Planococcus stackebrandtii sp. nov., isolated from a cold desert of the Himalayas, India

The taxonomic position of a bacterium isolated from a cold desert of the Himalayas, India, was analysed by using a polyphasic approach. The isolated strain, designated K22-03^T, had phenotypic characteristics that matched those of the genus Planococcus and it represents a novel species. The almost-complete 16S rRNA gene sequence (1464 bases) of the novel strain was compared with those of previously studied Planococcus type strains and confirmed that the strain belongs to the genus Planococcus. 16S rRNA gene sequence analysis indicated that strain K22-03^T differs from all other species of Planococcus by at least 2·5 %. DNADNA hybridization showed that it had low genomic relatedness with Planomicrobium mcmeekinii (MTCC 3704^T, 23 %), Planococcus psychrophilus (MTCC 3812^T, 61 %), Planococcus antarcticus (MTCC 3854^T, 45 %) and Planomicrobium okeanokoites (MTCC 3703^T, 51 %), the four species with which it was most closely related based on 16S rRNA gene sequence analysis (9797·5 % similarity). Therefore, strain K22-03^T should be recognized as a novel species, for which the name Planococcus stackebrandtii sp. nov. is proposed. The type strain is K22-03^T (=MTCC 6226^T=DSM 16419^T=JCM 12481^T).

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Published online ahead of print on 13 August 2004 as DOI 10.1099/ijs.0.63290-0.

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Planococcus stackebrandtii strain K22-03^T is AY437845.

The genus Planococcus was first established over 100 years ago by Migula (1894) and was later emended by Nakagawa et al. (1996). At present the genus comprises eight recognized species, Planococcus citreus (Migula, 1894), Planococcus kocurii (Hao & Komagata, 1985), Planococcus alkanoclasticus (Engelhardt et al., 2001), Planococcus antarcticus and Planococcus psychrophilus (Reddy et al., 2002), Planococcus maritimus (Yoon et al., 2003), Planococcus rifietoensis (Romano et al., 2003) and Planococcus maitriensis (Alam et al., 2003).

A bacterial strain, designated K22-03^T, was isolated from a cold desert of the Himalayas, northern India, using a dilution plating technique on trypticase soy agar (TSA; Himedia, India) and maintained as glycerol stocks at 70 °C. The reference strains of Planomicrobium mcmeekinii (MTCC 3704^T), Planomicrobium okeanokoites (MTCC 3703^T), Planococcus psychrophilus (MTCC 3812^T) and Planococcus antarcticus (MTCC 3854^T) were obtained from the Microbial Type Culture Collection (MTCC), Chandigarh, India.

Strain K22-03^T and the three closely related organisms given above were grown on TSA and tested for morphological, physiological and chemotaxonomic characteristics. These four strains were also checked for various biochemical properties, such as reaction to oxidase and catalase, aerobic or anaerobic growth, and hydrolysis of Tween 20, starch, casein and gelatin. Whole-cell sugars and amino acids were determined by TLC as described by Staneck & Roberts (1974). Phospholipids and menaquinones were extracted and analysed using the methods described by Minnikin & Goodfellow (1976) and Minnikin et al. (1978). Fatty acid methyl esters were extracted and analysed as described by Pandey et al. (2002).

The chromosomal DNA of strain K22-03^T was isolated according to the procedure described by Rainey et al. (1996). The 16S rRNA gene was amplified with primers 827f (5'-AGAGTTTGATCCTGGCTCAG-3') and 1500r (5'-AGAAAGGAGGTGATCCAGGC-3'). The amplified DNA fragment was separated on a 1 % agarose gel, eluted from the gel and purified using the Qiaquick gel extraction kit (Qiagen). The purified PCR product was sequenced with four forward and three reverse primers: 827f (5'-AGAGTTTGATCCTGGCTCAG-3'), 357f (5'-CTCCTACGGGAGGCAGCAG-3'), 704f (5'-TAGCGGTGAAATGCGTAGA-3'), 1114f (5'-GCAACGAGCGCAACC-3'), 685r (5'-TCTACGCATTTCACCGCTAC-3'), 1110r (5'-GGGTTGCGCTCGTTG-3') and 1500r (5'-GAAAGGAGGTGATCCAGGC-3') (Escherichia coli numbering system). The rRNA gene sequence was determined by the dideoxy chain-termination method using the BigDye terminator kit and an ABI 310 Genetic Analyser (Applied Biosystems). DNADNA hybridization was performed by the membrane filter method (Tourova & Antonov, 1987) as described by Shivaji et al. (1992) and Reddy et al. (2000).

The 16S rRNA gene sequence of strain K22-03^T generated here (1464 bases) was aligned with those of other Planococcus and Planomicrobium species retrieved from the GenBank database. A sequence similarity search was performed on these using BLASTN (Altschul et al., 1997). Sequences of closely related taxa were retrieved and aligned using the CLUSTAL_X program (Thompson et al., 1997); the alignment was corrected manually. For the neighbour-joining analysis (Saitou & Nei, 1987), distances between sequences were calculated using Kimura's two-parameter model (Kimura, 1980). Bootstrap analysis was performed to assess the confidence limits of the branching (Felsenstein, 1985).

Colonies grown on TSA were orange-coloured and about 12 mm in diameter, circular, smooth and convex. Biochemical and chemotaxonomic characteristics confirmed strain K22-03^T to be a member of the genus Planococcus. Test reactions are given in Table 1 together with those of three closely related taxa. Strain K22-03^T differs from these three closely related species in acid production from different sugars. The diagnostic cell-wall amino acid is L-lysine and the diagnostic cell-wall sugars are D-glucose and D-ribose. The fatty acid profile of strain K22-03^T is similar to those of the three closely related species except for the presence of C_{17 : 0} and C_{16 : 1}ω11c in K22-03^T (Table 2). The major menaquinones are MK-7 and MK-8 and the strain has a type II phospholipid pattern. The DNA G+C content of strain K22-03^T is 40 mol%. To determine the phylogenetic position of strain K22-03^T, the 16S rRNA gene sequence was compared with those of type strains of species of the genera Planococcus and Planomicrobium retrieved from the GenBank database. Sequence similarity of K22-03^T with the genera Planococcus and Planomicrobium ranges from 95·4 to 97·6 %. A rooted phylogenetic tree based on the neighbour-joining method is shown in Fig. 1. Strain K22-03^T formed a separate line among the genus Planococcus. Strain K22-03^T is moderately related to Planococcus psychrotoleratus (97·6 %), Planomicrobium okeanokoites (97·5 %) Planococcus psychrophilus (97·4 %) and Planococcus antarcticus (97·0 %), and distantly related to Planomicrobium mcmeekinii (96·8 %), Planococcus maritimus (96·6 %), Planococcus alkanoclasticus (96·5 %), Planococcus maitriensis (96·3 %), Planococcus rifietoensis (96·2 %), Planococcus citreus (95·7 %) and Planococcus kocurii (95·4 %). Genomic relatedness, as shown by DNADNA hybridization, of strain K22-03^T with Planococcus mcmeekinii is 23 %, with Planococcus psychrophilus is 61·0 %, with Planococcus antarcticus is 45 % and with Planomicrobium okeanokoites is 51·0 %. At the level of 34 % dissimilarity in 16S rRNA gene sequences, members of other species of the genus can be excluded from DNADNA hybridization studies (Stackebrandt & Goebel, 1994). This conclusion is confirmed by DNA dissimilarity values distinctly lower than 70 % (Wayne et al., 1987) obtained from two members each of the genera Planococcus and Planomicrobium. On the basis of morphology, growth characteristics, biochemical and chemotaxonomic characteristics, molecular systematic studies including 16S rRNA gene sequence analysis, DNA G+C content and DNADNA hybridization analysis, strain K22-03^T is considered to represent a novel species of the genus Planococcus, for which the name Planococcus stackebrandtii sp. nov. is proposed.

Table 1. Comparison of the phenotypic characteristics of strain K22-03T (1) with those of the type strains of Planomicrobium okeanokoites (2), Planomicrobium mcmeekinii (3) and Planococcus psychrophilus (4) None of the four micro-organisms produces spores, hydrolyses starch or is acid-fast. They all produce acid from fructose and galactose, but not from rhamnose or salicin. They all produce an orange pigment. +, Positive; , negative; W, weakly positive.

Table 2. Comparison of the fatty acid profile (%) of strain K22-03T (1) with those of the type strains of Planomicrobium okeanokoites (2), Planomicrobium mcmeekinii (3) and Planococcus psychrophilus (4)

(26K): Fig. 1. Neighbour-joining tree based on 16S rRNA gene (1464 bases) sequences showing the phylogenetic relationship of Planococcus stackebrandtii strain K22-03T with other related species of the genera Planococcus and Planomicrobium. Bootstrap values (expressed as percentage of 1000 replications) greater than 50 % are given at the nodes. Bar, 0·5 % sequence variation.

Description of Planococcus stackebrandtii sp. nov.
Planococcus stackebrandtii (sta.cke.brand.ti'i. N.L. gen. n. stackebrandtii of Stackebrandt, to honour Erko Stackebrandt, a German microbiologist, for his valuable contributions to microbial taxonomy and molecular systematics).

Cells are Gram-positive, aerobic, non-spore-forming, motile and coccoid, occurring in pairs and in groups. Colonies on TSA are orange, opaque, smooth, convex, circular, entire and 12 mm in diameter. Growth occurs at 1530 °C (optimum 25 °C) and at pH 5·611·0. Can tolerate up to 7 % NaCl. Positive for citrate utilization, Tween 20 hydrolysis, gelatin hydrolysis, ornithine decarboxylase and catalase, but negative for oxidase, casein hydrolysis, starch hydrolysis, urease production, methyl red and VogesProskauer reaction, hydrogen sulfide production, nitrate reduction, arginine dihydrolase and lysine decarboxylase. No gas is produced from glucose medium. Acid is produced from galactose, fructose, lactose, raffinose and sucrose, but not from glucose, arabinose, mannitol, xylose, myo-inositol, rhamnose or salicin. As sole nitrogen source, it is negative for histidine, phenylalanine, valine, cysteine, hydroxy proline, arginine and serine. Cell-wall peptidoglycan contains L-lysine; cell-wall sugars are D-glucose and D-ribose. MK-7 and MK-8 are the major menaquinones. DNA G+C content is 40 mol%. Predominant fatty acids are anteiso-C_{15 : 0} and C_{16 : 1}ω7c alcohol, with minor amounts of C_{15 : 0}, C_{17 : 0} and C_{16 : 1}ω11c. Phospholipid is of type II, which has the predominant polar lipids phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine.

The type strain, K22-03^T (=MTCC 6226^T=DSM 16419^T=JCM 12481^T), was isolated from soil, 0·45 m below an ice glacier, at 4200 m elevation, Kibber village, Spiti valley, Himachal Pradesh, India.