Introduction

The genus Labrenzia was proposed by (Biebl et al. 2007) for Labrenzia alexandrii as the type species of the genus and three species of the genus Stappia (Uchino et al. 1998) reclassified as Labrenzia aggregata, Labrenzia marina, and Labrenzia alba, and emended by Bibi et al. (2014). Two species, Labrenzia suaedae (Bibi et al. 2014) and Labrenzia salina (Camacho et al. 2016), were subsequently described. At the time of writing the genus Labrenzia comprises six recognized species, which have been isolated from sea water, marine sediments, dinoflagellates, oysters, and halophytes (Kim et al. 2006; Biebl et al. 2007; Bibi et al. 2014; Camacho et al. 2016). In the present study, the taxonomic position of five novel strains isolated from the red alga Polysiphonia sp. specimen collected from the Sea of Japan seashore was defined. On the basis of the phenotypic characteristics, DNA–DNA relatedness and phylogenetic analysis the novel species of the genus Labrenzia, Labrenzia polysiphoniae sp. nov. is described to accommodate five red alga-associated bacteria.

Materials and methods

Bacterial strains

A group of six bacteria designated rh32, rh44, rh45, rh46T, rh51, and rh52 was isolated from red alga Polysiphonia sp. (Polysiphonia Greville, 1823, family Rhodomelaceae) specimen, which was collected from the Peter the Great Bay, the Sea of Japan, Russia, in September 2015 as described previously (Romanenko et al. 2017). The strains were grown aerobically on marine 2216 agar (MA) or in marine broth (MB) (all BD Difco), and stored at − 80 °C in the liquid MB supplemented with 30% (v/v) glycerol. The strains rh32, rh44, rh45, rh46T, rh51, and rh52 have 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, under numbers KMM 9685 (rh32), KMM 9697 (rh44), KMM 9698 (rh45), KMM 9699T (rh46T), KMM 9704 (rh51), and KMM 9705 (rh52), respectively. The type strain KMM 9699T (= rh46T) has been deposited in the Korean Agricultural Culture Collection (KACC), National Institute of Agricultural Science and Technology (NIAST), Suwon, Korea, as KACC 19711T. The type strain Labrenzia suaedae KACC 13772T was kindly provided by the Korean Agricultural Culture Collection (KACC) and used in the phenotypic and genotypic analyses as a reference strain.

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 negatively stained with a 1% phosphotungstic acid was examined by electronic transmission microscopy [Libra 200 (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] using cells grown in MB on carbon-coated 200-mesh copper grids. The following physiological tests, including hydrolysis of starch, casein, gelatin, Tween 80, DNA, l-tyrosine, chitin, xanthine, hypoxanthine, nitrate reduction (sulfanilic acid/α-naphthylamine test), and formation of H2S from thiosulfate, were studied using artificial sea water (ASW) as described in the previous papers (Romanenko et al. 2011, 2013). Growth at various concentrations of NaCl [0, 0.5, at 1.0–12.0% (in increments of 1.0%), w/v] was tested by adding appropriate concentrations of NaCl on ASW-based medium containing (per litre) 10.0 g Bacto peptone, 2.0 g yeast extract, 0.028 g FeSO4, and 15.0 g agar. Growth at temperatures 5, 7, 15, 25, 28, 30, 35, 36, 37, 38, 39, 40, 41, and 42 °C was determined on MA for 4 days. Growth at pH 5.0–10.5 (at intervals of 0.5 pH units) was determined in MB adjusted with appropriate buffers as described by Hameed et al. (2014). Biochemical tests were carried out using API 20NE, API ID32 GN, API ZYM test kits (bioMérieux, France) as described by the manufacturer except the cultures were suspended in ASW. Antibiotic susceptibility of strains studied was examined using commercial paper discs (Research Centre of Pharmacotherapy, St. Petersburg, Russia) impregnated with the following antibiotics (mg 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), tetracycline (30), and doxocycline (10). For polar lipid and fatty acid analyses, six novel strains and Labrenzia suaedae KACC 13772T were grown on MA 2216 at 28 °C. Lipids were extracted using the extraction 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-17A 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 (Standard bacterial acid methyl ester mix 47080-U, Supelco). In addition, FAMEs were analyzed using a GLC–MS Shimadzu GC–MS model QP5050 (column MDM-5S, the temperature program from 140 to 250 °С, at a rate of 2 °С/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). DNA base composition was determined according to the methods of Marmur and Doty (1962) and Owen et al. (1969). DNA–DNA relatedness was measured by the hybridization method of De Ley et al. (1970) using a Cary 100 Bio UV–VIS spectrophotometer (Varian, Australia).

16S rRNA gene sequencing and phylogenetic analysis

Genomic DNA of five new isolates was extracted using a commercial genomic DNA extraction kit (Fermentas; EU) following the manufacturer’s instructions. The 16S rRNA genes were PCR-amplified (Devereux and Wilkinson 2004) with the universal bacterial primers 8F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1522R (5′-AAGGAGGTGATCCAGCCGCA-3′) (Edwards et al. 1989). The purified PCR fragments were sequenced using the ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems) and by the Big Dye v. 3.1 sequencing kit (Applied Biosystems).

The 16S rRNA gene sequences of five new isolates were compared with 16S rRNA gene sequences retrieved from the EMBL/GenBank/DDBJ databases using the FASTA program (Pearson and Lipman 1988). 16S rRNA gene sequences similarities for strains were calculated using Eztaxon service (Kim et al. 2012). Phylogenetic analysis was conducted using Molecular Evolutionary Genetics Analysis (MEGA, version 6.0) (Tamura et al. 2013). Phylogenetic trees were constructed by the neighbor-joining and the maximum-likelihood methods and the distances were calculated according to the Kimura two-parameter model. The robustness of phylogenetic trees was estimated by the bootstrap analysis of 1000 replicates.

Antimicrobial assay

To examine antimicrobial compound production, strains KMM 9697, KMM 9699T, and KMM 9704 were cultivated in 50 ml of MB for 3 days at 25 °C followed by extraction twice with an equal volume of ethyl acetate, washing with H2O, drying over Na2SO4, and concentrating under reduced pressure. Subsequently, obtained extracts were examined for the presence of antimicrobial activity against Staphylococcus aureus CIP 65.8T by an agar diffusion method as described previously (Romanenko et al. 2007).

Results and discussion

Phylogenetic analysis

Comparative 16S rRNA gene sequence analysis showed that six novel strains belonged to the genus Labrenzia, while five of them, KMM 9697, KMM 9698, KMM 9699T, KMM 9704, and KMM 9705, shared the highest gene sequence similarities of 98.1–98.4% with the type strain of Labrenzia suaedae KACC 13772T, and strain KMM 9685 was close to Labrenzia alba CECT 5094T (99.9% sequence similarity). In the phylogenetic trees generated by the different algorithms strains, KMM 9697, KMM 9698, KMM 9699T, KMM 9704, and KMM 9705 positioned as a distinct cluster adjacent to Labrenzia suaedae KACC 13772T within the genus Labrenzia (Fig. 1, Supplementary Fig. S1). The 16S rRNA gene sequence similarities obtained between these strains and the remaining Labrenzia species were lower the threshold similarity value of 97% proposed by Stackebrandt and Goebel (1994) and re-evaluated to 98.7–98.6% by Stackebrandt and Ebers (2006) and Kim et al. (2014), indicating that these novel strains could be assigned to the genus Labrenzia as an individual species.

Fig. 1
figure 1

Neighbor-joining phylogenetic tree based on the 16S rRNA gene sequences available from the GenBank/EMBL/DDBJ databases (accession numbers are given in parentheses) showing phylogenetic position of the novel Labrenzia isolates and members of the genus Labrenzia and related taxa. Bootstrap values based on 1000 replications are given as percentages at the branching points. Numbers indicate percentages greater than 70%. Bar, 0.02 substitutions per nucleotide position

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DNA–DNA hybridization

The DNA–DNA reassociation means between five novel strains were 83–91%. The DNA–DNA hybridization values between two of the novel strains, KMM 9699T and KMM 9704, and the closest neighbor Labrenzia suaedae KACC 13772T were measured to be 26% and 36%, respectively (Table 1). The DNA relatedness values confirmed an assignment of the novel isolates to the same separate species of the genus Labrenzia according to the value of 70% proposed by Wayne et al. (1987) for the bacterial species delineation.

Table 1 DNA–DNA relatedness among novel Labrenzia strains and Labrenzia suaedae KACC 13772T
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Phenotypic characterization

Cultural, physiological, and metabolic properties of novel strains are listed in Table 2 and in the species description. Novel isolates were aerobic, Gram-negative, ovoid- or rod-shaped bacteria motile by means of 2–5 subpolar and/or lateral flagella (Fig. 2, Supplementary Fig. S2). Cells with a single flagellum were rarely observed. Some bacteria are capable of producing extracellular material (Supplementary Fig. S2). In addition, cells could be observed as weakly motile or non-motile due to their property to lose flagella easily. Novel isolates were characterized by the inability to assimilate most organic substrates as gave negative results for the assimilation of carbon sources in API 32GN and API 20NE tests. The novel strains differed from each other in the reaction of tyrosine hydrolysis, β-galactosidase and trypsin API ZYM tests and antibiotic resistance profiles. Strain KMM 9685 differed from other novel isolates in many traits, as shown in Table 2, and was included to the present study for the comparative purpose.

Table 2 Phenotypic characteristics of novel Labrenzia strains, Labrenzia suaedae KACC 13772T, and Labrenzia alba CECT 5084T
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Fig. 2
figure 2

Transmission electron micrographs of strains: a KMM 9699T; b, c KMM 9697; d KMM 9704 grown in MB for 24–36 h. Bars a, c 500 nm; b, d 1 µm

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Fatty acid profiles were similar with a large proportion of C18:1ω7c followed by 11-Methyl C18:1ω7c, and C14:0 3-ОН found in the novel isolates KMM 9697, KMM 9698, KMM 9699T, KMM 9704, and KMM 9705. Unlike strain Labrenzia suaedae KACC 13772T the novel strains contained a small amount of C18:2 and C20:1, twice more 11-Methyl C18:1ω7c and did not contain C18:0 3-ОН (Table 3). The major isoprenoid quinone of novel strains was ubiquinone Q-10. The polar lipid composition of strains tested was found to be similar in major components and included phosphatidylcholine, PC, phosphatidylethanolamine, PE, phosphatidylmonomethylethanolamine, PMME, phosphatidylglycerol, PG, an unknown aminolipid, AL, and an unknown phospholipid, PL. Labrenzia suaedae KACC 13772T contained additionally an unknown lipid (Supplementary Fig. S3). Five novel strains and L. suaedae KACC 13772T did not contained sulphoquinovosyldiacylglyceride, (SQDG), which has been found in all other members of the genus Labrenzia (Biebl et al. 2007). This finding is congruent with the original description of Bibi et al. (2014) who reported lack of SQDG in Labrenzia suaedae KACC 13772T and emended description of the genus in terms of the variability of glycolipid SQDG in Labrenzia species (Bibi et al. 2014). Unlike all strains tested strain KMM 9685 contained SQDG that supported chemotaxonomically its assignment to the member of L. alba lineage.

Table 3 Fatty acid composition (%) of novel Labrenzia strains and Labrenzia suaedae KACC 13772T
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Chemotaxonomic characteristics, including ubiquinone Q-10, the predominance of C18:1ω7c, and the presence of PC, PMME, PE, PG, Al, PL, and the DNA G + C content values of 56.5–58.0 mol% obtained for five novel strains supported their assignment to the genus Labrenzia (Biebl et al. 2007; Bibi et al. 2014; Camacho et al. 2016).

Some of novel strains were found to inhibit growth of one or two test cultures including S. aureus, E. faecium (weakly), S. epidermidis (weakly), and Xanthomonas sp. pv. badrii as described in a previous paper (Romanenko et al. 2017). Subsequently, antimicrobial activity against S. aureus was revealed in ethyl acetate extracts of liquor cultures as shown by the example of three strains, KMM 9697, KMM 9699T, KMM 9704, pointing to the presence of low-molecular-weight metabolites, which could be responsible for their activity (Supplementary Fig. S4). It should be noted that the activity of some isolates was not consistently stable and changed with respect to test cultures. Further studies should be done to determine the structure of the antibacterial compounds and search for appropriate genes in the genome sequence of the respective strains.

It is evident from the results obtained that novel isolates can be assigned to the genus Labrenzia on the basis of their physiological, biochemical, and chemotaxonomic characteristics. Five novel isolates could be distinguished from the most Labrenzia members which are monotrichous, in the number and flagella arrangement forming 2–5 subpolar and/or lateral flagella, in not being able to assimilate most compounds which are included to the API 20 NE and API 32GN strips, as well as in enzymes activities in API ZYM, in the absence of SQDG in polar lipids and antibiotic sensitivity profiles. These strains differed from L. suaedae KACC 13772T in the requirement of sodium ions for growth, in the inability to grow in 9% NaCl and at 41 °C, to reduce nitrate to nitrite, to produce H2S from thiosulfate, to hydrolyze gelatin, to assimilate glucose, arabinose, mannitol, N-acetylglucosamine, malate and citrate, and to produce N-acetyl-β-glucosaminidase. Differential phenotypic characteristics are listed in Table 1 and Supplementary Table 1. Based on the results obtained, it is proposed to assign five novel strains KMM 9697, KMM 9698, KMM 9699T, KMM 9704, and KMM 9705 to the genus Labrenzia as representing a novel species, Labrenzia polysiphoniae sp. nov. with the type strain KMM 9699T.

Description of Labrenzia polysiphoniae sp. nov

Labrenzia polysiphoniae (po.ly.si.pho’ni.ae. N.L. fem. gen. n. polysiphoniae of Polysiphonia, pertaining to the isolation source of red alga Polysiphonia sp.)

Gram-negative, aerobic, oxidase-positive, catalase-positive, ovoid- or rod-shaped bacteria motile by means of 2–5 subpolar and/or lateral flagella cells, 0.6–0.8 µm in diameter and 1.5–2.0 µm in length. Bacteria are capable of producing extracellular material. Cells could be observed as weakly motile or non-motile due to their property to lose flagella easily. Cells occurred as a single or formed star-shaped aggregates. On Marine Agar 2216 hemi-transparent, lightly cream pigmented, shiny and smooth colonies of 2–3 mm diameter with the regular edges are produced. Grows on tryptic soya agar supplemented with sea water. Bchl a is not produced. Required NaCl for growth; growth occurred between 1 and 8% (w/v) NaCl with an optimum of 3–4% NaCl. The temperature range for growth was 7–39 °C with an optimum of 28–30 °C. The pH range for growth was 5.5–10.0 (optimal pH 6.5–7.5). Some strains could grow at pH 5.0–10.5. Negative for hydrolysis of casein, gelatin, chitin, starch, Tween 80, xanthine, hypoxanthine and production of H2S in conventional tests. Positive for DNA hydrolysis; hydrolysis of l-tyrosine is strain-dependent (the type strain reaction is slow). Negative for nitrate reduction in routine and API 20NE tests. According to the API 20NE, positive for PNPG test; and negative for aesculin and gelatin hydrolysis, nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease, assimilation of d-glucose, l-arabinose, d-mannose, d-mannitol, N-acetylglucosamine, maltose, d-gluconate, l-malate, caprate, adipate, citrate, and phenylacetate. According to the ID32 GN, strains could not assimilate any of substrates included to the ID32 GN gallery. Positive API ZYM test results were obtained for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase; and negative for lipase (C14), cystine arylamidase, valine arylamidase, α-chymotrypsin, α-galactosidase, β-glucosidase, α-glucosidase, β-glucuronidase, N-acetyl-β-glucosaminidase, α-mannosidase, and α-fucosidase; β-galactosidase and trypsin tests are variable (reactions are positive for the type strain). The major isoprenoid quinone is ubiquinone Q-10. Polar lipids consisted of phosphatidylcholine, phosphatidylmonomethylethanolamine, phosphatidylethanolamine, phosphatidylglycerol, an unknown aminophospholipid, and an unknown phospholipid. Fatty acid C18:1ω7c was predominant followed by 11-Methyl C18:1ω7c, and C14:0 3-ОН. Strains were susceptible to gentamicin, rifampicin, ofloxacin, streptomycin, nalidixic acid, neomycin, erythromycin, tetracycline, and resistant to ampicillin, benzylpenicillin, lincomycin, oxacillin, oleandomycin, and polymyxin. Susceptibility to vancomycin, kanamycin, carbenicillin, doxocycline, cephazolin, and cephalexin was strain-dependent (type strain was resistant to above antibiotics). The DNA G + C content is 56.5–58.0 mol%, the DNA G + C content of the type strain KMM 9699T is 57.3 mol%. The type strain of the species is strain KMM 9699T (= rh46T = KACC 19711T) isolated from red alga Polysiphonia sp. specimen, collected from the Sea of Japan seashore, Russia.