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Int J Syst Evol Microbiol 54 (2004), 783-789; DOI  10.1099/ijs.0.02851-0
© 2004 International Union of Microbiological Societies

Gulosibacter molinativorax gen. nov., sp. nov., a molinate-degrading bacterium, and classification of ‘Brevibacterium helvolum’ DSM 20419 as Pseudoclavibacter helvolus gen. nov., sp. nov.

Célia M. Manaia1, Balbina Nogales2,3,{dagger}, Norbert Weiss4 and Olga C. Nunes5

1 Escola Superior de Biotecnologia, Universidade Católica Portuguesa, 4200-072 Porto, Portugal
2 Division of Microbiology, GBF – German Research Centre for Biotechnology, Braunschweig, Germany
3 Area de Microbiologia, Universitat de les Illes Balears, Palma de Mallorca, Spain
4 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen, Mascheroder Weg 1b, D-38124 Braunschweig, Germany
5 LEPAE – Departamento de Engenharia Química, Faculdade de Engenharia, Universidade do Porto, R. Dr Roberto Frias, 4200-465 Porto, Portugal

Correspondence
Olga C. Nunes
opnunes{at}fe.up.pt


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
A Gram-positive, molinate-degrading bacterium, strain ON4T (=DSM 13485T=LMG 21909T), was isolated from a mixed bacterial culture able to mineralize the herbicide molinate. The strain was strictly aerobic, oxidase- and catalase-positive and non-acid-fast, with a growth temperature of 10–41 °C. It contained the major menaquinone MK-9 and a cell-wall peptidoglycan based on D-ornithine. 16S rDNA sequence analysis revealed that the strain formed a distinct line of descent in the family Microbacteriaceae, showing the highest 16S rDNA similarity (~95 %) to members of the genus Curtobacterium and ‘Brevibacterium helvolum DSM 20419 (=ATCC 13715). The latter was reported to have the cell-wall peptidoglycan type B2{gamma} and the major menaquinone MK-9, which are typical of Clavibacter, but it is clearly separated from this genus at the phylogenetic level. Based on low values of 16S rDNA sequence similarity to previously described genera and their distinctive phenotypic characteristics, it is proposed that strains ON4T and ‘B. helvolum’ DSM 20419 be classified as two novel genera and species, with the respective names Gulosibacter molinativorax gen. nov., sp. nov. and Pseudoclavibater helvolus gen. nov., sp. nov.


Abbreviations: DAB, 2,4-diaminobutyric acid

Published online ahead of print on 28 November 2003 as DOI 10.1099/ijs.0.02851-0.

The GenBank/EMBL/DDBJ accession number for the 16S rDNA sequence of isolate ON4T is AJ306835.

Micrographs of cells of the novel species and details of fatty acid compositions are available as supplementary material in IJSEM Online.

{dagger}Present address: Area de Microbiologia, Universitat de les Illes Balears, Palma de Mallorca, Spain. Back


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The family Microbacteriaceae (Park et al., 1993Go; Stackebrandt et al., 1997Go) embraces a large group of actinomycetes with cell-wall peptidoglycan of the B-type (Schleifer & Kandler, 1972Go). The family currently contains 16 genera, distinguished at the phenotypic level by a number of chemotaxonomic characteristics, including peptidoglycan diamino acids and respiratory menaquinone composition (Collins & Bradbury, 1992Go; Evtushenko & Takeuchi, 2003Go; Sheridan et al., 2003Go).

Brevibacterium helvolum’ DSM 20419 (=ATCC 13715), an organism isolated from butter, has peptidoglycan B2{gamma}, [L-dab]–D-glu–D-DAB (DAB is 2,4-diaminobutyric acid), unsaturated menaquinone with nine isoprene units (MK-9) (Schleifer & Kandler, 1972Go; Sasaki et al., 1998Go) and the polyamines 2,3-diaminopropane and cadaverine (Altenburger et al., 1997Go). Based on comparative analysis of 16S rDNA, Rainey et al. (1994)Go showed that ‘B. helvolum DSM 20419 formed a separate subline of descent within the radiation of actinomycetes belonging to the family Microbacteriaceae. According to Rainey et al. (1994)Go, this strain could represent the nucleus of a novel genus.

Strain ON4T was isolated from a microbial mixed culture enriched in molinate, which also included four strains of the genera Pseudomonas, Stenotrophomonas and Achromobacter (Barreiros et al., 2003Go). The strain used molinate as the only carbon, nitrogen and energy source and grew at herbicide concentrations up to 2 mM, although it did not perform its complete degradation (Barreiros et al., 2003Go). It exhibited the highest 16S rDNA similarity to the misclassified strain ‘B. helvolum DSM 20419 and could not be affiliated to any previously described genus. In this paper, we describe the morphological, physiological, chemotaxonomic and phylogenetic characteristics of these organisms and propose to accommodate them into two novel genera.


    METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Determination of morphological, growth and biochemical characteristics.
Wet mounts and staining methods followed by optical microscopic examination were used to assess cell morphology, Gram and Ziehl–Neelsen reactions, production of spores, accumulation of poly-hydroxybutyrate granules and motility, according to procedures described previously (Doetsch, 1981Go; Smibert & Krieg, 1981Go). Cell morphology of isolate ON4T was further studied by electron microscopy after negative staining with 2 % (w/v) sodium phosphotungstate (pH 7·0) using a Hitachi H-600 transmission electron microscope at 75 kV.

Growth temperature, pH and salinity ranges were examined by measuring turbidity (at 610 nm) of cultures in 250 ml screw-capped Erlenmeyer flasks containing 50 ml LB medium (Carlton & Brown, 1981Go) incubated in an orbital water-bath shaker. The pH range for growth was examined in medium buffered with 12 mM MES (Sigma) at pH 5·0–6·0 or with 15 mM CAPS (Sigma) at pH 9·5–11·0. Anaerobic growth of strains and oxidase (tetramethyl-p-phenylenediamine), catalase, nitrate reductase and Tweenase reactions were examined at 30 °C as described by Smibert & Krieg (1981)Go using 3-day cultures grown on LB agar (LA). Other enzymic activities were tested using the API ZYM system, following the instructions of the manufacturer. The nutritional pattern was characterized using the API 50CH system and a defined medium B (Manaia et al., 2003Go). The ability of strain ON4T to use API 50CH carbon sources was also tested in mineral medium supplemented with different concentrations of yeast extract (0·01–0·2 %, w/v). Additionally, the aerobic dissimilation of 95 carbon sources was tested using the Biolog GP microplate system, according to the manufacturer's indications.

Determination of chemotaxonomic characteristics.
The polar lipids of strains ON4T and DSM 20419 were characterized using cultures grown in LB medium, at 30 °C, at the late exponential phase of growth. Lipid extractions were performed as described previously (Tindall, 1990Go). Individual polar lipids were separated by two-dimensional TLC as described by Minnikin et al. (1977)Go. For the analysis of methylated fatty acids (FAMEs) of strains ON4T and DSM 20419, cells were grown for 3 days on LA medium at 30 °C. FAME extraction and analysis were performed as described by Moreira et al. (2000)Go. For the analysis of respiratory quinones, strains ON4T and DSM 20419 were cultured on LA medium, harvested, freeze-dried and extracted according to Tindall (1989)Go, and the extracts were analysed as described by Moreira et al. (2000)Go. The peptidoglycan of strain ON4T was analysed as described previously (Schleifer & Kandler, 1972Go), using TLC on cellulose sheets instead of paper chromatography. L- and D-ornithine could be distinguished by their small but significant mobility in the solvent system used for the characterization of diamino acids. For the determination of DNA base composition of strains ON4T and DSM 20419, genomic DNA was isolated as described by Cashion et al. (1977)Go and the DNA G+C content was analysed by HPLC (Mesbah et al., 1989Go).

16S rRNA sequence determination and phylogenetic analysis.
The sequence of the 16S rRNA gene of isolate ON4T was determined after PCR amplification from total DNA extracts using primers 27f and 1492r (Lane, 1991Go) as described previously (Nogales et al., 2001Go). The nucleotide sequence of purified PCR products was determined by using the BigDye Terminator cycle sequencing kit and ABI377 and 310 sequencers (Applied Biosystems), according to the manufacturer's instructions. The nucleotide sequence was aligned with reference sequences using the ARB package (http://www.arb-home.de). Phylogenetic trees were constructed using the ARB package and the distance methods of neighbour-joining and Fitch [using the correction of the Jukes & Cantor (1969)Go for calculation of evolutionary distances] as well as parsimony methods. Bootstrap analysis (1000 replicates) was done using the PHYLIP package (Felsenstein, 1989Go). Ambiguous nucleotide positions were excluded from the calculations.


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Morphological, biochemical and growth characteristics
On LA medium, strain ON4T formed white, opaque colonies, 1 mm in diameter, while colonies of strain DSM 20419 were yellow and 2 mm in diameter. Both strains formed irregularly rod-shaped cells (see Supplementary Figure in IJSEM Online), and strain ON4T had a tendency to form short filaments. Strains ON4T and DSM 20419 stained as Gram-positive, were non-acid-fast and non-spore-forming, had no visible deposits of poly-{beta}-hydroxybutyrate and were non-motile and catalase-positive. Both organisms were observed to be strictly aerobic and positive for oxidase, although a very weak reaction was observed for strain DSM 20419. Isolate ON4T was able to reduce nitrate under both aerobic and anaerobic conditions, although growth did not occur in the absence of oxygen (Table 1Go).


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Table 1. Phenotypic characteristics of strains ON4T and DSM 20419T

Both strains produced alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase. Neither strain produced trypsin, chymotrypsin, {alpha}-galactosidase, {beta}-glucosidase, {beta}-glucuronidase, N-acetyl-{beta}-glucosaminidase, {alpha}-mannosidase, {alpha}-fucosidase or amylase. Both strains were positive or showed weak reactions for the utilization of pyruvic acid, methyl pyruvate, L-glutamic acid, 2,3-butanediol, adenosine, 2'-deoxyadenosine, inosine, thymidine, uridine, adenosine 5'-monophosphate, thymidine 5'-monophosphate and uridine 5'-monophosphate. Neither strain was able to use the following carbon sources: adonitol, dulcitol, arbutin, starch, methyl {beta}-D-glucoside, salicin, {alpha}- and {beta}-cyclodextrin, glycogen, inulin, mannan, N-acetyl-D-mannosamine, amygdalin, D- and L-arabinose, D- and L-arabitol, erythritol, D- and L-fucose, D-galactose, D-galacturonic acid, maltotriose, D-lyxose, D-melezitose, D-melibiose, L-xylose, methyl {alpha}- and {beta}-D-galactoside, methyl {beta}-xyloside, 3-methyl-D-glucose, methyl {alpha}-D-glucoside, methyl {alpha}-D-mannoside, palatinose, L-rhamnose, D-ribose, L-sorbose, sedoheptulosan, stachyose, D-tagatose, xylitol, acetic acid, {alpha}- and {beta}-hydroxybutyric acid, {alpha}-ketoglutaric acid, {alpha}-ketovaleric acid, lactamide, D-lactic acid methyl ester, D- and L-malic acid, monomethyl succinate, propionic acid, succinamic acid, succinic acid, N-acetyl-L-glutamic acid, alaninamide, D-alanine, L-alanyl glycine, L-asparagine, glycyl L-glutamic acid, L-pyroglutamic acid, L-serine, fructose 6-phosphate, glucose 1-phosphate, glucose 6-phosphate and DL-{alpha}-glycerol phosphate. w, Weak.

 
The growth temperature optima were 28–30 °C for strain DSM 20419 and 35–37 °C for strain ON4T, although the latter also grew well at 28–30 °C. Metabolic activities of strain ON4T studied in the presence of vitamins or yeast extract were restricted to only 15 of 110 compounds tested and, of the few carbon sources utilized, most contained nitrogen. A slow reaction was observed with the only sugar, {alpha}-D-glucose. Strain DSM 20419 was much more versatile metabolically and utilized a variety of organic compounds, including sugars, alcohols, organic acids, amino acids and nucleotides. Details of the physiological and biochemical characteristics of the strains are given in Table 1Go and in the species descriptions.

Chemotaxonomic characteristics
Cell-wall analysis revealed that strain ON4T had a B-type peptidoglycan based upon D-ornithine. Strains ON4T and DSM 20419 had the major isoprenoid quinone MK-9 (about 95 %), with MK-8 as a minor component. The polar lipid patterns of both strains were composed of diphosphatidylglycerol, phosphatidylglycerol and one unknown minor glycolipid. The predominant cellular fatty acids detected in strains ON4T and DSM 20419 were anteiso-C15 : 0 (46·9 and 45 %), iso-C16 : 0 (33·2 and 23·3 %) and anteiso-C17 : 0 (9·7 and 19·4 %) (see Supplementary Table in IJSEM Online). The DNA G+C contents were 64·5 mol% for strain ON4T and 67·0 mol% for DSM 20419. The above characteristics are typical of the family Microbacteriaceae (Collins & Bradbury, 1992Go; Evtushenko & Takeuchi, 2003Go).

16S rDNA sequence analysis
Nearly the complete nucleotide sequence of the 16S rRNA gene (1430 nt) of isolate ON4T was determined. Phylogenetic analysis based on 16S rDNA sequences supported the affiliation of the strain to the family Microbacteriaceae. The strain formed a distinct lineage adjacent to ‘B. helvolum’ DSM 20419 (Fig. 1Go) when a different set of strains and different algorithms were used to generate the phylogenetic trees, although the stability of the branch was not confirmed by a high bootstrap resampling value (not shown). Pairwise sequence similarities calculated for 16S rDNA sequences in the region between positions 52 and 1459 (Escherichia coli numbering) revealed that ON4T exhibited the highest similarity to ‘B. helvolum and representatives of Curtobacterium flaccumfaciens (~95 %). The 16S rDNA sequence of ‘B. helvolum’ was most similar to the sequence of Curtobacterium luteum (95·4 %).



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Fig. 1. Phylogenetic relationships of the 16S rDNA sequences of strains ON4T and ‘B. helvolum’ DSM 20419 with related genera within the family Microbacteriaceae. Evolutionary distance matrices were calculated using the correction of Jukes & Cantor (1969)Go. The dendrogram was constructed using the neighbour-joining algorithm (Saitou & Nei, 1987Go), using a 50 % conservation filter (total positions compared, 1331). Multifurcations were introduced using the ARB package for those branches that did not show a stable position when different algorithms were used for the analysis, which corresponded to branches with low bootstrap values (1000 replicates were done). The 16S rDNA sequence of Arthrobacter globiformis DSM 20124T was used as the outgroup.

 
Taxonomic conclusions
The similarities of the 16S rDNA sequences of strains ON4T and B. helvolum’ DSM 20419 and each of these strains and other members of the Microbacteriaceae are within the range of 16S rRNA gene sequence similarities observed between those of the genera of the family. The organisms are also distinguished from most genera of the family by a combination of their menaquinone and peptidoglycan composition, which are salient chemotaxonomic markers of the genera belonging to the family Microbacteriaceae (Sasaki et al., 1998Go; Evtushenko & Takeuchi, 2003Go). Although strain ON4T is similar in these characteristics to Curtobacterium, it can be differentiated from this genus by positive oxidase reaction, a higher optimal temperature for growth, a lower G+C content and the specific nutritional pattern (Table 2Go). Strain DSM 20419 has the peptidoglycan and the major menaquinone typical of the genus Clavibacter (Sasaki et al., 1998Go). However, a distinctive polyamine pattern (Altenburger et al., 1997Go) along with some physiological properties clearly separate it from Clavibacter species. Thus, based on both molecular genetic and phenotypic data, it is proposed to classify the studied organisms in two novel genera and species, Gulosibacter molinativorax gen. nov., sp. nov. (type strain ON4T=DSM 13405T=LMG 21909T) and Pseudoclavibacter helvolus gen. nov., sp. nov. (type strain DSM 20419T=ATCC 13715T).


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Table 2. Differentiating characteristics of strains ON4T and DSM 20419T and related genera of the family Microbacteriaceae

Taxa: 1, strain ON4T; 2, strain DSM 20419T; 3, Curtobacterium; 4, Clavibacter. Data were obtained from Collins & Bradbury (1992)Go, Altenburger et al. (1997)Go, Sasaki et al. (1998)Go, Behrendt et al. (2002)Go and this study. Characteristics are scored as: +, positive; –, negative; NA, no data available; W, weak; d, variable characteristic among the genus. Abbreviations: DAP, 2,3-diaminopropane; CAD, cadaverine; SPD, spermidine; SPM, spermine.

 
Description of Gulosibacter gen. nov.
Gulosibacter (Gu.lo'si.bac.ter. L. adj. gulosus fond of titbits, dainty feeder, N.L. masc. n. bacter rod; N.L. masc. n. Gulosibacter rod fond of titbits).

Irregular rod-shaped cells; may tend to form short filaments and to branch. Gram-positive. Non-spore-forming. Aerobic. Catalase-positive. Chemo-organotrophic. The diamino acid of peptidoglycan is D-ornithine. The major respiratory quinone is menaquinone MK-9. Polar lipids are diphosphatidylglycerol, phosphatidylglycerol and an unknown glycolipid. The predominant fatty acids are 12-methyl-tetradecanoic acid (anteiso-C15 : 0), 14-methyl-pentadecanoic acid (iso-C16 : 0) and 14-methyl-hexadecanoic acid (anteiso-C17 : 0). The DNA G+C content is about 65 mol%. The type species is Gulosibacter molinativorax.

Description of Gulosibacter molinativorax sp. nov.
Gulosibacter molinativorax [mo.li.na'ti.vo.rax. N.L. n. molinas molinate (a herbicide); L. adj. vorax devouring, ravenous, voracious; N.L. masc. adj. molinativorax molinate-degrading].

Irregular rod-shaped cells, 0·8–1·0 µm long and 0·5–0·6 µm wide, with tendency to form short filaments and branching. Non-motile. Colonies grown on LA medium are white and about 1 mm in diameter after 48–72 h of growth. Gram-positive. Strictly aerobic. Oxidase test is positive. Mesophilic: growth occurs at 10–41 °C, with optimum growth at 35–37 °C. The pH growth range is 5·5–10·5. Maximal growth rate is observed in the presence of 1 % (w/v) NaCl; 8 % NaCl inhibits growth. Nitrate is reduced to nitrite. A few organic compounds, including putrescine, methyl pyruvate, p-hydroxyphenylacetic acid, adenosine, inosine, thymidine and uridine, are utilized. Growth occurs in mineral medium supplemented with the thiocarbamate herbicide molinate. The DNA G+C content is 64·5 mol%.

The type strain, ON4T (=DSM 13485T=LMG 21909T), was isolated from a mixture of contaminated soil and water collected from a site of effluent discharge of a molinate-producing chemical plant in southern Portugal.

Description of Pseudoclavibacter gen. nov.
Pseudoclavibacter (Pseu.do.cla'vi.bac.ter. Gr. adj. pseudes false; N.L. masc. n. Clavibacter a bacterial generic name; N.L. masc. n. Pseudoclavibacter false Clavibacter).

Forms rod-shaped cells. Non-spore-forming. Gram-positive. Aerobic, catalase-positive. Chemo-organotrophic; various organic compounds are used as carbon and energy sources, including sugars, alcohols, organic acids and nitrogenated bases. The peptidoglycan is B2{gamma} [L-DAB]–D-glu–D-DAB (Schleifer & Kandler, 1972Go; Sasaki et al., 1998Go). Menaquinone MK-9 is the major respiratory quinone. Polar lipids are diphosphatidylglycerol, phosphatidylglycerol and an unknown glycolipid. The predominant fatty acids are 12-methyl-tetradecanoic acid (anteiso-C15 : 0), 14-methyl-pentadecanoic acid (iso-C16 : 0) and 14-methyl-hexadecanoic acid (anteiso-C17 : 0). Major polyamines are 1,3-diaminopropane and cadaverine (Altenburger et al., 1997Go). The G+C content of DNA is about 67 mol%. The type species is Pseudoclavibacter helvolus.

Description of Pseudoclavibacter helvolus sp. nov.
Pseudoclavibacter helvolus (hel.vo'lus. L. adj. helvolus pale yellow, yellowish).

Irregular rod-shaped cells, 0·9–1·1 µm long and 0·4–0·5 µm wide. Non-motile. Colonies grown on LA medium are yellow and about 2–3 mm in diameter after 48–72 h of growth. Gram-positive. Strictly aerobic. Oxidase reaction is weakly positive. Mesophilic; growth occurs at 10–35 °C, with optimum growth at 28–30 °C. The pH growth range is 6–10. Maximal growth rate is observed in the presence of 1 % (w/v) NaCl; 8 % NaCl inhibits growth. Aesculin, L-alanine, {alpha}-D-glucose, D-fructose, {alpha}-D-lactose, D-maltose, sucrose, D-raffinose, N-acetyl-D-glucosamine, glycerol, D-mannitol, pyruvic acid, methyl pyruvate, Tween 80, adenosine, 2'-deoxyadenosine, inosine, thymidine and uridine are utilized. Lipases are produced. The DNA G+C content is 67 mol%.

The type strain is DSM 20419T (=ATCC 13715T), isolated from butter.


    ACKNOWLEDGEMENTS
 
This work was supported financially by the National Foundation for Science and Technology from Ministry of Science and Technology (project PRAXIS/P/BIO/12193/1998). Dr F. Hierro and Professor J. Lalucat are acknowledged for their help during electron microscopy observations.


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