Introduction

The genus Marinobacter was established by Gauthier et al. (1992) as a member of the family Alteromonadaceae of the gammaproteobacteria. Currently, it comprises 54 species with validly published names (https://lpsn.dsmz.de/genus/marinobacter). Members of this genus were isolated from a wide variety of environments (Ahmad et al. 2020; Zhang et al. 2015b, 2020), and are halophilic or halotolerant. Marinobacter species can degrade a wide variety of hydrocarbons (Gauthier et al. 1992; Chernikova et al. 2020). Marinobacter can have a close association with various marine phytoplanktons including diatoms and dinoflagellates (Ahmed and Holmstrom 2014; Amin et al. 2012; Green et al. 2006; Romanenko et al. 2005; Kaeppel et al. 2012; Zhang et al. 2015b, 2020). The phycosphere is a distinctive microscopical niche which hosts unique microbiota with dynamic interactions between the algae and its closely associated bacteria consortium (Amin et al. 2012; Seymour et al. 2017; Zhang et al. 2015a, b, 2020). Understanding the complex structures and roles of the phycosphere microbiota (PM) derived from toxic harmful algal blooms (HAB) dinoflagellates is important to unveil the nature of their inter-kingdom associations (Amin et al. 2012; Duan et al. 2020; Seymour et al. 2017; Zhou et al. 2021; Yang et al. 2018a, b, 2020a, b, c; Zhang et al. 2015b, 2020). These studies have resulted in the isolation of various new bacterial species, including marine species belonging to the genus Marinobacter (Ahmed and Holmstrom 2014; Green et al. 2006; Romanenko et al. 2005; Kaeppel et al. 2012; Zhang et al. 2015b, 2020).

In order to obtain more insights, we have initiated the Phycosphere Microbiome Project (PMP) where we combine the culture-dependent and multi-omics approaches (Duan et al. 2020; Yang et al. 2018a, b, 2020a, b; Zhou et al. 2021; Zhang et al. 2015a, b, 2020). During the subsequent studies, a novel mobile, faint yellow-pigmented bacterium, strain LZ-8T, was isolated from toxic HAB dinoflagellate Alexandrium catenella LZT09 which produces high levels of paralytic shellfish poisoning toxins (PSTs). The new isolate showed obvious growth-promoting potential toward its algal host. Here, we describe the polyphasic characterization of strain LZ-8T which represents a novel species of the genus Marinobacter.

Materials and methods

Bacterial strains isolation and maintenance

Strains LZ-8 was isolated from A. catenella LZT09 using our optimized isolation procedure by spreading the algal culture on a modified marine 2216 medium supplemented with algal culture extract of LZT09 at approximate 0.05 mg/L (Yang et al. 2018a, b, 2020a, b). The isolated strain was purified and maintained on marine agar (MA, Difco) and preserved as a glycerol suspension (20%, v/v) at -80 °C for long term preservation. For the comparative analysis, three reference type strains, M. sediminum R65T (= DSM 15400T = KMM 3657T) obtained from German Collection of Microorganisms and Cell Cultures (DSMZ, Germany), and M. salinus Hb8T (= JCM 31416T = KCTC 52255T) and M. similis A3d10T (KMM 7501T = CIP 110589T = JCM 19398T) obtained from Japan Collection of Microorganisms (JCM, Japan) were used. All the strains were maintained routinely on marine agar at 25 °C, and cultured under the same conditions as strain LZ-8T.

Phenotypic analysis

Morphological characteristics were recorded via light (CX21, Olympus, Tokyo, Japan) and transmission electron microscopy (JEM-1200, JEOL, Tokyo, Japan) using cultures grown on MA at 25 °C for 3 days. Gram-staining test was determined as described previously (Beveridge et al. 2007). Motility was tested microscopically under the phase-contrast mode by the hanging drop technique. Growth at different temperatures from 5 to 50 °C at 5 °C increments, pH range from 4.0 to 12.0 in increments of 0.5 pH unit) were determined as reported previously (Zhou et al. 2021; Yang et al. 2018b; Zhang et al. 2020). NaCl tolerance was determined in marine 2216 medium supplemented with 0–10% NaCl (w/v) at 25 °C for 14 days on a rotary shaker. Utilization of carbon sources and enzyme activity tests of the strains were performed using API 20NE, ZYM (bioMérieux, Marcy-l'Étoile, France) and GEN III Microplate systems (BIOLOG) following the manufacturer’s instructions.

Chemotaxonomic characteristics

Bacterial biomass for chemotaxonomic analysis was prepared by growing strain LZ-8T and the reference type strains in shake flasks with marine broth (MB) at 180 r.p.m., 25 °C for 3 days, and thereafter the harvested cells were freeze-dried. The polar lipids were determined by two-dimensional TLC according to the method described by Minnikin et al. (1984). The respiratory quinone was isolated, purified and identified according to Hiraishi et al. (1996). Cellular fatty acids were extracted, methylated and analyzed following the instructions of the Microbial Identification System (MIDI) (Sherlock version 6.1; MIDI database TSBA6).

Phylogenetic analysis

Genomic DNA extraction, PCR amplification and sequencing of the 16S rRNA gene were performed as described previously (Yang et al. 2018b). The almost-complete 16S rRNA gene sequence was compared with the sequences of the type strains available from GenBank using the BLAST program and online Ez-Taxon server (https://www.ezbiocloud.net). Multiple sequence alignments and phylogenetic analysis of the 16S rRNA gene, housekeeping rpoD gene sequences from strain LZ-8T and the related reference type strains (taken from the GenBank database) were performed using MEGA version 7 (Kumar et al. 2016). Phylogenetic trees were reconstructed using neighbour-joining (NJ), maximum likelihood (ML) and maximum parsimony (MP) algorithms using the bootstrap method with 1000 replications (Zhou et al. 2021; Zhang et al. 2020; Yang et al. 2020a). A distance matrix was generated by using Kimura’s two-parameter model (Kimura 1980).

Genomic sequencing and phylogenomic calculations

The genome sequencing, assembling and annotation of strain M. sediminum R65T was performed as reported previously (Zhang et al. 2020) at Major Bioscience (Shanghai, China) using an Illumina HiSeq 4000 system. The genome was assembled using SPAdes v3.5.0 (Anton et al. 2012). Gene prediction and genomic annotation were performed using NCBI PGAP v1.2.1 (Tatusova et al. 2016). The average nucleotide identity (ANI) and digital DNA-DNA genome hybridization (dDDH) values between strain LZ-8T and the close relative were calculated using the ANI/AAIMatrix genome-based distance matrix calculator (http://enve-omics.ce.gatech.edu/g-matrix) and online genome-to-genome distance calculator (version 2.1) (http://ggdc.dsmz.de). The genome-based phylogenetic tree using an up-to-date bacterial core gene set (UBCG) was constructed according to the method described by Na et al (2018).

Results and discussion

Phenotypic characteristics

Strain LZ-8T was isolated by spreading the algal culture on our modified isolation medium. The morphological characteristics of strain LZ-8T were consistent with its classification as a member of the genus Marinobacter. Colonies of strain LZ-8T grown on MA were faint yellow and creamy. Cells were 2.0–4.5 × 0.4–0.5 μm in size, rod-shaped, Gram-stain-negative, strictly aerobic, oxidase- and catalase-positive and motile by a flagellum (Fig. S1). Strain LZ-8T grew at pH 5.5–10 (optimum, 7.5) and 15–40 °C (optimum, 25 °C) in the presence of 0.5–9.5% (w/v) NaCl (optimum, 3.5%). Compared to its closest relative M. sediminum R65T, strain LZ-8T was positive for the utilization of D-fructose, glycerol, D-malic acid, β-hydroxy-D, L-butyric acid, propionic acid, and acetic acid, but negative for the utilization of D-cellobiose, α-D-glucose, D-mannose, or α-keto-butyric acid. In contrast to strain M. sediminum R65T, strain LZ-8T was positive for esterase (C4), valine arylamidase, cystine arylamidase, acid phosphatase and α-glucosidase, while negative for N-acetyl-β-glucosaminidase, and fermentation of glucose. These phenotypic characteristics can clearly distinguish strain LZ-8T from M. sediminum R65T (Table 1 and S1). The novel isolate did show growth-promoting potential toward its algal host LZT09 (Fig. S2).

Table 1 Differential characteristics of strain LZ-8T and close type strains of the genus Marinobacter with validly published names
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Phylogenetic characteristics

Strain LZ-8T showed high 16S rRNA gene sequence similarity values of 99.0% with M. sediminum R65T (Romanenko et al. 2005), and 98.67% with M. similis A3d10T (Ng et al. 2014), and low levels (< 98.0%) of similarities with other type strains of the genus Marinobacter (Ahmad et al. 2020). Based on 16S rRNA gene sequences analysis, strain LZ-8T formed a phyletic line on the periphery of the 16S rRNA gene subclade of the type strains of M. sediminum and M. similis (Fig. 1), which was also supported by the MP and ML trees (Figs. S3 and S4). Additionally, comparison of the housekeeping rpoD (RNA polymerase sigma factor) gene sequences showed only 93.5% similarity between strains LZ-8T and M. sediminum R65T (Romanenko et al. 2005). Phylogenetic analysis using the housekeeping genes is beneficial because of its advantage of avoiding the possibility of nucleotide polymorphisms of 16S rRNA gene (Cilia et al. 1996). Phylogenetic tree constructed based on rpoD gene sequences showed strain LZ-8T formed a separate branch outside the cluster formed by M. sediminum R65T and M. similis A3d10T (Figs. 2 and S5).

Fig. 1
figure 1

Neighbor-joining (NJ) phylogenetic tree based on almost-complete 16S rRNA gene sequences showing the taxonomic position of strain LZ-8T and related taxa of the genus Marinobacter. Filled circles indicate nodes that were also recovered in maximum-parsimony (MP) tree and maximum-likelihood (ML) tree based on the same sequences. Numbers at branch points indicate bootstrap percentages (based on 1000 replicates), only bootstrap values (≥ 50%) are shown. 16S rRNA gene sequences of Alteromonas macleodii ATCC 27126T is used as outgroup. Bar, 0.01 nucleotide substitutions per site

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Fig. 2
figure 2

Neighbor-joining phylogenetic tree based on the housekeeping rpoD gene sequences, showing the position of strain LZ-8T with respect to other members of the genus Marinobacter. GenBank accession numbers are indicated in parentheses. Numbers at branch points indicate bootstrap percentages (based on 1000 replicates), only bootstrap values (≥ 50%) are shown. Alteromonas macleodii ATCC 27126T is included as outgroup. Bar, 0.1 nucleotide substitutions per site

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Due to the high 16S rRNA gene sequence similarity between strains LZ-8T and M. sediminum R65T, a genome-scale comparison between the two strains was carried out. Therefore, the genome of M. sediminum R65T was sequenced and submitted to GenBank with the accession no. JAEMQH000000000. Its genome size was 3.719 Mbp consisting of 11 contigs with N50 value of 1,453 kbp. It had 3,187 protein-coding genes with the DNA G+C content of 57.9 mol% calculated from the genome. As shown in Table 2, the genome of strain LZ-8T (GenBank accession no. SWKM00000000) (Zhang et al. 2020) had various different features compared to those of strains M. sediminum R65T and M. similis A3d10T. For strains LZ-8T and M. sediminum R65T, 3059 genes (77.1%) and 2681 genes (79.9%), respectively, were functionally annotated within COG database. However, only 2146 (54.1%) and 1558 genes (46.5%) were annotated by KEGG for metabolic pathways, indicating that a number of functional genes were still indistinct (Fig. S6). Additionally, several key biosynthetic gene clusters (BGCs) responsible for biosynthesis of bacterial secondary metabolites including photosynthesis and antioxidant carotenoids, and beneficial siderophores that may involve in algae–bacterial interactions (Amin et al. 2012; Green et al. 2006) were only identified in the genome of strain LZ-8T.

Table 2 Genomic features and signatures between strain LZ-8T and its two close relatives, M. sediminum R65T and M. similis A3d10T
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Based on the phylogenomic calculations (Fig. S7), the ANI and dDDH values between strains LZ-8T and M. sediminum R65T, strains LZ-8T and M. similis A3d10T were 89.1 and 38.3%, 89.8% and 39.1%, respectively. All the phylogenomic values were below the threshold values for species delineation (Chun et al. 2018). Additionally, based on the constructed phylogenomic tree using the up-to-date bacterial core gene (UBCG) set (Fig. S8), strain LZ-8T formed a separate branch while strain M. sediminum R65T clustered with strain M. similis A3d10T. This taxonomic feature was similar to the phylogenetic analysis based on the housekeeping rpoD gene (Figs. 2 and S5). Therefore, the phylogenomic analysis indicates that strain LZ-8T represents a novel species within the genus Marinobacter.

Chemotaxonomic profiles

The major fatty acids of strain LZ-8T were determined as C16:0 (21.4%), C18:1 ω9c (13.5%), C12:0 3-OH (14.9%), summed feature 3 (9.9%), C16:1 ω9c (9.5%), C12:0 (6.3%) and summed feature 9 (5.5%). The common fatty acids profiles were similar to other members of the genus Marinobacter, with some minor differences listed in Table 3. Both C18:1 ω9c and C16:0 were determined as the major fatty acids components (Table 3). The major isoprenoid quinone was ubiquinone-9 (Q-9). Polar lipids of strain LZ-8T were determined as diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), unidentified phospholipid (PL), two unidentified aminolipids (ALs) and six unidentified polar lipids (Ls) (Fig. S9), which were also observed in strain M. sediminum R65T (Romanenko et al. 2005).

Table 3 Cellular fatty acid composition of strain LZ-8T and reference type strains of the genus Marinobacter with validly published names
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Taxonomic conclusion

Based on the polyphasic characterization, phylogenetic and genome comparison, strain LZ-8T clearly represents a novel species of the genus Marinobacter, for which the name Marinobacter alexandrii sp. nov. is proposed.

Description of Marinobacter alexandrii sp. nov.

Marinobacter alexandrii (a.le.xan´dri.i. N.L.gen. n. alexandrii of the dinoflagellate Alexandrium catenella, the source of isolation of the type strain).

Cells are Gram-stain-negative, strictly aerobic, oval or slightly curved rods, motile by flagellum with size 0.4–0.5 × 2.0–4.5 μm. They form faint yellow-pigmented, creamy, smooth-rounded colonies of about 1.0–2.0 mm in diameter on MA after 36 h incubation at 25 °C. Temperature, salt and pH ranges for growth are 15–40 °C, 0.5–9.5% (w/v) NaCl and pH 5.5–10, respectively. Optimal growth occurs at 4% (w/v) NaCl, 25 °C and pH 7.5. Positive for hydrolysis of Tween 40, but negative for hydrolysis of aesculin and gelatin. Oxidase and catalase activities are positive, and nitrate reduction to nitrite is variable. In the API ZYM tests, the following activities/tests are positive: alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-glucosidase; lipase (C14), trypsin, α-chymotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, β-glucosidase, nacetyl-β-glucosaminidase, α-mannosidase and α-fucosidase are absent. In the API 20NE tests, negative for arginine dihydrolase, indole production, urease, oxidation and fermentation of D-glucose, assimilation of glucose, arabinose, mannose, mannitol, N-acetyl-glucosamine, maltose, potassium gluconate, capric acid, adipic acid, trisodium citrate, phenylacetic acid, malate. Able to grow by using the following sole carbon sources in the Biolog GNIII MicroPlate: d-fructose, glycerol, l-arginine, glucuronamide, l-lactic acid, d-malic acid, l-malic acid, Tween 40, β-hydroxy- d, l-butyric acid, acetoacetic acid, propionic acid, and acetic acid; and it can survive in environments which have potassium tellurite, aztreonam and sodium butyrate. The dominant fatty acids are C18:1 ω9c, C16:0, C12:0 3-OH and summed feature 3 (comprises C16:1 ω7c and/ or C16:1 ω6c). Minor amounts (2–6%) of C17:0, summed feature 1 (comprises C13:0 3-OH and/or iso-C15:1 H) and summed feature 3 (comprises C16:1 ω7c and/ or C16:1 ω6c) are also detected. The polar lipid profile comprises diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, unidentified phospholipid, two unidentified aminolipids and six unidentified polar lipids. The genomic DNA G+C content is 57.4 mol%.

The type strain, LZ-8T (= CCTCC AB 2018386T = KCTC 72198T), was isolated from the cultivable phycosphere microbiota of highly-toxic dinoflagellate Alexandrium catenella LZT09 which was collected in Zhoushan Archipelago area in the East China Sea, China, during an algal bloom occurred in July of 2018, and routinely cultured in the ABI Laboratory. The GenBank/EMBL/DDBJ accession numbers for 16S rRNA gene sequence and draft genome sequence of strain LZ-8T are MK092986 and SWKM00000000, respectively.