Micrococcus endophyticus sp. nov., isolated from surface-sterilized Aquilaria sinensis roots

Recent studies have indicated that endophytic bacteria are common among the resident microflora of the healthy inner tissues of various species of plants (Coombs et al., 2004; Gu et al., 2006; Trujillo et al., 2006; Tian et al., 2007), in which these micro-organisms play an important role in plant growth. The genus Micrococcus was first described by Cohn (1872) and since then the description of the genus has been revised repeatedly (Stackebrandt et al., 1995; Wieser et al., 2002). Members of the genus Micrococcus are aerobic, Gram-positive and catalase-positive. Cells are spherical, non-motile and do not produce endospores. The G+C contents of the DNA range from 66.3 to 73.3 mol%. At the time of writing, the genus Micrococcus encompasses four species: Micrococcus luteus, Micrococcus lylae, Micrococcus antarcticus and Micrococcus flavus (Stackebrandt et al., 1995; Kloos et al., 1974; Liu et al., 2000; Liu et al., 2007). These species are phylogenetically closely related to each other. The aim of this study was to determine the exact taxonomic position of a novel Micrococcus strain by means of a polyphasic characterization including phenotypic and chemotaxonomic determinations and a detailed phylogenetic analysis based on 16S rRNA gene sequences.

Strain YIM 56238^T was isolated from the roots of a plant, Aquilaria sinensis (traditionally used in Chinese medicine), collected in the tropical rainforest of Yunnan Province in south-west China. Samples were washed in running water to remove soil particles and were sterilized using an established procedure (Coombs & Franco, 2003). After being surface-sterilized, the root samples were sliced into pieces for plating on tap water-yeast extract agar (TWYE) plates, which were incubated at 28 °C for 3–7 weeks. The micro-organisms were observed around the root samples under a light microscope. Pure cultures were obtained by means of repeated streaking on plates containing TWYE agar. The purified strain (YIM 56238^T) was picked and then maintained on trypticase soy agar (TSA; BD) slants at 4 °C and as 20 % (w/v) glycerol suspensions at −20 °C. Biomass for chemical and molecular studies was obtained by means of cultivation according to Stackebrandt et al. (1995).

Gram staining was carried out using the standard Gram reaction and was confirmed with the KOH test (Moaledj, 1986). Morphology and motility were examined by using light microscopy (BH-2; Olympus) and scanning electron microscopy (LEO 1455VP; Zeiss) with cells from exponentially growing cultures. Colony morphology was observed on TSA after incubation at 28 °C for 3 days. Physiological characteristics of strain YIM 56238^T and its closest neighbours were tested in parallel by using the following procedures. Growth was tested on TSA at 4, 15, 20, 28, 37, 45, 50 and 55 °C. Growth at various NaCl concentrations (0, 1, 3, 5, 7, 10, 15 and 20 %, w/v) was tested. The pH range and optimum for growth were investigated at pH 4.0–10.0 using the buffer system described by Xu et al. (2005) and trypticase soy broth as the basal medium. Metabolic properties were determined using API 50 CH test kits according to the manufacturer's instructions (bioMérieux). Other physiological and biochemical tests were performed as described previously (Barrow & Feltham, 1993; Wieser et al., 2002).

Cells of strain YIM 56238^T were Gram-positive, aerobic, non-motile, non-endospore-forming and coccoid (0.5–0.7 μm in diameter) (see Supplementary Fig. S1, available in IJSEM Online). The colonies were yellow, circular, slightly convex, opaque and had a maximum diameter of about 2 mm after incubation at 28 °C for 48 h on TSA. No diffusible pigments were produced on any of the media tested. The novel isolate could grow at temperatures in the range 15–37 °C and at pH 6–9. Optimal growth was observed at 28 °C and at around pH 7.0–8.0. The isolate was catalase-positive and gave a positive oxidase reaction. Detailed physiological and biochemical properties are given in Table 1⇓ and in the species description. It is evident from Table 1⇓ that there were some phenotypic differences between YIM 56238^T and recognized species of the genus Micrococcus.

Peptidoglycan was purified and the cell-wall amino acids and peptides in cell-wall hydrolysates were analysed by two-dimensional ascending TLC on cellulose plates using the solvent system of Schleifer & Kandler (1972). The cell-wall sugars were analysed according to the procedures developed by Hasegawa et al. (1983). Phospholipids were extracted, examined by two-dimensional TLC and identified using previously described procedures (Minnikin et al., 1984). Menaquinones were isolated as described by Minnikin et al. (1984) and separated by HPLC (Kroppenstedt, 1982). Cellular fatty acid analysis was performed using the Sherlock Microbial Identification System (MIDI) according to the manufacturer's instructions. The results indicated that the cell-wall peptidoglycan of strain YIM 56238^T contained lysine, glutamic acid, alanine and glycine. The phospholipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and an unknown ninhydrin-negative phospholipid. The menaquinones were MK-8(H₂) (63.6 %) and MK-7(H₂) (21.1 %). The major fatty acids were iso-C_15 : 0 and anteiso-C_15 : 0, the profile being very similar to those described for recognized species of the genus Micrococcus (Table 2⇓).

Extraction of genomic DNA, PCR amplification and sequencing of the 16S rRNA gene were performed as described by Li et al. (2007). Multiple alignments with sequences from the most closely related actinobacteria and calculations of sequence similarity were carried out using clustal_x (Thompson et al., 1997). A phylogenetic tree (Fig. 1⇓) was constructed by means of the neighbour-joining method of Saitou & Nei (1987), from K_nuc values (Kimura, 1980), using mega, version 3.1 (Kumar et al., 2004). Alignment positions with insertions or deletions were excluded from the calculations. The topology of the phylogenetic tree was evaluated by using the bootstrap resampling method of Felsenstein (1985) with 1000 replicates.

The almost-complete 16S rRNA gene sequence (1438 bp) of strain YIM 56238^T was determined. Phylogenetic analysis revealed that the closest relatives of strain YIM 56238^T were M. luteus DSM 20030^T, M. antarcticus T2^T, M. lylae DSM 20315^T and M. flavus LW4^T, which showed 16S rRNA gene sequence similarities of 99.06, 98.05, 97.56 and 97.79 %, respectively. The results of the comparative 16S rRNA gene sequence analysis clearly demonstrated that strain YIM 56238^T is a member of the genus Micrococcus. The chemotaxonomic characteristics of strain YIM 56238^T, such as peptidoglycan type, menaquinones, major fatty acids, phospholipids and DNA G+C content, were consistent with its assignment to the genus Micrococcus.

DNA–DNA relatedness was studied using the fluorometric micro-well method (Ezaki et al., 1989; Christensen et al., 2000; He et al., 2005). Fluorescence intensities were measured using a fluorescence microplate reader (SpectraMax Gemini XPS; Molecular Devices). Hybridizations were performed using six replications for each sample. The DNA hybridization rate was calculated according to the method of He et al. (2005). The DNA–DNA hybridization values for strain YIM 56238^T with respect to M. luteus DSM 20030^T, M. antarcticus CGMCC 1.2372^T, M. lylae DSM 20315^T and M. flavus CGMCC 1.5361^T were 27.3, 50.3, 30.1 and 54.2 %, respectively. The G+C content of genomic DNA was determined by using the HPLC method (Mesbah et al., 1989) with Escherichia coli JM-109 as the reference strain, and a value of 72.9 mol% was derived.

The values for DNA–DNA relatedness among strains YIM 56238^T, M. luteus DSM 20030^T, M. antarcticus CGMCC 1.2372^T, M. lylae DSM 20315^T and M. flavus CGMCC 1.5361^T were all below 70 %, which indicates that the novel isolate represents a distinct genospecies (Wayne et al., 1987) of the genus Micrococcus. Thus, on the basis of the differential phenotypic properties mentioned above and the characteristics shown in Tables 1⇑ and 2⇑, strain YIM 56238^T represents a novel species of the genus Micrococcus, for which the name Micrococcus endophyticus sp. nov. is proposed.