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1 The School of Health, Natural and Social Sciences, University of Sunderland, Sunderland SR1 3SD, UK
2 School of Applied Sciences, Northumbria University, Ellison Building, Newcastle upon Tyne NE1 8ST, UK
Correspondence
Stephen P. Cummings
stephen.cummings{at}sunderland.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Published online ahead of print on 4 October 2002 as DOI 10.1099/ijs.0.02271-0.
The GenBank accession numbers for the 16S rDNA sequences of Cellvibrio japonicus NCIMB 10462T, Cellvibrio mixtus subsp. mixtus ACM 2601T, Cellvibrio vulgaris NCIMB 8633T and Cellvibrio fulvus NCIMB 8634T are respectively AF452103, AF448515, AF448513 and AF448514.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. The bacterium produces an array of plant cell wall-degrading enzymes (glycosyl hydrolases), a characteristic it shares with several other Gram-negative soil bacteria such as Cellvibrio mixtus subsp. mixtus ACM 2601T and plant pathogens such as Pseudomonas syringae. Much of the work on ‘P. fluorescens subsp. cellulosa’ has focused on the molecular and structural characterization of these modular enzymes, with particular modules involved in substrate binding or catalysis. Many glycosyl hydrolases, such as cellulases and xylanases, share a common ability to bind to plant cell walls and not form aggregates (Hazlewood et al., 1992). Xylanases E and F and arabinofuranosidase (XYLC) from ‘P. fluorescens subsp. cellulosa’ contain carbohydrate-binding modules similar to those of xylanases A and B from ‘Cellvibrio vulgaris’ NCIMB 8633. Therefore, it was hypothesized that there had been a recent transfer of genes between these bacteria (Kellett et al., 1990; Millward-Sadler et al., 1995). However, high similarity between the 
-glucuronidase genes from the two bacteria has recently been demonstrated (H. J. Gilbert, personal communication). This led to speculation that ‘P. fluorescens subsp. cellulosa’ was misclassified.
The phylogenetic status of ‘P. fluorescens subsp. cellulosa’ NCIMB 10462 has been largely overlooked, despite the large body of work on the plant cell wall-degrading enzymes of this bacterium. It was addressed by Dees et al. (1995), whose study was based on biochemical and chemotaxonomic data. They concluded, on the basis of the fatty acid profile similarities, that the organism was a member of Pseudomonas. However, the similarity was rather low and the study was not supported by genotypic data. It was proposed that the organism be classified as the novel species ‘Pseudomonas cellulosa’, but this was never validated and the bacterium remained inadequately classified and was excluded from the recent revision of the genus Pseudomonas (Anzai et al., 2000). The results of the analysis presented here are conclusive in demonstrating that ‘P. fluorescens subsp. cellulosa’ is not a member of the genus Pseudomonas, but is most closely related to C. mixtus subsp. mixtus ACM 2601T.
The genus Cellvibrio was proposed by Winogradsky (1929) and it originally included ‘Cellvibrio ochraceus’ (as the type species of the genus) and ‘Cellvibrio flavescens’. Later, two more species, ‘C. vulgaris’ and ‘Cellvibrio fulvus’, were added to the genus (Stapp & Bortels, 1934). The genus appeared in this form in the 7th edition of Bergey's Manual of Determinative Bacteriology (Winogradsky et al., 1957). Regrettably, ‘C. ochraceus’ and ‘C. flavescens’ were lost from culture and the genus was dropped from the Approved Lists of Bacterial Names (Skerman et al., 1980) on the basis that there was no longer an officially recognized type species. The genus Cellvibrio was relegated to species incertae sedis within the genus Pseudomonas (Doudoroff & Palleroni, 1974). The genus was subsequently revived, with a new type species, C. mixtus, containing two subspecies, C. mixtus subsp. mixtus ACM 2601T and C. mixtus subsp. dextranolyticus ACM 1666T (Blackall et al., 1985). However, ‘C. vulgaris’ and ‘C. fulvus’ were not included in the newly defined genus.
This study sought to establish whether ‘P. fluorescens subsp. cellulosa’ was a member of the genus Cellvibrio; as compelling evidence has shown that this is the case, it is proposed that it be classified as the type strain of the novel species Cellvibrio japonicus sp. nov. It shall be referred to by this name subsequently. It was resolved to clarify the phylogeny of the genus Cellvibrio, using all the bacteria currently available in culture that are presently, or have been historically, associated with the genus. Employing a polyphasic approach, it was demonstrated that isolates NCIMB 8633 and NCIMB 8634, respectively described as ‘C. vulgaris’ and ‘C. fulvus’, are indeed members of the genus Cellvibrio and hence, they are designated as the novel type strains C. vulgaris NCIMB 8633T and C. fulvus NCIMB 8634T. They will be referred to by these new names throughout the rest of this article. It was also found that C. mixtus subsp. dextranolyticus ACM 1666T is no longer available for analysis, since the cultures from the Australian Collection of Microorganisms (ACM) have been found to be non-viable.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Approved Voges–Proskauer and methyl red tests were employed (Collins et al., 1991). The KOH solubilization test was done according to an accepted method (Gregersen, 1978). Curdlan-type polysaccharide was detected using aniline blue dye (Wako) and an appropriate staining method (Nakanishi et al., 1976). Hydrolysis of neutral and alkaline pectate gels was determined using previously described methods (Hildebrand, 1971).
Acid production from cellobiose, melezitose, rhamnose, glycerol, mannitol and raffinose was examined according to Smibert & Krieg (1984), with incubation at 25 °C for 7 days and pH checks after visible growth and then every day. The substrate-utilization profile of the bacterium was determined in triplicate at 25 °C in minimal medium containing 0·5 % (w/v) substrate. The minimal medium used was M9 (Sambrook et al., 1989); for substrate-utilization testing, the additive required did not contain glucose. Endo-1,3-
-glucanase (laminarinase) activity was examined by growing the test bacterium in 0·5 % (w/v) carboxymethyl curdlan (CML; Megazyme) in an M9 broth with additive. The culture supernatant was then assayed for activity against CML using the dinitrosalicylic acid method (Miller, 1959). The test for agar degradation was done on M9 with additive and 1·5 % (w/v) purified agar (L28; Oxoid). Pullulanase and dextranase activities were tested for by incorporating 0·5 % (w/v) red pullulan or AZCL-dextran (both from Megazyme), respectively, with an equal concentration of dialysed soluble starch, into M9 agar plates with additive. The cultures were incubated at 25 °C for 7 days; broths were aerated at 150 r.p.m.
Duplicate API 20NE and API ZYM identification strips were set up, as indicated by the manufacturer (bioMérieux), and incubated at 25 °C for 3 days. API ZYM strips were set up for C. japonicus NCIMB 10462T C. mixtus subsp. mixtus ACM 2601T, C. vulgaris NCIMB 8633T and C. fulvus NCIMB 8634T. All the bacteria tested were grown on TSA at 25 °C. The API 20NE strips were incubated at 25 °C for 24 h and then developed; utilization tests that were inconclusive were re-examined at 48 h. The API ZYM strips were incubated at 25 °C and then developed and read after 45 h.
Antibiotic-sensitivity tests.
Stocks of the antibiotics ampicillin (sodium salt), erythromycin, neomycin sulphate, kanamycin A, chloramphenicol, gentamicin sulphate, streptomycin sulphate, tetracycline hydrochloride and rifampicin (all from Sigma) were made up at an appropriate concentration range (Humphry et al., 2001) in TSA plates for testing. The test bacteria were spread onto the plates and incubated at 25 °C for 7 days. Duplicate antibiotic-sensitivity tests were also done using Mastring-S M11, M14 and M43 (Mast Diagnostics) disc sensitivity kits. These discs were placed on TSA plates spread with test culture and incubated at 25 °C for 7 days.
PCR amplification of 16S rDNA.
The 16S rDNA primers used to obtain the initial PCR product were identical to the Escherichia coli 16S rDNA sequence between positions 8 and 27 (forward direction; 5'-AGAGTTTGATCCTGGCTCAG-3') and from 1509 to 1491 (reverse direction; 5'-GGHTACCTTGTTACGACTT-3') (Weisburg et al., 1991). The PCR mixtures and conditions have been described previously (Brown et al., 2001).
Cloning and sequencing of PCR products.
The methods and equipment used have been described previously (Humphry et al., 2001). The two plasmid inserts were sequenced completely, initially using the M13 forward and reverse primers designed on the basis of the sequence of the pGEM-T ‘Easy’ plasmid; subsequently, oligonucleotide primers were designed on the basis of the sequence of the ends of the resulting sequences. For C. japonicus NCIMB 10462T, C. mixtus subsp. mixtus ACM 2601T, C. vulgaris NCIMB 8633T and C. fulvus NCIMB 8634T, these forward and reverse primers were respectively 5'-TCACACTGGAACTGAGAC-3' and 5'-GGTCGTAAGGGCCATGAT-3', 5'-AGGCAGCAGTGGGGAATA-3' and 5'-TCACCGGCAGTCTCCTTA-3', 5'-TCAGTGGGGAGAAAGGTC-3' and 5'-TCACCGGCAGTCTCCTTA-3', and 5'-AGGCAGCAGTGGGGAATA-3' and 5'-TTAGAGTTCCCGCCATTA-3'.
16S rDNA sequence analysis.
The 16S rDNA sequences of C. japonicus NCIMB 10462T, C. mixtus subsp. mixtus ACM 2601T, C. vulgaris NCIMB 8633T and C. fulvus NCIMB 8634T were respectively 1507, 1494, 1501 and 1494 bases long, with no non-base characters. The Cellvibrio 16S rDNA sequences were individually aligned against that from C. japonicus NCIMB 10462T, all columns containing non-base characters were removed and the percentage similarities were then calculated. The sequence was also aligned against nine other sequences at once, using the CLUSTAL method in the MEGALIGN program (DNAStar), and all non-base characters were then removed manually. This reduced the length of the sequences analysed by the PHYLIP and MEGALIGN programs to 1450 bases. The six other 16S rDNA sequences were taken from the National Institutes of Health GenBank database (Benson et al., 1998). Programs used for phylogenetic analysis included SEQBOOT, DNADIST, NEIGHBOR, CONSENSE (PHYLIP 3.5c package; Felsenstein, 1993) and TREEVIEW (Page, 1996). Computerized phylogenetic analysis was done as described by Brown et al. (2001).
Genomic DNA G+C content.
The genomic DNA of the bacterium was extracted using a Puregene DNA isolation kit (Gentra Systems). The G+C content was determined in triplicate, using a spectrophotometer (LKB) and an HPLC method (Mesbah et al., 1989). The mobile phase was 0·4 M NH4H2PO4 and acetonitrile in a 50 : 1 ratio. Calibration of the method was done using non-methylated lambda DNA (Sigma D3654) with a G+C content, determined using genome sequencing data, of 49·858 mol% (Sanger et al., 1982).
Electron microscopy.
Samples of trypticase soy broth (TSB) cultures in exponential growth phase were prepared and examined as described previously (Humphry et al., 2001).
DNA–DNA hybridizations.
Comparisons were made by the DSMZ. The DNA was isolated by chromatography (Cashion et al., 1977), and DNA–DNA hybridizations were then performed as described by De Ley et al. (1970) with the modification of Huß et al. (1983) and Escara & Hutton (1980) on a Gilford System model 2600 spectrometer equipped with a Gilford model 2527-R thermoprogrammer and plotter. Finally, renaturation rates were computed using the program TRANSFER.BAS (Jahnke, 1992).
Chemotaxonomic lipid analyses.
Total lipids were extracted with chloroform and methanol by the method of Bligh and Dyer (Kates, 1986). Individual lipids were separated by TLC on Merck 60H silica-gel plates and quantified by phosphorus analysis (Russell et al., 1985). Phospholipids were identified by comparison of their Rf values with those of authentic standards (Sigma) in a one-dimensional solvent system containing chloroform/methanol/acetic acid/water (85 : 15 : 10 : 3·5). Lipids were visualized using iodine vapour (general stain), ninhydrin (for free amino groups) and molybdate reagent (for phosphate groups) as described by Kates (1986). Quantification was established as mol% lipid phosphorus; the standard deviations of replicates were less than 5 % (see Table 2).
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). Fatty acid methyl esters were analysed by GLC (Sutcliffe, 1994). Analyses were performed in triplicate on three independent cultures; the standard deviations were less than 1 %. |
RESULTS AND DISCUSSION |
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Physiological characteristics
C. japonicus NCIMB 10462T, C. mixtus subsp. mixtus ACM 2601T, C. vulgaris NCIMB 8633T and C. fulvus NCIMB 8634T are mesophilic, neutrophilic, aerobic bacteria. Catalase and cytochrome oxidase tests on distinct colonies were positive. Nitrates were reduced to nitrites by C. japonicus NCIMB 10462T; none of the other strains reduced nitrates. None of the four bacteria showed indole production, arginine dihydrolase activity or urease, but there was -galactosidase activity. The bacteria could hydrolyse aesculin (-glucosidase activity) but not gelatin (protease activity). The Voges–Proskauer and methyl red tests for C. japonicus NCIMB 10462T were negative.
C. japonicus NCIMB 10462T produced acid from cellobiose and mannitol, but not from glucose, mannose, fructose, galactose, maltose, sucrose, lactose, xylose, arabinose, dextrin, glycerol, sorbitol, starch or cellulose. A comparison of the main phenotypic properties of C. japonicus NCIMB 10462T with C. mixtus subsp. mixtus ACM 2601T, C. vulgaris NCIMB 8633T and C. fulvus NCIMB 8634T is given in Table 1. Differentiating test results of the API ZYM strips for these bacteria are also shown in Table 2
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). For example, C. mixtus subsp. mixtus ACM 2601T can be differentiated from other Cellvibrio isolates on the basis of its ability to utilize mannose but not curdlan or chitin. Unique to C. japonicus NCIMB 10462T is the ability to hydrolyse 6-bromo-2-naphthyl -D-galactopyranoside; it also has -glucuronidase activity and can grow on medium B (King et al., 1954). C. vulgaris NCIMB 8633T is distinguishable by the ability to hydrolyse 2-naphthyl phosphate at pH 5·4 and by the utilization of both raffinose and fructose as sole carbon sources. The unique characteristics of C. fulvus NCIMB 8634T among members of the genus Cellvibrio are its ability to utilize casein acid hydrolysate, but not amylose, as a growth substrate.
Antibiotic-sensitivity tests
C. japonicus NCIMB 10462T was resistant to ampicillin, neomycin and streptomycin (all at 10 µg ml-1). It showed sensitivity to kanamycin, erythromycin, gentamicin, chloramphenicol, tetracycline and rifampicin (all at 10 µg ml-1). C. vulgaris NCIMB 8633T had the same resistance pattern apart from having sensitivity to ampicillin, neomycin and streptomycin (all at 10 µg ml-1). C. fulvus NCIMB 8634T had the same profile as C. japonicus NCIMB 10462T, apart from being resistant to 100 µg ampicillin ml-1 and 200 µg streptomycin ml-1. C. mixtus subsp. mixtus ACM 2601T had the same profile as C. vulgaris NCIMB 8633T except that it was resistant to 10 µg neomycin ml-1 and 200 µg streptomycin ml-1.
C. japonicus NCIMB 10462T showed zones of inhibition greater than 3 mm when tested with the following antibiotic discs: chloramphenicol (25 µg), erythromycin (5 µg), novobiocin (5 µg), streptomycin (10 µg), tetracycline (10 µg), colistin sulphate (25 µg), gentamicin (10 µg), sulphatriad (200 µg), cotrimoxazole (25 µg), trimethoprim (1·25 U) and sulphamethoxazole (25 µg). The fusidic acid (10 µg), methicillin (10 µg), penicillin G (1 U), ampicillin (10 µg), cephalothin (5 µg) and clindamycin (2 µg) discs caused no growth inhibition. For the antibiotic-sensitivity patterns of the other Cellvibrio species, see Table 2.
Membrane composition
The phospholipid types present in C. japonicus NCIMB 10462T are phosphatidylglycerol (15·9 %), phosphatidylethanolamine (84·1 %) and trace amounts of phosphatidylserine and diphosphatidylglycerol. The other bacteria showed similar phospholipid profiles (Table 1). The fatty acid profiles were also similar: the predominant fatty acids were unsaturated C16 : 1, C18 : 1 and saturated C16 : 0 in all the isolates. The cluster of bacteria tentatively identified as Cellvibrio by Lednická et al. (2000) also had fatty acid profiles that were similar to four strains of C. mixtus (LMG 2847T, LMG 2848, LMG 2849 and ACM 2603) and had the major fatty acids observed in this study in the profiles for C. japonicus NCIMB 10462T, C. mixtus subsp. mixtus ACM 2601T, C. vulgaris NCIMB 8633T and C. fulvus NCIMB 8634T (Table 1).
DNA G+C content
The G+C content of the genomic DNA of C. japonicus NCIMB 10462T was 53·3 mol%. The G+C contents of C. mixtus subsp. mixtus ACM 2601T, C. vulgaris NCIMB 8633T and C. fulvus NCIMB 8634T DNA were determined by Blackall et al. (1985). For further characterization of the genotypic characteristics of the four strains, the G+C contents of genomic DNA from the four strains were compared. The range was less than 10 %, from 44·6 to 53·3 %, which is acceptable for a defined genus (Vandamme et al., 1996). C. vulgaris NCIMB 8633T and C. fulvus NCIMB 8634T clustered together at the lowest end of this range, whereas C. japonicus NCIMB 10462T and C. mixtus subsp. mixtus ACM 2601T had very similar ratios, around 53 mol% (Table 1).
16S rDNA analysis
Phylogenetic analysis has shown that C. japonicus NCIMB 10462T falls within members of the genus Cellvibrio (Fig. 1), as most recently defined (Blackall et al., 1985). The closest 16S rDNA sequence belongs to C. mixtus subsp. mixtus ACM 2601T, whose sequence is 95·5 % similar (67 different bases) over 1494 bases. C. vulgaris NCIMB 8633T and C. fulvus NCIMB 8634T were respectively 94·0 and 93·6 % similar, with 89 and 95 base differences over 1495 and 1491 bases. The analysis also demonstrates that the 16S rDNA sequences of four isolates tentatively identified as members of Cellvibrio (Lednická et al., 2000), two of which belong to the newly described species Cellvibrio fibrivorans and Cellvibrio gandavensis (Mergaert et al., 2003), and that of C. mixtus subsp. mixtus ACM 2603 cluster with C. japonicus NCIMB 10462T, C. mixtus subsp. mixtus ACM 2601T, C. vulgaris NCIMB 8633T and C. fulvus NCIMB 8634T in a group that is well supported by bootstrap resampling. This degree of similarity between the 16S rDNA sequences indicates that all the sequences belong to bacteria of the same genus. Fatty acid profiles and biochemical data presented for the four strains tentatively identified by Lednická et al. (2000) are also very similar to those presented in this study and by Blackall et al. (1985). These isolates are worthy of further study to elaborate their taxonomic status within the genus Cellvibrio.
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). The genotypic data are indicative of a group of organisms that are members of the same genus but sufficiently distinct to be classified as distinct species; on this basis, isolate NCIMB 10462 should be reassigned to the genus Cellvibrio as the type strain of a novel species, Cellvibrio japonicus sp. nov.
To the best of our knowledge, this study represents the first re-examination of isolates NCIMB 8633T and NCIMB 8634T since they were originally described and respectively proposed to represent C. vulgaris and C. fulvus (Stapp & Bortels, 1934). The genotypic and phenotypic data presented here demonstrate that the two organisms are closely related to C. mixtus subsp. mixtus ACM 2601T and should be reinstated as species in their own right within the genus Cellvibrio as C. vulgaris sp. nov., nom. rev. and C. fulvus sp. nov., nom. rev.
Emended description of Cellvibrio (ex Winogradsky 1929) Blackall et al. 1986
The genus is as described by Blackall et al. (1985), with the following additional characteristics. The organisms show no indole production, no arginine dihydrolase activity and no urease activity, but there is -galactosidase activity. The bacteria can hydrolyse aesculin (-glucosidase activity) but not gelatin. The G+C content of the DNA is 48·8±4·7 mol%. Can utilize xylan, xylose, glucose, maltose, tryptone, carboxymethyl cellulose, pectin, starch, amylopectin, trehalose, lactose, arabinose and galactose as sole carbon and energy sources. However, there is no growth on succinate, ribose, pyruvate, mannitol, agar, acetate, dextran, glutamate, Tween 80, inositol, lactate, gluconate, caprate, adipate, malate, citrate, phenyl acetate, gelatin or casein. All isolates can grow in TSB (CM129; Oxoid) and on sucrose/peptone agar (ACM medium 416).
Description of Cellvibrio japonicus sp. nov.
Cellvibrio japonicus (ja.po'ni.cus. N.L. adj. japonicus pertaining to Japan, where the type strain was originally isolated).
The description is based on those given by Ueda et al. (1952) and Dees et al. (1995); it is based on one strain, i.e. no. 107. The description is as for the genus with the following additional traits. Cells are non-sporulating rods, 1·2–3 µm long and 0·5–0·7 µm wide. Mesophilic and has xylanase, arabinofuranosidase, esterase, cellulase, pullulanase, pectate lyase, mannanase and laminarinase activities. Cells are motile by two polar flagella. The G+C content of the genomic DNA is 53·3 mol%. Growth occurs over the temperature range 17–47 °C and at pH 6–9. There is growth on PYEA and medium B (King et al., 1954). Nitrate is reduced to nitrite; Voges–Proskauer and methyl red tests are negative. Produces acid from cellobiose and mannitol, but not from glucose, mannose, fructose, galactose, maltose, sucrose, lactose, xylose, arabinose, dextrin, glycerol, sorbitol, starch or cellulose. Resistant to the following antibiotics in TSA (µg ml-1): ampicillin (10), neomycin (10) and streptomycin (10). Sensitive to kanamycin (10), erythromycin (10), gentamicin (10), chloramphenicol (10), tetracycline (10) and rifampicin (10). The following antibiotic discs caused zones of inhibition greater than 3 mm on TSA (µg unless stated): chloramphenicol (25), erythromycin (5), novobiocin (5), streptomycin (10), tetracycline (10), colistin sulphate (25), gentamicin (10), sulphatriad (200), cotrimoxazole (25), trimethoprim (1·25 U) and sulphamethoxazole (25). Has a chemo-organotrophic metabolism and utilizes fructose, CML, amylose and chitin as sole carbon and energy sources. Grows in GYE broth with 2 %, but not 4 %, NaCl (w/v). Hydrolyses the following substrates when grown on TSA (API ZYM): 2-naphthyl phosphate (pH 8·5), 2-naphthyl butyrate, 2-naphthyl caprylate, L-leucyl 2-naphthylamide, naphthol-AS-BI-phosphate, 6-bromo-2-naphthyl -D-galactopyranoside, 2-naphthyl -D-glucopyranoside, 2-naphthyl -D-glucopyranoside and 1-naphthyl N-acetyl--D-glucosaminide. The plasma membrane, when grown in TSB (CM129; Oxoid), is composed of the following fatty acids: C14 : 0, 1·67 %; C16 : 0, 14·69 %; C16 : 1, 42·47 %; C17 : 0, 1·26 %; C18 : 0, 3·54 %; and C18 : 1, 24·63 %. Phospholipids detected are phosphatidylglycerol and phosphatidylethanolamine; trace amounts of diphosphatidylglycerol are detected.
The single and type strain, NCIMB 10462T (=NCDO 2697T), was isolated in 1948 from field soil in Saitama-ken, Japan.
Description of Cellvibrio vulgaris sp. nov., nom. rev.
Cellvibrio vulgaris (vul.gar'is. L. adj. vulgaris commonly occurring).
This description is based on data presented in this study and from work by Blackall et al. (1985) and Winogradsky et al. (1957); it is as for the genus, but with the following additional characteristics. Cells are curved rods, 2·9–4 µm long and 0·3 µm wide. Mesophilic and has xylanase, arabinofuranosidase, esterase, cellulase, pullulanase, pectate lyase, mannanase and laminarinase activities. Cells are motile by a single polar flagellum. The G+C content of the genomic DNA is 44·9 mol%. Growth occurs over the temperature range 5–35 °C. No growth on PYEA or medium B. Nitrate is not reduced to nitrite; there is -glucuronidase activity. Produces acid from cellobiose, melezitose and raffinose, but not from glycerol, rhamnose or mannitol. Has the same antibiotic resistance pattern as C. japonicus NCIMB 10462T apart from sensitivity to ampicillin, neomycin and streptomycin (all at 10 µg ml-1). Has a chemo-organotrophic metabolism and utilizes fructose, CML, amylose, chitin and raffinose as sole carbon and energy sources. No growth in GYE broth with 2 or 4 % (w/v) NaCl. When grown on TSA, can hydrolyse only the following substrates on API ZYM galleries: 2-naphthyl phosphate (pH 5·4 and 8·5), 2-naphthyl butyrate, 2-naphthyl caprylate, L-leucyl 2-naphthylamide, naphthol-AS-BI-phosphate and 1-naphthyl N-acetyl--D-glucosaminide. The single and type strain, NCIMB 8633T (=LMG 2848T), was isolated from beech leaf matter in Waldstreu (Faithful Forest), Germany (Stapp & Bortels, 1934).
Description of Cellvibrio fulvus sp. nov., nom. rev.
Cellvibrio fulvus (ful'vus. L. adj. fulvus mixture of yellow and grey, pertaining to the colour of the colonies on solid growth medium).
The description is as for the genus, but with the following additional characteristics. Cells are curved rods, 1·5–3 µm long and 0·3–0·4 µm wide. Aerobic, mesophilic and has xylanase, esterase, cellulase, pectate lyase and laminarinase activities. Cells are motile with a single polar flagellum. The G+C content of the genomic DNA is 44·6 mol%. Growth occurs over the temperature range 5–35 °C. Nitrate is not reduced to nitrite; there is -glucuronidase activity. Produces acid from cellobiose, melezitose and raffinose, but not from glycerol, rhamnose or mannitol. Has a chemo-organotrophic metabolism and utilizes sucrose, CML, casein acid hydrolysate, N-acetylglucosamine and chitin as sole carbon and energy sources. No growth in GYE broth with 2 or 4 % (w/v) NaCl. When grown on TSA, can hydrolyse the following substrates (API ZYM galleries): 2-naphthyl butyrate, 2-naphthyl caprylate, L-leucyl 2-naphthylamide, naphthol-AS-BI-phosphate, 6-bromo-2-naphthyl -D-glucopyranoside, 2-naphthyl -D-glucopyranoside and 1-naphthyl N-acetyl--D-glucosaminide. The single and type strain, NCIMB 8634T (=LMG 2847T), was isolated from beech leaf matter in Waldstreu (Faithful Forest), Germany (Stapp & Bortels, 1934).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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