Cocleimonas flava gen. nov., sp. nov., a gammaproteobacterium isolated from sand snail (Umbonium costatum)

Marine coastal environments serve as a harbour for a wide array of hydrobionts, including molluscs, most of which live by burrowing into the shallow sand or mud. Marine molluscs can be considered to be natural accumulators of specific microbial communities due to their filter-feeding habit and provide an untapped potential source of novel micro-organisms. During a survey of bacteria associated with marine invertebrates inhabiting shallow sediments of the Sea of Japan, we have isolated and characterized several interesting bacterial groups. Of these, two unique pink-red-pigmented bacteria isolated from the sand snail Umbonium costatum were recently proposed to represent a novel genus and species, Umboniibacter marinipuniceus, in the class Gammaproteobacteria (Romanenko et al., 2010). Strain KMM 3898^T was simultaneously isolated from the same snail specimen.

Strain KMM 3898^T was isolated from an internal tissue of a marine mollusc, the sand snail Umbonium costatum (Gastropoda, Trochidae), collected from the shallow sediments of the Sea of Japan, Russia as described previously (Romanenko et al., 2005, 2008). Strain KMM 3898^T was maintained under aerobic conditions on marine 2216 agar (MA) or in marine broth (MB) at 25–30 °C and stored at –80 °C in MB supplemented with 30 % (v/v) glycerol.

Growth under anaerobic conditions was determined following incubation for 1 week in MB after removing air by flushing with N₂/CO₂ (4 : 1, v/v). Motility was observed by the hanging-drop method as described by Gerhardt et al. (1994). Cell morphology and sulfur inclusions were observed by phase-contrast microscopy (AX70; Olympus). Gram-staining, oxidase and catalase activities, H₂S formation and hydrolysis of gelatin, casein, chitin, CM-cellulose, DNA, starch and Tweens 20, 40 and 80 were tested according to the standard methods described by Smibert & Krieg (1994). The oxidation/fermentation medium of Leifson (1963) was used to determine acid production from carbohydrates with 1 % (w/v) of each compound. The ability to grow in the presence of organic substrates as sole carbon and energy source was tested for 3 weeks on artificial seawater medium (containing l⁻¹: 30 g NaCl, 4.9 g MgCl₂, 3.9 g Na₂SO₄, 1.1 g CaCl₂, 0.66 g KCl, 0.2 g NaHCO₃, 0.096 g KBr, 0.026 g H₃BO₃, 0.024 g SrCl₂ and 0.003 g NaF) supplemented with (l⁻¹) 2 g NH₄Cl, 0.5 g yeast extract and 0.4 % carbon source. The ability to grow without additional organic growth factors was tested on the basal medium of Baumann & Baumann (1981) supplemented with 0.1 % glycerol, 0.1 % potassium acetate and 0.1 % potassium succinate. Growth at 4–40 °C, at pH 4.5–14.0, with 0–20 % NaCl and with antibiotics was studied as described previously (Romanenko et al., 2005, 2008). Additional biochemical tests were carried out using the API 20 NE, API 32 GN and API ZYM systems (bioMérieux) according to the manufacturer's instructions, except that the culture was suspended in artificial seawater.

For polar lipid and fatty acid analyses, strain KMM 3898^T was cultivated on MA at 28 °C for 3 days. Lipids were extracted using chloroform/methanol (Bligh & Dyer, 1959) and analysed as described by Vaskovsky & Terekhova (1979). Fatty acid methyl esters were obtained by alkaline methanolysis (15 % NaOH/methanol), extracted by hexane and analysed using GLC-MS (model 6890; Hewlett Packard) equipped with a HP 5 MS 5 % phenyl-methyl-siloxane capillary column (30 m×250 μm×0.25 μm) and connected to a mass spectrometer (model 5973; Hewlett Packard). Cells for respiratory lipoquinone analysis were obtained after culture in MB at 28 °C. Isoprenoid quinones were extracted using chloroform/methanol (2 : 1, v/v), purified by preparative TLC on silica gel 60 ADAMANT plates (Fluka) and analysed by HPLC (series 1100; Agilent) using a reversed-phase column (4.6×250 mm Hypersil ODS, 5 μm; Agilent) with methanol/2-propanol (65 : 35) as the mobile phase. Quinones were detected by monitoring absorbance at 270 nm. Pigments were extracted using hexane and then chloroform/methanol (2 : 1, v/v) and analysed with a CECIL 7250 spectrophotometer. The base composition of DNA isolated by the method of Marmur (1961) was determined as described by Marmur & Doty (1962) and Owen et al. (1969).

The 16S rRNA gene sequence (1512 nt) was determined for strain KMM 3898^T as described by Shida et al. (1997) and compared with sequences retrieved from public databases using fasta (Pearson & Lipman, 1988). After multiple sequence alignment using clustal x version 1.83 (Thompson et al., 1997), phylogenetic analysis was performed using mega4 (Tamura et al., 2007). Phylogenetic trees were reconstructed by the neighbour-joining and maximum-parsimony methods and distances were calculated according to the Kimura two-parameter model. The robustness of the trees was estimated by bootstrap analysis using 1000 replicates.

Strain KMM 3898^T was a Gram-stain-negative, aerobic, yellow pigmented, heterotrophic, sulfur-oxidizing, non-motile bacterium. The NaCl concentration and temperature ranges that supported optimal growth were narrow. Cells of strain KMM 3893^T were rods when on MA and in MB (Fig. 1a⇓). In addition, oval-shaped and swollen cells with black precipitations and sulfur inclusions were observed during static cultivation in MB supplemented with 20 mM thiosulfate or 1 % (w/v) elemental sulfur (Fig. 1b, c⇓). Chemotaxonomic analysis revealed Q-8 as the major isoprenoid quinone and phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and an unknown phospholipid as the polar lipids (Supplementary Fig. S1, available in IJSEM Online). The predominant fatty acids were C_18 : 1ω7c (25.8 %), C_16 : 1ω7c (25.5 %) and C_16 : 0 (16 %) and the minor fatty acids were C_18 : 1ω9c (8.4 %), C_14 : 0 (5.2 %), C_18 : 0 (5.1 %), iso-C_17 : 1 (2.4 %), iso-C_15 : 1 (1.8 %) and C_16 : 1ω9c (1.7 %). The DNA G+C content was 43.4 mol%. Physiological, biochemical and metabolic characteristics of strain KMM 3898^T are listed in the genus and species descriptions and in Table 1⇓.

Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain KMM 3898^T formed a deeply branching lineage in a cluster containing members of the genus Leucothrix in the family Leucotrichaceae (Brock, 1974) of the class Gammaproteobacteria (Fig. 2⇓ and Supplementary Fig. S2). Strain KMM 3898^T shared 89.2 % 16S rRNA gene sequence similarity with Leucothrix mucor DSM 2157^T and 96.9 % 16S rRNA gene sequence similarity with an uncultured clone from crab (Shinkaia crosnieri) collected from hydrothermal vents of Okinawa (X. Ding & W. J. Yang, unpublished data). Members of the genus Thiothrix shared 86.7–88.5 % 16S rRNA gene sequence similarity with strain KMM 3898^T.

It is conceivable that strain KMM 3898^T was intimately associated with its host as it displayed intrinsic morphological, physiological, cultural and metabolic properties and a phylogenetic position distinctly different from its closest neighbours. The species Leucothrix mucor represents an obligatory aerobic, heterotrophic, non-pigmented micro-organism that is widespread in marine environments and fresh water as a seaweed epiphyte. Morphologically, L. mucor is characterized by the presence of filaments, gonidia with gliding ability, rosettes, knots and holdfasts and it can be directly observed under the microscope because the filaments form attachments to the surface of algal fronds (Brock, 1966; Kelly & Brock, 1969; Bland & Brock, 2005). The genus Thiothrix is physiologically distinguishable from the genus Leucothrix and comprises colourless, filamentous, sulfur-oxidizing bacteria capable of depositing sulfur intracellularly. Their habitats include sulfide-containing waters and activated-sludge wastewater treatment plants (Larkin, 1989; Howarth et al., 1999; Aruga et al., 2002). Members of the genus Thiothrix share a characteristic deletion in helix 18 of the 16S rRNA gene (Howarth et al., 1999), which was not found in strain KMM 3898^T (Table 1⇑).

The phenotypic characteristics of strain KMM 3898^T and the low 16S rRNA gene sequence similarity values (86.7–89.2 %) suggest that strain KMM 3898^T should be classified in a novel genus and species, for which the name Cocleimonas flava gen. nov., sp. nov. is proposed.