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1 Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon, Korea
2 National Research Laboratory of Molecular Ecosystematics, Institute of Probionic, Probionic Corporation, Bio-venture Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon, Korea
Correspondence
Jung-Hoon Yoon
jhyoon{at}kribb.re.kr
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Published online ahead of print on 3 December 2004 as DOI 10.1099/ijs.0.03051-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains SW-62T, SW-74, SW-63T and SW-72 are AY445073, AY445074, AY445075 and AY445076, respectively.
Transmission electron micrographs showing the appendages found on most cells of strains SW-62T, SW-74, SW-63T and SW-72 are available as supplementary material in IJSEM Online.
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with the single species Muricauda ruestringensis. Phylogenetic analysis based on 16S rRNA gene sequences showed that the genus Muricauda is a member of the family Flavobacteriaceae within the Cytophaga–Flavobacterium–Bacteroides (CFB) complex (Bruns et al., 2001). At the time of writing, there is only one Muricauda species with a validly published name (Bruns et al., 2001). The aim of the present study was to investigate the possibility that strains SW-62T, SW-74, SW-63T and SW-72 were novel species of the genus Muricauda. In this work, we describe the morphological, phenotypic, phylogenetic and genomic characteristics of strains SW-62T, SW-74, SW-63T and SW-72.
Strains SW-62T, SW-74, SW-63T and SW-72 were isolated by using the standard dilution plating technique on marine agar 2216 (MA; Difco). For isoprenoid quinone analysis and DNA extraction, cell biomass of the four isolates and M. ruestringensis DSM 13258T was obtained after cultivation in marine broth 2216 (MB; Difco) for 2 days at 30 °C. For fatty acid methyl ester analysis, cell mass of the four isolates and M. ruestringensis DSM 13258T was obtained from agar plates after incubation for 3 days at 30 °C on MA. M. ruestringensis DSM 13258T was obtained from the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany). Cell morphology was examined by using light microscopy (E600 apparatus; Nikon) and transmission electron microscopy (CM-20 apparatus; Philips). Gram reactions were determined using the bioMérieux Gram stain kit according to the manufacturer's instructions. Growth under anaerobic conditions was determined after incubation in an anaerobic chamber on MA and MA supplemented with nitrate, both of which had been prepared anaerobically using nitrogen. Growth in the absence of NaCl was investigated in trypticase/soy broth (Difco) lacking NaCl. Growth at various NaCl concentrations was investigated for 14 days at 30 °C in MB. Growth at various temperatures was measured on MA at 4–50 °C. Catalase and oxidase activities and hydrolysis of casein and starch were determined as described by Cowan & Steel (1965). Hydrolysis of aesculin, urea and gelatin and nitrate reduction were studied as described previously (Lanyi, 1987) with the modification that artificial sea water was used. The artificial sea water contained the following (per litre of distilled water): 23·6 g NaCl, 0·64 g KCl, 4·53 g MgCl2.6H2O, 5·94 g MgSO4.7H2O and 1·3 g CaCl2.2H2O (Bruns et al., 2001). Hydrolysis of Tweens 20, 40, 60 and 80 was determined as described by Cowan & Steel (1965) or tested on MA using the substrate concentrations described by Cowan & Steel (1965). Hydrolysis of hypoxanthine, tyrosine and xanthine was tested on MA plates with the substrate concentrations described previously (Cowan & Steel, 1965). Hydrolysis of birchwood xylan (Sigma) was tested on solid marine salts basal medium (Baumann & Baumann, 1981) supplemented with 0·5 % (w/v) xylan as the sole carbon source. H2S production was tested as described previously (Bruns et al., 2001). Acid production from carbohydrates was determined as described by Leifson (1963). Utilization of various substrates for growth was determined as described by Yurkov et al. (1994).
Isoprenoid quinones were analysed as described by Komagata & Suzuki (1987), using reversed-phase HPLC. For quantitative analysis of cellular fatty acid compositions, a loop of cell mass was harvested and fatty acid methyl esters were extracted and prepared according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification System (Sasser, 1990). Chromosomal DNA was isolated and purified according to the method described previously (Yoon et al., 1996), with the exception that ribonuclease T1 was used with ribonuclease A. The DNA G+C content was determined by the method of Tamaoka & Komagata (1984). DNA was hydrolysed and the resultant nucleotides were analysed by reversed-phase HPLC. The 16S rRNA gene was amplified by a PCR using two universal primers, as described previously (Yoon et al., 1998). Sequencing of the amplified 16S rRNA gene and phylogenetic analysis were performed as described by Yoon et al. (2003). DNA–DNA hybridization was performed fluorometrically according to the method of Ezaki et al. (1989), using photobiotin-labelled DNA probes and microdilution wells. Hybridization was performed using five replications for each sample. Of the values obtained, the highest and lowest for each sample were excluded. DNA–DNA relatedness values are the means of the remaining three values.
The colonies of strains SW-62T SW-74, SW-63T and SW-72 were golden yellow in colour on MA, whereas those of M. ruestringensis DSM 13258T were yellow on MA. Acid production from D-melezitose was observed only for strains SW-62T and SW-74. Other morphological, cultural, physiological and biochemical characteristics are shown in Table 1 or are given in the species descriptions (see below). Strains SW-62T SW-74, SW-63T and SW-72 contained unsaturated menaquinone with six isoprene units (MK-6) as the predominant isoprenoid quinone. In this study, the predominant isoprenoid quinone of M. ruestringensis DSM 13258T was also analysed and found to be MK-6. Strains SW-62T SW-74, SW-63T and SW-72 had cellular fatty acid profiles containing large amounts of straight-chain, branched and hydroxy fatty acids; the major fatty acids were iso-C17 : 0 3-OH, iso-C15 : 1 and iso-C15 : 0 (Table 2). These fatty acid profiles were similar to that of M. ruestringensis DSM 13258T (analysed in this study; see Table 2). For some fatty acids, particularly C15 : 0, the proportions present in M. ruestringensis DSM 13258T differed between this study and the study of Bruns et al. (2001), perhaps because of differences in cultivation conditions. The DNA G+C contents of strains SW-62T, SW-74, SW-63T and SW-72 were 45·2, 45·4, 44·1 and 44·2 mol%, respectively.
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), strains SW-63T and SW-72 formed a coherent cluster with M. ruestringensis DSM 13258T at a bootstrap resampling value of 96·4 %; this cluster joined strains SW-62T and SW-74 at a bootstrap resampling value of 100 % (Fig. 1). Strains SW-62T and SW-74 and strains SW-63T and SW-72 exhibited 16S rRNA gene sequence similarity levels of 96·5 and 98·3 %, respectively, with respect to the type strain of M. ruestringensis (Fig. 1). The four isolates exhibited levels of 16S rRNA gene sequence similarity of less than 91·6 % (between strains SW-63T and SW-72 and Zobellia uliginosa ATCC 14397T) to other species used in the phylogenetic analysis (Fig. 1). Levels of DNA–DNA relatedness between strains SW-62T and SW-74 and between strains SW-63T and SW-72 were 90·7 and 93·6 %, respectively. The mean level of DNA–DNA relatedness between strains SW-62T and SW-74 and strains SW-63T and SW-72 was 9·2 %. Strains SW-62T and SW-63T exhibited DNA–DNA relatedness levels of 4·9 and 7·0 %, respectively, with respect to M. ruestringensis DSM 13258T.
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; Pinhassi et al., 1997; Llobet-Brossa et al., 1998; Glöckner et al., 1999; Barbeyron et al., 2001; Kirchman, 2002). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strains SW-62T, SW-74, SW-63T and SW-72 are most closely related to the genus Muricauda, a member of the CFB complex. Their morphological properties are similar to those of M. ruestringensis; in particular, all possess an appendage not found in the phylogenetically related genus Zobellia (Barbeyron et al., 2001; Bruns et al., 2001). The results of chemotaxonomic analyses are consistent with the results of phylogenetic and morphological analyses. The chemotaxonomic properties of the four strains are similar to those of M. ruestringensis (Bruns et al., 2001). The fatty acid profiles of the genus Zobellia, the four strains and M. ruestringensis DSM 13258T are similar, but there are differences in the proportions of some fatty acids, particularly iso-C15 : 1 (Barbeyron et al., 2001).
Strains SW-62T, SW-74, SW-63T and SW-72 were found to have almost identical cultural and physiological characteristics (Table 1
). There are no sequence differences in 16S rRNA gene sequences between strains SW-62T and SW-74 or between strains SW-63T and SW-72. DNA–DNA relatedness values indicate that strains SW-62T and SW-74 and strains SW-63T and SW-72 are members of two different genomic species (Wayne et al., 1987). Strains SW-62T and SW-74 and strains SW-63T and SW-72 are similar to M. ruestringensis in most physiological characteristics, except growth on some substrates and acid production from some sugars (Table 1). While M. ruestringensis grows in anaerobic conditions, the four isolates do not grow in anaerobic conditions on MA or on MA supplemented with nitrate. Strains SW-62T and SW-74 and strains SW-63T and SW-72 are phylogenetically and genetically distinguishable from M. ruestringensis (Wayne et al., 1987; Stackebrandt & Goebel, 1994). Therefore, on the basis of the data presented above, strains SW-62T and SW-74 and strains SW-63T and SW-72 should be placed in the genus Muricauda as two distinct novel species, for which the names Muricauda flavescens sp. nov. and Muricauda aquimarina sp. nov., respectively, are proposed.
Emended description of the genus Muricauda Bruns et al. 2001
The description is as given previously (Bruns et al., 2001), with the following amendments. Facultatively anaerobic or strictly aerobic. Optimal growth occurs between 20 and 30 °C or between 30 and 37 °C. The fatty acid profile is characterized by large amounts of branched and straight-chain fatty acids. The DNA G+C content is 41–45 mol%.
Description of Muricauda flavescens sp. nov.
Muricauda flavescens (fla.ves'cens. L. v. flavesco to become golden yellow; L. part. adj. flavescens becoming golden yellow).
Cells are non-motile, non-spore-forming rods that are 0·2–0·5 µm wide by 2·5–6·0 µm long. Gram-negative. Appendages are found on most cells (see Supplementary Figure in IJSEM Online). On MA, colonies are golden yellow in colour, glistening, circular, slightly convex and 0·8–1·2 mm in diameter after 3 days incubation at 30 °C. Optimal growth temperature is 30–37 °C. Growth occurs at 10 °C, but not at 4 °C. Maximum growth temperature is 44 °C. Optimal growth pH is around 7·0. Growth occurs weakly at pH 5·0, but not at pH 4·5. Optimal growth occurs in the presence of 2 % (w/v) NaCl. Growth occurs in the presence of 9 % (w/v) NaCl, but not without NaCl or in the presence of more than 10 % (w/v) NaCl. No growth occurs under anaerobic conditions on MA. The predominant respiratory lipoquinone is MK-6. The major fatty acids are iso-C17 : 0 3-OH, iso-C15 : 1, iso-C15 : 0 and C15 : 0. The DNA G+C content (determined by HPLC) is 45·2–45·4 mol%. Other phenotypic properties are given in Table 1.
The type strain, SW-62T (=KCCM 41645T=JCM 11812T), was isolated from a salt lake near Hwajinpo Beach of the East Sea in Korea. The reference strain is SW-74.
Description of Muricauda aquimarina sp. nov.
Muricauda aquimarina (a.qui.ma.ri'na. L. n. aqua water; L. adj. marinus of the sea; N.L. adj aquimarina pertaining to sea water).
Cells are non-motile, non-spore-forming rods that are 0·2–0·5 µm wide by 2·5–6·0 µm long. Gram-negative. Appendages are found on most cells (see Supplementary Figure in IJSEM Online). On MA, colonies are golden yellow in colour, glistening, circular, slightly convex and 0·8–1·2 mm in diameter after 3 days incubation at 30 °C. Optimal growth temperature is 30–37 °C. Growth occurs at 10 °C, but not at 4 °C. Maximum growth temperature is 44 °C. Optimal growth pH is around 7·0. Growth occurs weakly at pH 5·0, but not at pH 4·5. Optimal growth occurs in the presence of 2 % (w/v) NaCl. Growth occurs in the presence of 9 % (w/v) NaCl, but not without NaCl or in the presence of more than 10 % (w/v) NaCl. No growth occurs under anaerobic conditions on MA. The predominant respiratory lipoquinone is MK-6. The major fatty acids are iso-C15 : 0, iso-C15 : 1 and iso-C17 : 0 3-OH. The DNA G+C content (determined by HPLC) is 44·1–44·2 mol%. Other phenotypic properties are given in Table 1.
The type strain, SW-63T (=KCCM 41646T=JCM 11811T), was isolated from a salt lake near Hwajinpo Beach of the East Sea in Korea. The reference strain is SW-72.
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ACKNOWLEDGEMENTS |
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