Gillisia mitskevichiae sp. nov., a novel bacterium of the family Flavobacteriaceae, isolated from sea water

Footnotes

During June 2000, we isolated an unknown bacterium from a sea-water sample collected in Amursky Bay, Gulf of Peter the Great, Sea of Japan. A polyphasic taxonomic study of strain KMM 6034^T, cultured on marine agar 2216 at 25 °C for 48 h, indicates that the isolate represents a novel species of the genus Gillisia, for which the name Gillisia mitskevichiae sp. nov. is proposed.

Genomic DNA extraction, PCR and sequencing of the 16S rRNA gene followed previous procedures (Kim et al., 1998). The sequence data obtained were aligned with those of members of the family Flavobacteriaceae using PHYDIT version 3.2 (). Phylogenetic trees were inferred by using suitable programs of the PHYLIP package (Felsenstein, 1993). Phylogenetic distances were calculated from the model of Kimura (1980) and trees were constructed on the basis of the neighbour-joining (Saitou & Nei, 1987) and maximum-likelihood (Felsenstein, 1993) algorithms. Bootstrap analysis was performed with 1000 resampled datasets by using SEQBOOT and CONSENSE programs of the PHYLIP package.

Phylogenetic analysis of the almost-complete 16S rRNA gene sequence (1430 nucleotide positions) revealed that strain KMM 6034^T was a member of the family Flavobacteriaceae and formed a distinct lineage within the genus Gillisia (Fig. 1). The level of 16S rRNA gene sequence similarity between strains KMM 6034^T and G. limnaea LMG 21470^T was 96·7 %. 16S rRNA gene sequence similarity values of the strain studied with the next closest relatives Mesonia algae KMM 3909^T, [Cytophaga] latercula ATCC 23177^T, Salegentibacter salegens DSM 5424^T, Psychroflexus torquis ACAM 623^T and Kordia algicida OT-1^T were 91·4, 92·2, 93·1, 91·4 and 90·5 %, respectively.

Analysis of fatty acid methyl esters was carried out according to the standard protocol of the Microbial Identification System (Microbial ID). The cellular fatty acids comprising more than 1 % for strain KMM 6034^T were straight- and branched-chain unsaturated and saturated fatty acids, namely i-14 : 0 (1·2 %), i-15 : 1 (11·8 %), a-15 : 1 (2·4 %), i-15 : 0 (7·5 %), a-15 : 0 (5·1 %), 15 : 0 (4·4 %), 15 : 1ω6c (1·9 %), i-16 : 1 (6 %), i-16 : 0 (9·3 %), i-15 : 0 3-OH (1·1 %), 15 : 0 2-OH (2·4 %), i-17 : 1ω9c (4 %), a-17 : 1ω9c (2·1 %), 17 : 1ω6c (4·7 %), i-16 : 0 3-OH (7·2 %), 16 : 0 3-OH (1·2 %), i-17 : 0 3-OH (6·7 %), 17 : 0 2-OH (3·8 %), and summed feature 3 (11·1 %), comprising i-15 : 0 2-OH and/or 16 : 1ω7c, which corresponds to the fatty acid composition of G. limnaea (Van Trappen et al., 2004). Both the strain studied and G. limnaea strains contained similar amounts of branched fatty acids (>65 % of total).

Phenotypic analysis was performed by using previously described methods (Nedashkovskaya et al., 2003a, b). Gliding motility was determined as described by Bowman (2000).

The physiological, morphological and biochemical characteristics of strain KMM 6034^T are listed in the species description and Table 1. The similarities in phenotypic characteristics support the inclusion of the strain studied in the genus Gillisia. However, strain KMM 6034^T clearly differed from strains of G. limnaea by the NaCl requirement for growth, growth at 12 % NaCl, maximum growth temperature (31 °C), acid production from carbohydrates, utilization of mannitol and the ability to degrade casein, urea, DNA and Tweens 20 and 40 (Table 1).

Table 1. Phenotypic characteristics that distinguish Gillisia mitskevichiae sp. nov. from G. limnaea Data from Van Trappen et al. (2004) and this study. Strains of both species are Gram-negative and are positive for respiratory type of metabolism, oxidase, catalase and alkaline phosphatase activities, growth at 15 % NaCl and at 530 °C, hydrolysis of gelatin and Tween 80, utilization of glucose and sucrose and susceptibility to ampicillin, carbenicillin, lincomycin and oleandomycin. Strains of both species are negative for gliding motility, requirement for organic growth factors, flexirubin pigments, nitrate reduction, H2S, indole and acetoin production, degradation of agar, starch, cellulose (CM-cellulose, filter paper) and chitin, acid formation from arabinose, cellobiose, fucose, galactose, lactose, maltose, melibiose, raffinose, rhamnose, sorbose, xylose, inositol, mannitol, sorbitol, glycerol, adonitol, dulcitol and citrate, utilization of arabinose, lactose, mannose, inositol, sorbitol, malonate and citrate and susceptibility to gentamicin, kanamycin, neomycin and polymyxin B.

Description of Gillisia mitskevichiae sp. nov.
Gillisia mitskevichiae (mit.ske'vi.chi.ae. N.L. gen. n. mitskevichiae of Mitskevich, in honour of Irina N. Mitskevich, Russian marine microbiologist, for her contributions to the development of marine microbiology).

Cells are Gram-negative, strictly aerobic with respiratory metabolism, chemo-organotrophic, asporogenic rods, non-motile by gliding, 0·50·7 µm wide and 34 µm long. Oxidase-, catalase-, urease- and alkaline phosphatase-positive. Colonies are circular, convex, shiny with entire edges, 13 mm in diameter on marine agar 2216. Produces yellow non-diffusible pigments. No growth is observed without Na⁺; grows at 112 % NaCl. Flexirubin pigments are absent. Growth occurs at 431 °C. The pH range of growth is 5·510·0, with optimum growth occurring between pH 7·6 and 8·3. Casein, gelatin, DNA, urea and Tweens 40 and 80 are degraded; does not hydrolyse agar, starch, cellulose (CM-cellulose and filter paper), chitin or Tween 20. Forms acid from glucose, sucrose and N-acetylglucosamine, but not from arabinose, cellobiose, fucose, galactose, lactose, maltose, melibiose, raffinose, rhamnose, xylose, adonitol, dulcitol, glycerol, inositol or mannitol. Does not utilize arabinose, lactose, mannose, inositol, mannitol, sorbitol or citrate as sole sources of carbon and energy. Nitrate reduction is negative. H₂S, indole and acetoin (VogesProskauer reaction) are not produced. Susceptible to ampicillin, carbenicillin, oleandomycin, lincomycin, streptomycin and tetracycline; resistant to kanamycin, benzylpenicillin, neomycin, gentamicin and polymyxin B. The predominant cellular fatty acids are straight-chain unsaturated, branched-chain unsaturated and saturated, namely i-15 : 0 (7·5 %), a-15 : 0 (5·1 %), i-15 : 1 (11·8 %), i-16 : 1 (6 %), i-16 : 0 (9·3 %), i-16 : 0 3-OH (7·2 %), i-17 : 0 3-OH (6·7 %) and summed feature 3 (11·1 %), consisting of i-15 : 0 2-OH and/or 16 : 1ω7c (64·7 % of total). The G+C content of the DNA is 36·4 mol%.

The type strain is KMM 6034^T (=KCTC 12261^T=NBRC 100590^T=LMG 22575^T), isolated from sea-water collected in Amursky Bay, Gulf of Peter the Great, Sea of Japan.