Marinobacter guineae sp. nov., a novel moderately halophilic bacterium from an Antarctic environment

Two Gram-negative, cold-adapted, moderately halophilic, aerobic bacteria, designated strains M3B^T and M3T, were isolated from marine sediment collected from the South Shetland Islands, Antarctica. The organisms were rod-shaped, catalase- and oxidase-positive, and motile by means of polar flagella. These two psychrotolerant strains required Na⁺ and grew at NaCl concentrations of 1–15 % and temperatures between 4 and 42 °C. 16S rRNA gene sequence analysis placed strains M3B^T and M3T within the genus Marinobacter. DNA–DNA hybridization experiments between the Antarctic isolate M3B^T and type strains of phylogenetically related species, namely Marinobacter lipolyticus, Marinobacter flavimaris, Marinobacter sediminum, Marinobacter algicola, Marinobacter maritimus and Marinobacter koreensis, revealed levels of relatedness lower than 32 %. Strain M3T showed 99 % DNA relatedness to strain M3B^T. The DNA G+C contents of M3B^T and M3T were 57.1 and 57.4 mol%, respectively, and their major isoprenoid quinone was ubiquinone-9. Several phenotypic characteristics, together with data on cellular fatty acid composition, served to differentiate strains M3B^T and M3T from strains of related Marinobacter species. On the basis of the polyphasic taxonomic evidence presented in this study, it can be concluded that strains M3B^T and M3T belong to the same genospecies and represent a novel species of the genus Marinobacter, for which the name Marinobacter guineae sp. nov. is proposed. The type strain is M3B^T (=LMG 24048^T=CECT 7243^T).

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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain M3B^T is AM503093.

Fatty acid composition data and DNA–DNA relatedness data for M3B^T and M3T and strains of related species are available with the online version of this paper.

The genus Marinobacter, first established by Gauthier et al. (1992), belongs to the Gammaproteobacteria and comprises moderate halophilic bacteria from marine-related environments. At the time of writing, this genus included 16 species. During a taxonomic investigation of cold-adapted bacteria from samples collected in the Antarctic area of the South Shetland Islands, two strains, M3B^T and M3T, were isolated that were able to grow at 15 °C on marine agar (MA; Difco), but not on tryptic soy agar (TSA; Difco). In this study, the taxonomic status of these two strains was investigated by using a combination of phenotypic characterization, 16S rRNA gene sequencing, DNA G+C content determination, DNA–DNA hybridization, and cellular fatty acid and isoprenoid quinone analysis.

Strains M3B^T and M3T were isolated from a marine sediment collected from Deception Island (South Shetland Islands, Antarctica). Sample aliquots were removed with a platinum loop and diluted in a saline solution (pH 7) containing the following salts (g l^–1): 0.56, NaCl; 0.027, KCl; 0.03, CaCl₂; 0.01, NaHCO₃. MA and TSA plates (both Difco) were inoculated with loopfuls of several sample dilutions by using the streak-plate method to obtain isolated colonies. Plates were incubated for 7 days at 15 °C. Growth was observed only on MA. Isolates were maintained aerobically on MA slopes at 4 °C and also at –80 °C on cryo-beads (AES Laboratoire).

Morphology, cell size and shape of cells grown on MA at 15 °C were determined by means of negative staining and transmission electron microscopy. Motility was determined by phase-contrast microscopy. The temperature range for growth was determined on MA incubated for 14 days at temperatures from 4 to 45 °C. NaCl tolerance was measured on nutrient agar (Cultimed) containing 0–20 % (w/v) NaCl; plates were incubated at 30 °C for 14 days. The pH range for growth was established on marine broth (Difco) at pH 4.0–10.0 (increments of 0.5 pH units) at 30 °C for 14 days. Anaerobic growth was evaluated on MA and on MA in the presence of KNO₃ (0.1 %) after incubation in an anaerobic chamber at 30 °C for 14 days. Oxidase, catalase and urease activities, nitrate reduction, and hydrolysis of casein, lecithin, gelatin, DNA, starch and Tween 80 were determined according to Barrow & Feltham (1993). Acid production from carbohydrates, enzyme production and additional characteristics were determined by using the API 50CH, API ZYM and API 20NE strips (bioMérieux). API strips were inoculated with colonies suspended in a solution of 3 % NaCl (w/v). For API 50CH, media were supplemented with a solution of sea salts (Sigma) and 0.85 % NaCl (w/v).

Results of the morphological and phenotypic characterization are given in the species description and in Table 1. These phenotypic studies showed that the isolates displayed characteristics that are consistent with those of members of the genus Marinobacter.

Table 1. Differential characteristics between the novel species and its closest phylogenetic neighbours Strains: 1, M3BT; 2, M3T; 3, M. lipolyticus DSM 15157T (Martín et al., 2003); 4, M. flavimaris DSM 16070T (Yoon et al., 2004); 5, M. sediminum KMM 3657T (Romanenko et al., 2005); 6, M. algicola DG893T (Green et al., 2006); 7, M. maritimus CK 47T (Shivaji et al., 2005); 8, M. koreensis DSM 17924T (Kim et al., 2006). All strains are positive for motility, catalase and oxidase, leucine arylamidase, naphthol-AS-BI-phosphohydrolase and N-acetyl-β-glucosaminidase. All strains are negative for α-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, α-mannosidase and α-fucosidase. –, Negative; +, positive; W, weakly positive; ND, no data available.

Cellular fatty acid and isoprenoid quinone contents were determined for cell mass grown on MA for 4 days at 15 °C as described previously (Bozal et al., 2002). The mean fatty acid compositions of strains M3B^T and M3T, together with those of type strains of the closest phylogenetic neighbours, are shown in Supplementary Table S1 (available in IJSEM Online). The most abundant fatty acids were summed feature 3 (C_{16 : 1}ω7c and/or iso-C_{15 : 0} 2-OH), C_{16 : 0}, C_{18 : 1}ω9c, C_{12 : 0} 3-OH and C_{16 : 1}ω9c. The isolates had cellular fatty acid profiles that were similar to those of other phylogenetically closely related Marinobacter type strains, although there were some differences in fatty acid composition: strains M3B^T and M3T had increased levels of summed feature 3 and reduced levels of C_{16 : 0}. The major isoprenoid quinone was ubiquinone-9, which is consistent with that found in other Marinobacter species, with the exception of Marinobacter lutaoensis, which contains ubiquinone-8 (Shieh et al., 2003).

Total DNA for complete 16S rRNA gene sequence analysis was prepared according to the protocol of Niemann et al. (1997). Phylogenetic analyses were carried out by using the neighbour-joining method as described previously by Bozal et al. (2002) with the software package BIONUMERICS (Applied Maths). For DNA–DNA hybridizations and determination of G+C content, total DNA was prepared according to a modification of the procedure of Wilson (1987). The G+C content was determined by using the HPLC technique as described by Mesbah et al. (1989). DNA–DNA hybridizations were performed at 47 °C according to a modification (Goris et al., 1998; Cleenwerck et al., 2002) of the method described by Ezaki et al. (1989).

16S rRNA phylogenetic studies confirmed that the Antarctic strains M3B^T and M3T were members of the genus Marinobacter. The highest level of 16S rRNA gene sequence similarity (99.7 %) was found with a partially determined sequence corresponding to Marinobacter arcticus, which is not significant for taxonomic purposes. The name of this strain has not yet been validly published and the article in which M. arcticus was studied (Button & Robertson, 2001) does not report any taxonomic description of this strain. Moreover, the strain is not available in any public culture collection and could not be supplied by the authors who studied it. Lower levels of similarity (97.3–98.4 %) were found with other Marinobacter species (Fig. 1). Strain M3T showed 100.0 % 16S rRNA gene sequence similarity to M3B^T, indicating that these strains probably belong to the same species. To verify the taxonomic position of strain M3B^T, DNA–DNA hybridizations were performed with Marinobacter lipolyticus LMG 23831^T, Marinobacter flavimaris LMG 23834^T, Marinobacter sediminum LMG 23833^T, Marinobacter algicola LMG 23835^T, Marinobacter maritimus LMG 23847^T and Marinobacter koreensis DSM 17924^T. Strain M3B^T showed less than 32 % DNA relatedness with all assayed Marinobacter strains (see Supplementary Table S2, available in IJSEM Online). The low DNA–DNA reassociation values and the 16S rRNA gene sequence data showed that strain M3B^T occupies a distinct position within the genus Marinobacter (Wayne et al., 1987). Strain M3T showed 99 % DNA relatedness to M3B^T and it can be concluded that they belong to the same genospecies. The DNA G+C contents of M3B^T and M3T (57.1 and 57.4 mol%, respectively) lie within the range described for members of the genus Marinobacter.

(35K): Fig. 1. Phylogenetic tree obtained by neighbour-joining analysis of 16S rRNA gene sequences, showing the position of the Antarctic isolates M3BT and M3T among neighbouring species of the genus Marinobacter and related genera. Bootstrap values >50 % (based on 1000 replications) are shown at branch points.

The morphological, physiological, chemotaxonomic and phylogenetic data show that strains M3B^T and M3T belong to the genus Marinobacter. DNA–DNA hybridization analyses clearly distinguished strain M3B^T from other related Marinobacter species. Therefore, on the basis of data from this polyphasic study, it is proposed that strains M3B^T and M3T represent a novel species of the genus Marinobacter, for which the name Marinobacter guineae sp. nov. is proposed.

Description of Marinobacter guineae sp. nov.
Marinobacter guineae (gui.ne'ae. N.L. gen. masc. n. guineae in honour of the late Professor Jesús Guinea, a prominent Spanish microbiologist, who isolated this strain).

Cells are rod-shaped (0.4 µm wide and 1.4–4.0 µm long), Gram-negative and non-spore-forming. Cells are motile by means of polar flagella. After 72 h incubation at 30 °C on MA, colonies are 1.5–2.0 mm in diameter, smooth, round, convex and white. Growth occurs between 4 and 42 °C and at pH 5.0–9.5. The NaCl range for growth is 1–15 % (w/v). No growth occurs in the absence of Na⁺. Growth is observed under anaerobic conditions in the presence of KNO₃. Nitrate and nitrite are reduced. Tween 80 is hydrolysed, but DNA, lecithin, casein and starch are not. Using the API 20NE test, aesculin hydrolysis is positive, but gelatin hydrolysis, indole production, glucose fermentation, arginine dihydrolase and urease are negative. Assimilates N-acetylglucosamine and phenylacetic acid, but not D-glucose, L-arabinose, D-mannose, D-mannitol, maltose, potassium gluconate, capric acid, adipic acid or trisodium citrate. Using the API 50CH test, only glycerol, D-glucose and D-fructose are fermented; none of the other organic substrates included in the API 50CH test are fermented. Positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase and N-acetyl-β-glucosaminidase, but negative for α-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, α-mannosidase and α-fucosidase (API ZYM). The main fatty acids are summed feature 3 (C_{16 : 1}ω7c and/or iso-C_{15 : 0} 2-OH), C_{16 : 0} and C_{18 : 1}ω9c. Ubiquinone-9 is the major isoprenoid quinone. The DNA G+C content is 57.1 mol%.

The type strain, M3B^T (=LMG 24048^T=CECT 7243^T), was isolated from a marine sediment collected from Deception Island (South Shetland Islands, Antarctica).