Planococcus donghaensis sp. nov., a starch-degrading bacterium isolated from the East Sea, South Korea

The genus Planococcus was first established by Migula (1894) to accommodate Gram-positive, motile cocci of the family Micrococcaceae. However, the genus name Planococcus was validly published by Kocur et al. (1970) for the first species, Planococcus citreus. Yoon et al. (2001) created the novel genus Planomicrobium on the basis of menaquinone profiles and phylogenetic data, as well as cellular morphology. Consequently, Planococcus okeanokoites (Nakagawa et al., 1996), Planococcus mcmeekinii (Junge et al., 1998), Planococcus alkanoclasticus (Engelhardt et al., 2001) and Planococcus psychrophilus (Reddy et al., 2002) were transferred to the genus Planomicrobium (Yoon et al., 2001; Dai et al., 2005). At present, the genus Planococcus comprises eight recognized species (Euzéby, 1997). In this study, the taxonomic status of a novel bacterial strain, JH1^T, was investigated by using a combination of phenotypic properties, phylogeny based on 16S rRNA gene sequence and genomic relatedness. The polyphasic evidence presented in this paper indicates that strain JH1^T represents a novel species of the genus Planococcus.

During screening of starch-degrading bacteria, strain JH1^T was isolated from marine sediment of the East Sea, South Korea, and selected to determine its exact taxonomic position. A bottom sediment sample was retrieved at a depth of 200–500 m, placed in a sterile tube and diluted serially with filtered deep-sea water. An aliquot of each dilution was spread on 100-fold-diluted Marine agar (MA; Difco) containing 1 % starch and incubated at 17 °C for 3–10 days. Colonies showing a halo on the medium were selected as starch-degrading bacteria. Single colonies with the ability to degrade starch were purified by streaking onto new plates and subjected to additional incubation for 3 days at 25 °C. Cultures were stored at −80 °C in Marine broth (MB; Difco) supplemented with 25 % (v/v) glycerol. Strain JH1^T was grown routinely on MA or trypticase soy agar (TSA) or broth (TSB; Difco). Planococcus kocurii DSM 20747^T, Planococcus antarcticus DSM 14505^T, Planomicrobium koreense KCTC 3684^T and Planomicrobium okeanokoites KCTC 3672^T were used as reference strains for some analyses. For polar lipid analysis, menaquinone and cell-wall analyses and DNA extraction, strain JH1^T was cultured in MB at 25 °C. For fatty acid analysis, cells were grown on MA plates at 25 °C.

Morphology and cell size were determined by phase-contrast microscopy (80i; Nikon). Gram staining was performed with a BD Gram stain kit, according to the instructions of the manufacturer, and with a non-staining method, as described by Buck (1982). Catalase and oxidase activities were determined by using a bioMérieux Orientation test kit according to the manufacturer's instructions after cultivation for 2 days. Acid production from carbohydrates, single carbon-source assimilation and additional physiological characteristics were determined by using the API 20E, API 20NE and API ID32 galleries according to the instructions of the manufacturer (bioMérieux). Tolerance to NaCl was measured on nutrient agar (Difco) containing 0–14 % (w/v) NaCl. Plates were incubated at 25 °C for 10 days. The temperature range for growth was determined on TSA incubated for 6 days at 0, 4, 10, 15, 20, 25, 30, 35, 37, 40, 45 and 50 °C.

Cellular fatty acid profiles were determined for strains grown on MA for 2 days. The cellular fatty acids were saponified, methylated and extracted according to the protocol of the Sherlock Microbial Identification system (MIDI). The fatty acids were then analysed by gas chromatography (6890; Hewlett Packard) using the Microbial Identification software package (Sasser, 1990). Isoprenoid quinones were extracted with chloroform/methanol (2 : 1), evaporated under vacuum conditions and re-extracted in n-hexane/water (1 : 1). Then, the crude menaquinone in n-hexane was purified by using Sep-Pak Vac Cartridges Silica (Waters) and subsequently analysed by HPLC as described by Hiraishi et al. (1996). Peptidoglycan structure was determined according to Schleifer & Kandler (1972). Polar lipids were extracted by a biphasic mixture of petroleum ether and methanolic saline and examined by two-dimensional TLC (Minnikin et al., 1984).

For phylogenetic analysis of strain JH1^T, DNA was extracted by using a commercial genomic DNA extraction kit (Solgent). The 16S rRNA gene was amplified from the chromosomal DNA by using the universal bacterial primer set 27F and 1492R, and the purified PCR products were sequenced by Solgent, South Korea (Park et al., 2006). The full sequences of the 16S rRNA gene were compiled by using SeqMan software (dnastar). The 16S rRNA gene sequences of related taxa were obtained from GenBank. Multiple alignments were performed by using the clustal_x program (Thompson et al., 1997). Gaps were edited in the BioEdit program (Hall, 1999). Evolutionary distances were calculated by using the Kimura two-parameter model (Kimura, 1980). A phylogenetic tree was constructed by using the neighbour-joining method (Saitou & Nei, 1987) in the mega3 program (Kumar et al., 2004), with bootstrap values based on 1000 replications (Felsenstein, 1985).

For measurement of the G+C content of the chromosomal DNA, the extracted genomic DNA was degraded enzymically into nucleosides and the DNA G+C content was determined as described by Mesbah et al. (1989) by using reverse-phase HPLC. DNA–DNA hybridization experiments were performed between strain JH1^T and Planococcus antarcticus DSM 14505^T, Planococcus kocurii DSM 20747^T, Planomicrobium koreense KCTC 3684^T and Planomicrobium okeanokoites KCTC 3672^T, using photobiotin-labelled DNA probes and microdilution wells as described by Ezaki et al. (1989). Hybridization was performed with five replications for each sample. The highest and lowest values obtained for each sample were excluded and the means of the remaining three values were quoted as the DNA relatedness value.

Strain JH1^T appeared as Gram-positive, aerobic and motile cocci in all growth phases, as shown by all members of the genus Planococcus. Morphological, physiological, biochemical and chemotaxonomic characteristics are given in Table 1⇓. Phenotypically, strain JH1^T could hydrolyse starch and could not hydrolyse gelatin, characteristics that are unique in the genera Planococcus and Planomicrobium. In addition, strain JH1^T differed from related species by having positive reactions for oxidase and hydrolysis of aesculin. The amino acids in the cell wall were lysine, glutamic acid and alanine. Accordingly, the peptidoglycan type of strain JH1^T was A4α, based on l-Lys–d-Glu, as described by Schleifer & Kandler (1972). This result shows that strain JH1^T had an identical cell-wall peptidoglycan type (l-Lys–d-Glu) to Planococcus species, whereas Planomicrobium species, except for Planomicrobium koreense, have cell-wall peptidoglycan based on l-Lys–d-Asp (Yoon et al., 2001). The major menaquinones in strain JH1^T were found to be similar amounts of MK-7 and MK-8, as in most other members of the genus Planococcus (Hao & Komagata, 1985).

Strain JH1^T contained anteiso-C_15 : 0 (43.8 %) as the major fatty acid and significant amounts of anteiso-C_17 : 0 (15.5 %) and C_16 : 0 (6.3 %) (Table 2⇓). The cellular phospholipids found in strain JH1^T were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The genomic DNA G+C content of strain JH1^T was 47 mol%.

To determine the phylogenetic position of strain JH1^T, the 16S rRNA gene sequence (1452 nt) was compared with those of type strains of species of the genera Planococcus and Planomicrobium retrieved from GenBank. Sequence similarity of strain JH1^T to species in the genera Planococcus and Planomicrobium ranged from 96.0 to 98.2 %. Phylogenetic analyses based on the neighbour-joining method (Fig. 1⇓) and maximum-parsimony method (data not shown) showed that strain JH1^T falls within the evolutionary radiation of the genus Planococcus, with moderate bootstrap support (Fig. 1⇓). In addition, at positions 183 and 190 (Escherichia coli numbering), JH1^T had the signature nucleotides of the genus Planococcus (T and A), rather than the C and G nucleotides characteristic of the genus Planomicrobium (Dai et al., 2005). Strain JH1^T falls within the cluster comprising Planococcus antarcticus and Planococcus kocurii. JH1^T exhibited highest levels of sequence similarity to the type strains of these species (98.2 and 97.2 %, respectively).

DNA–DNA relatedness values of strain JH1^T are 9 % with Planococcus antarcticus DSM 14505^T, 22 % with Planococcus kocurii DSM 20747^T, 6 % with Planomicrobium okeanokoites KCTC 3672^T and 24 % with Planomicrobium koreense KCTC 3684^T, suggesting clearly that it is a novel species. When 16S rRNA gene sequence similarity is ≥97 %, members of other species of the genus can be excluded from DNA–DNA hybridization studies (Stackebrandt & Goebel, 1994). The DNA–DNA relatedness between the two strains is <70 %, indicating a level of DNA relatedness appropriate for defining a species in current bacterial systematics (Wayne et al., 1987). Levels of DNA–DNA relatedness provide decisive evidence that strain JH1^T is genetically different from the phylogenetically related species Planococcus antarcticus, Planococcus kocurii, Planomicrobium koreense and Planomicrobium okeanokoites (Wayne et al., 1987). Therefore, on the basis of morphology, growth characteristics, biochemical and chemotaxonomic characteristics, molecular systematic studies including 16S rRNA gene sequence analysis, and DNA–DNA hybridization analysis, strain JH1^T should be placed in the genus Planococcus as the type strain of a novel species, for which the name Planococcus donghaensis sp. nov. is proposed.