–3% NaCl). The pH range for growth is 6.0–10.0 with an optimum of 7.0–8.0. Negative for hydrolysis of gelatin, casein, tyrosine, starch, Tweens 20, 40, 80, chitin, xanthine and hypoxanthine, and H2S production. Positive for DNA and urea hydrolysis. Negative for acid production from d-glucose, d-fructose, maltose, lactose, d-galactose, cellobiose, sucrose, d-xylose, d-raffinose, and l-arabinose. According to the API 20NE, positive for aesculin hydrolysis and PNPG test. In the API 20E tests positive for urease production and oxidation of l-rhamnose, d-sucrose (weakly), and amygdalin. In the API ZYM tests positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, and naphthol-AS-BI-phosphohydrolase. Susceptible to (content per disc): ampicillin (10 µg), benzylpenicillin (10 U), vancomycin (30 µg), kanamycin (30 µg), carbenicillin (100 µg), chloramphenicol (30 µg), lincomycin (15 µg), neomycin (30 µg), oxacillin (10 mg), oleandomycin (15 µg), ofloxacin (5 µg), rifampicin (5 µg), streptomycin (30 µg), tetracycline (30 µg), cephazolin (30 µg), cephalexin (30 µg), erythromycin (15 µg); and resistant to gentamicin (10 µg), nalidixic acid (30 µg), and polymyxin B (300 U). The major isoprenoid quinone is ubiquinone Q-10. Major fatty acid is C18:1ω7c followed by C16:0. The polar lipids include phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, an unidentified phospholipid, two unidentified aminolipids and five unidentified lipids. The DNA G+C content of 61.8% is calculated from the genome sequence.

The type strain of the species is strain KMM 3653T (= KCTC 82575T) isolated from a shallow sediment sample collected from the Sea of Japan, Russia.