Introduction

The genus Cellulosimicrobium within the family Promicromonosporaceae was proposed by the reclassification of Cellulomonas cellulans based on its phylogenetic and chemotaxonomic evidences (Schumann et al. 2001). To date, three more species, Cellulosimicrobium variabile (Bakalidou et al. 2002), Cellulosimicrobium funkei (Brown et al. 2006) and Cellulosimicrobium terreum (Yoon et al. 2007), have been described within the genus. However, C. variabile was later reclassified to the novel genus Isoptericola within the family Promicromonosporaceae as Isoptericola variabilis based on distinct phylogenetic position and the amino acid composition of the peptidoglycan (Stackebrandt et al. 2004). The members of the genus Cellulosimicrobium have peptidoglycan type of A4α, with lysine as the diagnostic diamino acid and the predominant menaquinone is MK-9(H4). The type strains of C. cellulans and C. terreum were isolated from soils, while that of C. funkei was isolated from human blood.

Marine environments have attracted attention as an isolation source of actinobacteria, and it has been reported that marine-derived actinobacteria are useful for screening for novel bioactive substances (Blunt et al. 2007; Lam 2006). During the course of a study on the actinobacterial diversity in seashore environments, a novel actinobacterium was isolated from a sea sediment sample collected in Indonesia. Comparative 16S rRNA gene sequence analysis revealed that the isolate was phylogenetically related to the members of the genus Cellulosimicrobium. In this study, we determined the taxonomic position of the isolate using a polyphasic approach.

Materials and methods

Bacterial strain and isolation

Strain RS-7-4T was isolated from a sea sediment sample that had been collected from the foreshore of Rambut Island (5°58′634″S 106°41′596″E), DKI Jakarta, Indonesia. Approximately 1 g of the sample was diluted 10-, 100- and 1000-fold with artificial seawater (Wako) before 0.2 ml of each dilution was spread on plates of 1/5 NBRC medium 802 [0.2 % (w/v) polypeptone, 0.04 % (w/v) yeast extract, 0.02 % (w/v) MgSO4·7H2O and 1.5 % (w/v) agar; at pH 7.0] supplemented with 5.0 % (w/v) NaCl, 0.005 % (w/v) cycloheximide and 0.002 % (w/v) nalidixic acid. The plates were incubated at 30 °C for 4 days, and then visible colonies were transferred to new plates. After single-colony isolation, strain RS-7-4T was obtained. As the strain did not require NaCl for growth, full-strength NBRC medium 802 [1.0 % (w/v) polypeptone, 0.2 % (w/v) yeast extract, 0.1 % (w/v) MgSO4·7H2O and, if required, 1.5 % (w/v) agar, at pH 7.0] was used as the basal medium unless otherwise stated for this study. C. cellulans NBRC 15516T, C. funkei NBRC 104118T and C. terreum KCTC 19206T were used as the reference strains in this study.

Morphological, physiological and biochemical tests

Colony appearance was examined after cultivation at 28 °C for 2 days on an agar plate of NBRC medium 802. Morphological features were observed depending on the age (up to 5 days) under a light microscope (BX-51; Olympus) and a scanning electron microscope (JSM-6060; JEOL). The temperature range and optimum temperature for growth were determined by cultivating cultures at 5, 10, 15, 20, 25, 28, 37, 45 and 60 °C on agar plates of NBRC medium 802 for 4 days (15–60 °C) or 14 days (5 and 10 °C). The pH range and optimum pH for initial growth were established by using liquid NBRC medium 802 adjusted to pH 4–12 (in 1.0 pH unit intervals) with either 4 M HCl or 5 M KOH after 4 days incubation at 28 °C. Tolerance to NaCl was determined using liquid NBRC medium 802 adjusted to NaCl concentrations of 1, 3, 5, 7, 10 and 15 % (w/v) after 4-day incubation at 28 °C. Cell motility, oxidase and catalase activities, anaerobic growth and Gram staining were determined using the methods described by Hamada et al. (2012). Other physiological and biochemical tests were performed using API ZYM, API Coryne and API 50 CH systems (bioMérieux) according to the manufacturer’s instructions.

16S rRNA gene sequence determination and phylogenetic analysis

DNA was isolated using PrepMan Ultra Reagent (Applied Biosystems) according to the manufacturer’s instructions. The 16S rRNA gene was amplified by PCR using KOD FX (Toyobo) with the following primer pair: 9F (5′-GAGTTTGATCCTGGCTCAG) and 1541R (5′-AAGGAGGTGATCCAGCC). The amplified 16S rRNA gene was subjected to cycle sequencing using a BigDye Terminator version 3.1 cycle sequencing kit (Applied Biosystems) with the following primers: 9F, 785F (5′-GGATTAGATACCCTGGTAGTC), 802R (5′-TACCAGGGTATCTAATCC) and 1541R. The products were analyzed using an automated DNA sequencer (ABI PRISM 3730 Genetic Analyzer; Applied Biosystems). Phylogenetic neighbors were identified, and pairwise 16S rRNA gene sequence similarities were calculated using the EzTaxon-e server (Kim et al. 2012). The CLUSTAL_X program (Thompson et al. 1997) was used to align the almost-complete 16S rRNA gene sequence of strain RS-7-4T (1476 nt) with corresponding sequences of related taxa. Phylogenetic trees were reconstructed by the neighbor-joining (NJ) (Saitou and Nei 1987) and maximum likelihood (ML) (Felsenstein 1981) algorithms by using the MEGA 6.0 program (Tamura et al. 2013). The resultant tree topologies were evaluated by bootstrap analysis (Felsenstein 1985) based on 1000 replicates.

G+C content of genomic DNA and DNA–DNA hybridization

Genomic DNA was obtained using the method of Saito and Miura (1963). The DNA G+C content was determined by the method of Tamaoka and Komagata (1984). The microplate hybridization method (Ezaki et al. 1989) was used to determine DNA–DNA relatedness. DNA–DNA hybridizations were performed using five replications. After the highest and lowest values for each sample were excluded, the mean of the remaining three values was reported as the DNA–DNA relatedness value.

Chemotaxonomic tests

Biomass for chemotaxonomic studies, except for fatty acid analysis, was obtained by cultivating the strain in shake flasks at 28 °C and 100 r.p.m. for 48 h. Amino acids and their isomers in cell wall hydrolysate, cell wall sugars, isoprenoid quinones and DNA G+C content were determined according to the methods described by Hamada et al. (2012). Polar lipids were extracted using the method described by Minnikin et al. (1975) and analyzed by TLC with chloroform/methanol/water (65:25:4, by vol.) in the first direction and chloroform/acetic acid/methanol/water (80:18:12:5, by vol.) in the second. Lipids were visualized by spraying the TLC plate with 10 % molybdophosphoric acid. Anisaldehyde (sugar), ninhydrin (amino groups), Schiff’s reagent (glycol) and Dittmer–Lester reagent (phosphorous; Dittmer and Lester 1964) were also used as specific spray reagents for polar lipids. For fatty acid methyl ester analysis, strain RS-7-4T and the reference strains were cultivated on tryptic soy agar (Difco) for 24 h at 28 °C. Cellular fatty acid methyl esters were analyzed by GC (6890 N; Agilent Technologies) according to the standard protocol of the Sherlock Microbial Identification System (Sasser 1990) with Sherlock MIDI software (version 6.2) and the TSBA6 database (version 6.2).

Nucleotide sequence accession numbers

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain RS-7-4T is LC042213.

Results and discussion

Strain RS-7-4T was observed to form yellow, circular and smooth colonies that were approximately 0.5–1.0 mm in diameter after 2-day cultivation. The cells were Gram stain positive, non-motile and non-endospore forming. Strain RS-7-4T was found to develop branched vegetative hyphae (0.4–0.5 × 3–10 μm) in the early phase of growth, but these hyphae eventually fragmented into short rods to coccoid cells (0.4–0.5 × 0.4–1.0 μm) (Supplementary Fig. S1). The strain was catalase positive but oxidase negative. The strain grew at 15–37 °C but not at 5, 10, 45 or 60 °C. The pH range for growth was 6.0–11.0. Optimal growth was noted at 28 °C and pH 7.0–8.0. The strain exhibited good growth with NaCl concentrations of 0–5 % (w/v) and moderate growth with 7 % (w/v) NaCl; no growth was observed with 10 or 15 % (w/v) NaCl. Growth under anaerobic conditions was not observed. The results of other physiological and biochemical tests are summarized in the species description below.

Phylogenetic analysis based on the 16S rRNA gene sequences revealed that strain RS-7-4T was closely related to the members of the genus Cellulosimicrobium. In the NJ tree, strain RS-7-4T and the type strains of C. cellulans and C. funkei formed a monophyletic cluster with a bootstrap resampling value of 96 % (Fig. 1). This cluster was also recovered in the tree generated by the ML algorithm. The highest 16S rRNA gene sequence similarity was observed with C. funkei ATCC BAA-886T (99.10 %), followed by C. cellulans LMG 16121T (99.04 %), C. terreum DS-61T (98.08 %) and Luteimicrobium xylanilyticum W-15T (97.04 %). Strain RS-7-4T showed low DNA–DNA relatedness values with C. funkei NBRC 104118T (19–20 %), C. cellulans NBRC 15516T (20–30 %) and C. terreum KCTC 19206T (15–28 %) (Table 1). These values are well below the 70 % cutoff point of DNA–DNA relatedness, which is a criterion for the assignment of bacterial strains to the same genomic species (Wayne et al. 1987).

Fig. 1
figure 1

Phylogenetic tree derived from the 16S rRNA gene sequences of strain RS-7-4T and their taxonomic neighbors, reconstructed with the neighbor-joining algorithm. The 16S rRNA gene sequence of Brevibacterium linens DSM 20425T (X77451) was used as the outgroup. Bootstrap values (>70 %) based on 1000 replicates are shown at branch nodes. Filled circles indicate that the corresponding nodes were also recovered in the tree generated with the maximum likelihood algorithm. Bar, 0.01 K nuc substitutions per nucleotide position

Full size image
Table 1 DNA–DNA relatedness values (%) among strain RS-7-4T and Cellulosimicrobium species
Full size table

The peptidoglycan sample of strain RS-7-4T was found to contain alanine (Ala), aspartic acid (Asp), glutamic acid (Glu), lysine (Lys) and threonine (Thr) in a molar ratio of 2.2:1.0:1.0:0.9:0.7. Enantiomeric analysis of the peptidoglycan amino acids revealed the presence of d-Ala, l-Ala, d-Asp, d-Glu, l-Lys and l-Thr. These data strongly suggested that the peptidoglycan type of strain RS-7-4T is A4α l-Lys–l-Thr–d-Asp, A11.45 (Schleifer and Kandler 1972; Schumann 2011). This interpeptide bridge was the same as that of C. terreum but differed from those of C. cellulans and C. funkei (Yoon et al. 2007; Schumann et al. 2001; Schumann, personal communication). A small amount of glucose was detected as the only cell wall sugar. The predominant menaquinone was identified as MK-9(H4), with MK-8(H4) and MK-9(H6) present as minor components (92:5:3). The major cellular fatty acids of strain RS-7-4T were anteiso-C15:0 (41.4 %), iso-C15:0 (18.9 %) and anteiso-C17:0 (15.8 %) (Table 2). The profile was found to be qualitatively similar to those of the recognized species of the genus Cellulosimicrobium although quantitative differences were found. The polar lipids were found to be phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannosides (Supplementary Fig. S2). The DNA G+C content of strain RS-7-4T was determined to be 75.6 mol%.

Table 2 Cellular fatty acid compositions (%) of strain RS-7-4T and Cellulosimicrobium species
Full size table

The result of the phylogenetic analysis based on the 16S rRNA gene sequences suggested that strain RS-7-4T belonged to the genus Cellulosimicrobium, and the major chemotaxonomic characteristics of the strain also corresponded to those of the genus. Therefore, it is appropriate to regard the strain RS-7-4T as a member of the genus Cellulosimicrobium. Meanwhile, the results of physiological and biochemical tests enabled strain RS-7-4T to be distinguished from the type strains of the recognized species: C. funkei, C. cellulans and C. terreum (Table 3), and the DNA–DNA relatedness values between strain RS-7-4T and these type strains were also low (Table 1). Therefore, it is proposed that strain RS-7-4T should be classified as a representative of a novel species of the genus Cellulosimicrobium, with the name Cellulosimicrobium marinum sp. nov.

Table 3 Differential phenotypic characteristics of strain RS-7-4T and Cellulosimicrobium species
Full size table

Description of Cellulosimicrobium marinum sp. nov

Cellulosimicrobium marinum (ma.ri′num. L. neut. adj. marinum belonging to the sea, marine)

Cells are Gram stain positive, aerobic, non-motile and non-endospore forming, and develop branched vegetative hyphae (0.4–0.5 × 3–10 μm) in the early phase of growth, but eventually fragment into short rods to coccoid cells (0.4–0.5 × 0.4–1.0 μm). Colonies are yellow, circular and smooth. Catalase positive and oxidase negative. The temperature range for growth is 15–37 °C (optimum 28 °C), and the pH range for growth is 6.0–11.0 (optimum pH 7.0–8.0). Growth occurs in NaCl concentrations of 0–7 % (w/v). N-Acetyl-β-glucosaminidase, alkaline phosphatase, esterase (C4), esterase lipase (C8), α-glucosidase, leucine arylamidase, pyrrolidonyl arylamidase and trypsin are present, but acid phosphatase, chymotrypsin, cystine arylamidase, α-fucosidase, α- and β-galactosidases, β-glucosidase, β-glucuronidase, lipase (C14), α-mannosidase, phosphohydrolase, pyrazinamidase, urease and valine arylamidase are not. In API 50 CH assays, acid is produced from N-acetyl-glucosamine, amygdalin, d-cellobiose, d-fructose, d-galactose, gentiobiose, gluconate, d-glucose, glycerol, glycogen, d-lactose, d-maltose, d-mannose, methyl α-d-glucopyranoside, salicin, starch, d-sucrose, trehalose, turanose and d-xylose. Aesculin and gelatin are hydrolyzed. Nitrate is not reduced. The cell wall peptidoglycan is of the A4α l-Lys–l-Thr–d-Asp. The predominant menaquinone is MK-9(H4); MK-8(H4) and MK-9(H6) are present as the minor components. The major cellular fatty acids are anteiso-C15:0, iso-C15:0 and anteiso-C17:0, while C16:0, iso-C16:0, iso-C17:0 and C14:0 are found in minor amounts. The polar lipids are phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannosides.

The type strain, RS-7-4T (=NBRC 110994T = InaCC A726T), was isolated from a sea sediment sample collected from the foreshore of Rambut Island, Indonesia. The DNA G+C content of the type strain is 75.6 mol%.