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1 Ministry of the Flemish Community, Centre for Agricultural Research, Department of Animal Product Quality, Brusselsesteenweg 370, 9090 Melle, Belgium
2 Glasgow Caledonian University, School of Biological and Biomedical Sciences, Cowcaddens Road, Glasgow G4 0BA, UK
3 Ghent University, Faculty of Sciences, Laboratory of Microbiology (WE10), K. L. Ledeganckstraat 35, 9000 Gent, Belgium
Correspondence
Patsy Scheldeman
P.Scheldeman{at}clo.fgov.be
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Published online ahead of print on 9 February 2004 as DOI 10.1099/ijs.0.63095-0.
The GenBank accession numbers for the 16S rRNA gene sequences of B. farraginis R-8039, R-6538, R-6540T and R-6915, B. fortis R-6514T and B. fordii R-7190T are AY443034–AY443039, respectively.
Details of DNA–DNA hybridization results and photomicrographs of sporangia and vegetative cells of the type strains of B. farraginis, B. fortis and B. fordii are available as supplementary material in IJSEM Online.
Present address: Center for Microbial Ecology, Michigan State University, 540 Plant & Soil Science Building, East Lansing, MI 48824-1325, USA. 
Present address: Department of Bacteriology and Immunology, Veterinary and Agrochemical Research Centre, VAR-CODA-CERVA, Groeselenberg 99, 1180 Ukkel, Belgium.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Griffiths & Philips, 1990). Aerobic spore-forming bacteria of the genus Bacillus and related genera are commonly present in raw milk (Waes, 1976; Crielly et al., 1994; McGuiggan et al., 2002). Owing to their natural resistance properties, spores can survive during food processing. After germination and outgrowth to high levels, they may cause spoilage and even outbreaks of food-borne illness. They have been responsible for adversely affecting the quality of pasteurized milk and milk products (Guirguis et al., 1983; Meer et al., 1991) and ultra heat-treated (UHT) products (Hammer et al., 1995). Highly heat resistant spores of Bacillus sporothermodurans were detected in UHT products in different countries both inside and outside Europe (Guillaume-Gentil et al., 2002).
The presence of B. sporothermodurans spores in feed concentrate, silage, soy and pulp was demonstrated for individual dairy farms (de Silva et al., 1998; Vaerewijck et al., 2001; Scheldeman et al., 2002). In order to prevent contamination of milk and dairy products with aerobic spore-forming bacteria, it is important to ensure that contamination of raw milk is minimized. To achieve this, the nature and origin of spores and in particular of spores with a potential high heat resistance in raw milk must be better understood. In a survey of dairy farms, several strains were isolated from raw milk, the milking apparatus and animal feeds after 30 min heat treatment at 100 °C, as a selection procedure for spore-forming bacteria with potential high heat resistance (Scheldeman et al., 2002).
In this study, we elucidated the taxonomic positions of 48 potentially highly heat-resistant isolates from different sources at dairy farms. In addition to molecular typing, we used extensive phenotypic characterization as well as 16S rRNA gene sequence determinations and whole-genome hybridization studies to group and to identify these strains.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. All strains were isolated after 30 min heat treatment at 100 °C of samples taken at dairy farms (Scheldeman et al., 2002). All isolates were grown on brain heart infusion (BHI) (Oxoid) supplemented with bacteriological agar no. 1 (Oxoid) and filter-sterilized vitamin B12 (1 mg l–1) (Sigma).
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. For DNA–DNA reassociation experiments, high-quality total genomic DNA was purified using a modification of the method described by Gevers et al. (2001), as outlined by De Clerck et al. (2004).
rep-PCR genomic fingerprinting.
The conditions for genomic fingerprinting by means of repetitive element sequence-based PCR (rep-PCR), directed against the REP (repetitive extragenic palindromic) element or the (GTG)5 repetitive sequence, were previously described by Herman et al. (1998). The (GTG)5 primer (Versalovic et al., 1994) was used under the following PCR conditions. Amplification reactions were performed in a final volume of 25 µl containing PCR buffer (10 mM Tris/HCl, pH 8·3, and 50 mM KCl) (Applied Biosystems), 1·5 mM MgCl2, 0·2 mM of each dNTP (Pharmacia), 0·3 µg primer (Isogen), 1 U Goldstar DNA polymerase (Eurogentec) and 25 ng DNA template. The PCR program (Versalovic et al., 1994) was run on a DNA thermal cycler (Perkin Elmer 9600). To avoid inter-PCR differences, all samples were included in one single PCR run.
The rep-PCR amplicons, obtained with the REP- and (GTG)5 primers, were separated on a 1·5 % LSI LE agarose gel (Life Science International) (20x25 cm) for 4 h at a constant voltage of 4 V cm–1 in 1x TBE (100 mM Tris/HCl, 100 mM boric acid, 2 mM EDTA, pH 8·0). The rep-PCR profiles were visualized after staining with ethidium bromide, followed by digital image capturing using a GelDoc 2000 camera (Bio-Rad). A mixture of molecular mass markers VIII, IX and X (Roche) was used as reference for intra- and inter-gel comparison. Numerical analysis of the resulting fingerprints was performed using the GELCOMPAR II version 2.0 software package (Applied Maths). The similarity among digitized profiles was calculated using Pearson's correlation coefficient and an average linkage (UPGMA) dendrogram was derived.
Whole-cell protein profiling.
Cells used for whole-cell protein profiling through SDS-PAGE analysis were obtained after 24 h of growth on NAG (nutrient agar supplemented with 1 % glucose, pH 7·4). SDS protein extracts were prepared and electrophoresed according to Pot et al. (1994). Data collection and interpretation were conducted as described by Vauterin & Vauterin (1992).
16S rDNA sequencing and phylogenetic analysis.
The 16S rRNA genes of strains R-6514T, R-6521, R-6538, R-6540T, R-6915 and R-7190T were amplified by PCR using conserved primers pA (5'-AGAGTTTGATCCTGGCTCAG-3') and pH (5'-AAGGAGGTGATCCAGCCGCA-3'). The PCR products were purified and subsequently sequenced using an ABI 310 Sequencer (Applied Biosystems) as previously described (Scheldeman et al., 2002). A combination of the primers given by Coenye et al. (1999) was used to obtain a continuous stretch of the 16S rRNA gene sequence. Sequence assembly was implemented using the GENEBASE software (Applied Maths). The BIONUMERICS 3.0 software package (Applied Maths) was used for construction of a phylogenetic tree based on the neighbour-joining method.
DNA–DNA reassociation experiments.
DNA–DNA hybridizations were performed at 37 °C with photobiotin-labelled probes in microplate wells according to the method of Ezaki et al. (1989), using an HTS7000 Bio Assay Reader (Perkin Elmer) for the fluorescence measurements as described in detail by Willems et al. (2001).
DNA G+C content.
The DNA base composition was determined by HPLC using further specifications given by Logan et al. (2000).
Fatty acid analysis.
The culture conditions for fatty acid methyl ester (FAME) analysis were as described by Scheldeman et al. (2002). The methods used for fatty acid extraction, methyl ester preparation and separation by gas chromatography were as described by Vancanneyt et al. (1996).
Physiological and morphological characterization.
Phenotypic tests were performed as described by Logan & Berkeley (1984) and Heyrman et al. (2004); other characters were determined and the data analysed numerically using the general similarity coefficient of Gower (SG) as described by Logan et al. (2000). For observations on sporangia, cells were grown on tryptic soy agar (TSA) supplemented with 5 mg MnSO4 l–1 at 30 °C. Tests for assimilation of substrates as the sole carbon source were performed following the methods described by Heyndrickx et al. (1997).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. Three clusters could be delineated with an internal similarity of at least 90 %. The 32 strains grouping together in cluster A (and accordingly denominated in Table 1) mainly originated from feed concentrate for dairy cattle. Clusters B and C each included seven isolates and showed 83 % mutual similarity. Cluster B comprised four feed concentrate isolates and one isolate from raw milk, from the milking apparatus (cluster) and from soy. The seven strains from cluster C were isolated from the milking equipment [namely, the filter cloth (two strains) and the cluster (two)], from feed concentrate (two) and from raw milk (one). Only two strains occupied distinct positions in the dendrogram, although their profiles closely resembled those of cluster A. R-6521 still showed 87 % similarity to cluster A, whereas R-6952 only showed 79 % similarity to this cluster, probably due to one single additional dense band in the lower molecular mass region (data not shown).
Molecular typing by means of (GTG)5- and REP-PCR revealed respectively four and five groups of profiles among the strains studied (Table 1). The seven strains included in (GTG)5 cluster A corresponded to those included in SDS-PAGE cluster C. The 32 strains in (GTG)5 cluster B largely matched those in SDS-PAGE cluster A, with the exception of R-6952, which took a single position in the SDS-PAGE clustering (see above). Although included in SDS-PAGE cluster A, strain R-6685 held a distinct position in the (GTG)5 clustering. The five strains included in (GTG)5 cluster C were also included in SDS-PAGE cluster B. The two remaining strains of SDS-PAGE cluster B, R-6684 and R-7426, were respectively included in (GTG)5 cluster D or distinctly located in the clustering. Strain R-6521, with a separate position in the SDS-PAGE clustering, grouped together with R-6684 in (GTG)5 cluster D. Finally, R-6685 did not cluster with any of the (GTG)5 groups. Numerical analysis of the REP-PCR data revealed five clusters and three separate strains. The 27 and five strains of, respectively, REP clusters A and B corresponded to the 32 strains in (GTG)5 cluster B, and hence largely to those in SDS-PAGE cluster A. Although REP clusters A and B showed a mutual similarity of only 32 %, some common bands in the profiles of these two groups could be observed (data not shown). REP cluster C consisted of five strains, which also grouped together in SDS-PAGE cluster B. The two remaining strains in SDS-PAGE cluster B, R-6696 and R-7795, were respectively included in REP cluster D or distinctly located in the dendrogram. Finally, the seven strains included in SDS-PAGE cluster C and (GTG)5 cluster A were grouped together in REP cluster E, except for R-7810, which was grouped in REP cluster D. Overall, there was a good agreement between the three typing methods used, resulting in three groups of strains for which at least two of the three typing techniques supported the overall pattern of the group. Group I comprised 32 isolates corresponding to those in (GTG)5 cluster B, group II of seven isolates comprised all those included in SDS-PAGE cluster B and group III included all isolates of SDS-PAGE cluster C (Table 1). Ultimately, two strains could not be considered to belong to these delineated groups on these criteria and were therefore considered as single strains.
16S rDNA-based phylogeny and DNA–DNA relatedness
To investigate the taxonomic position of the three groups of strains, 16S rDNA sequence analysis and genomic DNA–DNA reassociation studies were performed. For the latter study, one representative of each group was included. In addition, the internal relatedness of the large group I was evaluated by including two strains (R-6540T and R-8039). A reassociation value of 87 % indicated that R-6540T and R-8039 (group I) indeed belong to the same species. The DNA–DNA reassociation values between the representatives of groups I, II and III proved to be low (
32 %; for more details see supplementary table in IJSEM Online). As a result, the three groups delineated on the basis of SDS-PAGE, REP- and (GTG)5-PCR can be considered to represent three separate species. The DNA base compositions presented in Table 1 for strains R-6540T, R-8039, r-6514T and R-7190T are in accordance with the DNA–DNA reassociation values (supplementary table in IJSEM Online). Moreover, the G+C value for R-6521 further confirmed its exclusion from groups I and II.
16S rRNA gene sequences for four group I strains (R-6538, R-6540T, R-6915, R-8039) and strains R-6514T and R-7190T, representatives of respectively groups II and III, were determined in the present study. The lowest pairwise similarity found within the 16S rDNA sequences of the four group I strains was 98·8 %, for strains R-6540T and R-8039. All other values ranged between 99·0 and 99·8 % (data not shown). The 16S rDNA sequence similarity of R-6514T (group II) with the strains from group I ranged from 96·8 % with R-6540T to 97·5 % with R-6538. For group III strain R-7190T a 16S rDNA sequence similarity with group I ranging from 95·8 % with R-6540T to 97·1 % with R-6538 was observed. The 16S rDNA sequence similarity of R-6514T and R-7190T, representing groups II and III, was 97·9 %. These data indicate the close relationship among the three groups.
The highest similarity found by a FASTA search (Pearson & Lipman, 1988) for strain R-7190T (group III) was 99·3 % with the 16S rDNA of Bacillus sp. 112442 JS2 (GenBank accession no. AF071857). The latter strain was isolated from silage after heating samples for 60 min at 100 °C (de Silva et al., 1998), also suggesting the possible formation of very heat-resistant spores by this strain. For group I and II strains R-6540T and R-6514T, the FASTA search revealed 96·0 and 99·1 % sequence similarity, respectively, with the low G+C Gram-positive bacterium M52 (AB116130) isolated from compost. Both strains R-6540T and R-6514T furthermore showed 95·8 and 98·0 % sequence similarity, respectively, with the 16S rDNA of the previously mentioned Bacillus sp. 112442 JS2. The highest 16S rDNA sequence similarity with a recognized species found for strain R-6514T was only 94·9 % with Bacillus lentus NCIMB 8773T (AB021189) and 94·5 % with B. sporothermodurans M215T (U49078). The 16S rDNA sequence of strain R-6540T showed only 94·5 % similarity with B. lentus NCIMB 8773T (AB021189) and 94·5 % with Bacillus galactosidilyticus LMG 17892T (AJ535638). For the 16S rDNA sequence of R-7190T, only 94·3 % similarity with B. lentus NCIMB 8773T, 94·3 % similarity with Bacillus firmus IAM 12464T (D16268) and 94·1 % with B. sporothermodurans M215T (U49078) was observed. It can therefore be concluded that groups I, II and III, represented by R-6540T, R-6514T and R-7190T, constitute three novel species within the genus Bacillus. The phylogenetic positions of these three novel species are presented in Fig. 1, by means of a neighbour-joining tree with a selection of recognized Bacillus species. All groups (I, II and III) were clearly separated from the other Bacillus species included. The four strains comprising group I clearly appeared as a well-separated branch in the phylogenetic tree, whereas R-7190T showed a high relatedness to Bacillus sp. 112442 JS2, as discussed above. Group II strain R-6514T was located in the same deeper branching group.
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). Most of the group I strains showed a reactive and consistent profile in which amino acids and organic acids were utilized as sole carbon and energy sources, but very few carbohydrates were assimilated. Members of groups II and III also attacked amino acids and organic acids and very few carbohydrates, but their ranges of substrates utilized were narrower than for group I strains, their reactions were weaker and considerable between-strain variation was observed. As the three novel species are unreactive in the API 20E and 50CHB kits, this distinguishes them from their closest phylogenetic neighbours (e.g. B. lentus), and so a more extended diagnostic table is of no informative or practical value.
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). Strains forming group II merged at 90 % SG, but this cluster contained one of the atypical group I strains (R-7351), and the other atypical group I strain (R-6952) joined this cluster at 87·5 % SG. These atypical strains could be recognized as members of group I on the basis of molecular data, but their phenotypic profiles were narrower and weaker than the other group I strains. Strain R-6952, however, was also found to be atypical in its whole-cell protein profile. Strains belonging to group III showed even greater within-group variation and all but one of the strains (R-7810) formed two subclusters at 90 %; one of these subclusters merged with group II at 85 % and the other, along with the remaining ungrouped strain (R-7810), joined at only 82·5 %. The atypical group III strain R-7810 also showed a different REP pattern to other members of this group.
The mean fatty acid profiles for the Bacillus strains examined are listed in Table 3. A predominance of iso-C15 : 0 could be observed for each novel species. Group I strains could be differentiated from group II and III by means of a larger amount of the branched fatty acids anteiso-C15 : 0 and anteiso-C17 : 0 on the one hand and a smaller amount of the saturated fatty acids C14 : 0 and C16 : 0 on the other hand. Discrimination between group II and groups I and III was possible based on a larger amount of the saturated fatty acids C15 : 0 and C17 : 0 for group II strains. Finally, group III strains could be distinguished from groups I and II on the basis of a larger amount of the unsaturated fatty acids C16 : 1
11c and C18 : 19c and the branched fatty acids iso-C17 : 0 and iso-C17 : 010c. These data are in accordance with the previous findings that groups I, II and III represent separate novel species within the genus Bacillus.
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Description of Bacillus farraginis sp. nov.
Bacillus farraginis (far.ra.gin'is. L. gen. fem. n. farraginis from mixed fodder for cattle, referring to feed concentrate for dairy cattle as the principal isolation source).
Cells are long, straight, round-ended, motile, strictly aerobic, Gram-negative rods, occurring singly, in pairs or filaments. Cell diameter is 0·5–0·8 µm and cell length 1·2–4 µm. Spores are ellipsoidal and occur paracentrally or subterminally in occasionally slightly swollen sporangia. Colonies grown for 3 days at 30 °C on nutrient agar are cream coloured or translucent, slightly raised, with irregular margins and granular, glossy surfaces. Colony diameter is no greater than 1 mm. Good growth occurs at 30 and 45 °C and weak growth occurs at 20 °C. Some strains are capable of growth at pH 9 but none grows at pH 5. Growth is not inhibited by 7 % (w/v) NaCl. Hydrolysis of starch and casein is not observed within 7 days of incubation at 30 °C, and growth in casein agar is poor or negative. Catalase and oxidase are positive. All strains are unreactive in the API 20E and API 50CHB test kits. In the Biotype100 kit using the Biotype 2 medium, nearly all strains (>83 %) belonging to the species are able to use the following substrates as sole carbon sources: 4-aminobutyrate, 5-aminovalerate, D- and L-alanine, fumarate, L-glutamate, glutarate, L-histidine, 3-hydroxybutyrate, 2-oxoglutarate, D- and L-malate, DL-lactate, L-proline, putrescine, succinate, L-tryptophan and L-tyrosine. Many strains (>45 %) are able to use L-aspartate, dulcitol, m-hydroxybenzoate, malonate, D-mannitol, D-ribose and D-sorbitol. Other substrates are used less frequently (17–44 %): L-arabinose, D-galactose, gentisate, D-glucuronate, p-hydroxybenzoate, myo-inositol and 
-L-rhamnose. The following substrates are seldom or never used as sole carbon sources: cis- and trans-aconitate, adonitol, D- and L-arabitol, benzoate, betaine, caprate, caprylate, D-cellobiose, citrate, m-coumarate, erythritol, aesculin, ethanolamine, 
-D-fructose, -L-fucose, D-galacturonate, -gentiobiose, D-gluconate, D-glucosamine, -D-glucose, DL-glycerate, glycerol, histamine, hydroxyquinoline -glucuronide, itaconate, 2- and 5-keto-D-gluconate, -lactose, lactulose, D-lyxose, maltitol, maltose, maltotriose, D-mannose, -D-melibiose, D-melezitose, methyl -galactopyranoside, methyl -galactopyranoside, 3-methyl D-glucopyranose, methyl -D-glucopyranoside, methyl -D-glucopyranoside, mucate, N-acetyl D-glucosamine, palatinose, phenylacetate, 3-phenylpropionate, propionate, protocatechuate, quinate, D-raffinose, D-saccharate, sucrose, L-serine, L-sorbose, D-tagatose, L-, meso- and D-tartrate, D-trehalose, D-turanose, tricarballylate, trigonelline, tryptamine, D-xylose and xylitol.
The type strain, R-6540T (=MB 1885T=LMG 22081T=DSM 16013T), utilizes the following substrates as sole carbon sources: 4-aminobutyrate, 5-aminovalerate, D- and L-alanine, L-aspartate, dulcitol, fumarate, D-glucosamine, L-glutamate, glutarate, histamine, L-histidine, m-hydroxybenzoate, 3-hydroxybutyrate, 2-oxoglutarate, D- and L-malate, malonate, DL-lactate, L-proline, putrescine, D-ribose, D-sorbitol, succinate, meso-tartrate, L-tryptophan and L-tyrosine. The major cellular fatty acids (>5 % of total cellular fatty acids) are iso-C15 : 0, anteiso-C15 : 0, anteiso-C17 : 0, iso-C16 : 0 and C16 : 17c alcohol. The DNA G+C composition of the type strain is 43·7 mol%.
Description of Bacillus fortis sp. nov.
Bacillus fortis (for'tis. L. adj. fortis strong, referring to the fact that the strains were isolated after heat treatment for 30 min at 100 °C).
Cells are straight, round-ended, motile, strictly aerobic, Gram-negative rods, occurring singly or in pairs. Cell diameter is 0·6–0·8 µm and length 1·0–3·5 µm. Spores are oval and occur centrally or paracentrally in slightly swollen sporangia. Colonies grown on nutrient agar at 30 °C for 3 days are cream coloured, raised with entire margins and smooth, glossy surfaces. Their maximum diameter is 1 mm. Good growth occurs at 30 and 45 °C and weak growth occurs at 20 °C. Growth does not occur at pH 9 or 5. Growth is not inhibited by 7 % (w/v) NaCl. Hydrolysis of starch and casein is not observed within 7 days of incubation at 30 °C, and growth on casein agar is poor or negative. Catalase and oxidase are positive. All strains are unreactive in the API 20E and API 50CHB test kits. In the Biotype100 kit using the Biotype 2 medium, strains use L-tryptophan and L-histidine as sole carbon sources. Most strains (>50 %) use the following substrates as sole carbon sources: 4-aminobutyrate, 5-aminovalerate, aesculin, ethanolamine, glutarate, hydroxyquinoline -glucuronide, 2-oxoglutarate, DL-lactate, malonate, phenylacetate, L-proline, putrescine, D-ribose and L-tyrosine. Some strains (<50 %) use the following substrates: citrate, erythritol, D-gluconate, D-glucosamine, -D-glucose, L-glutamate, DL-glycerate, histamine, m-hydroxybenzoate, 2- and 5-keto-D-gluconate, L- and D-malate, -D-melibiose, protocatechuate, L-sorbose and L-tartrate. Only the type strain uses D-glucuronate and no strain utilizes the following substrates as sole carbon sources: trans- and cis-aconitate, adonitol, D- and L-alanine, L-arabinose, D- and L-arabitol, L-aspartate, betaine, benzoate, caprate, caprylate, D-cellobiose, m-coumarate, dulcitol, -D-fructose, -L-fucose, fumarate, D-galactose, D-galacturonate, -gentiobiose, gentisate, glycerol, p-hydroxybenzoate, 3-hydroxybutyrate, myo-inositol, itaconate, -lactose, lactulose, D-lyxose, maltitol, maltose, maltotriose, D-mannitol, D-mannose, D-melezitose, methyl -galactopyranoside, methyl -galactopyranoside, 3-methyl D-glucopyranose, methyl -D-glucopyranoside, methyl -D-glucopyranoside, mucate, N-acetyl D-glucosamine, palatinose, 3-phenylpropionate, propionate, quinate, D-raffinose, -L-rhamnose, D-saccharate, sucrose, L-serine, D-sorbitol, succinate, D-tagatose, D- and meso-tartrate, D-trehalose, tricarballylate, trigonelline, tryptamine, D-turanose, xylitol and D-xylose.
The type strain is R-6514T (=LMG 22079T=DSM 16012T). Where results are variable, the type strain uses the following substrates: aesculin, D-gluconate, D-glucosamine, D-glucuronate, DL-glycerate, hydroxyquinoline--glucuronide, 2-oxoglutarate, 2- and 5-keto-D-gluconate, DL-lactate, malonate, phenylacetate, protocatechuate, L-tartrate and L-tyrosine. The major cellular fatty acids (>5 % of total cellular fatty acids) are iso-C15 : 0, anteiso-C15 : 0, iso-C16 : 0, C15 : 0 and anteiso-C17 : 0. The DNA G+C content of the type strain is 44·3 mol%.
Description of Bacillus fordii sp. nov.
Bacillus fordii (for'di.i. N.L. gen. n. fordii named after W. W. Ford, an American microbiologist working on aerobic spore-forming bacteria at the beginning of the twentieth century).
Cells are long, straight, round-ended, motile, strictly aerobic, Gram-negative rods, occurring singly or in pairs. Cell diameter is 0·6–0·8 µm and length 1·6–3·5 µm. Spores are ellipsoidal and occur paracentrally or subterminally in, occasionally, slightly swollen sporangia. Colonies grown on nutrient agar at 30 °C for 3 days are cream coloured, raised, with entire margins and smooth glossy surfaces. Their maximum diameter is 2 mm. Good growth occurs at 30 and 45 °C and weak growth occurs at 20 °C. Growth occurs at pH 9 and some strains grow at pH 5. Growth is not inhibited by 7 % (w/v) NaCl. Hydrolysis of starch and casein is not observed within 7 days of incubation at 30 °C. Growth on casein agar is coloured faint pink. Catalase and oxidase are positive. All strains are unreactive in the API 20E and API 50CHB test kits. In the Biotype100 kit using the Biotype 2 medium, all strains show very good production of biomass using malonate and L-tyrosine as sole carbon sources. Variable results with good production of biomass are obtained for L-histidine, 2-oxoglutarate, glutarate, DL-lactate, 5-aminovalerate and L-tryptophan. All or most strains are capable of weak growth from the following carbon sources: aesculin, gentisate, protocatechuate, meso-tartrate, D-glucosamine, DL-glycerate, quinate, ethanolamine, D-glucuronate, p-hydroxybenzoate, hydroxyquinoline -glucuronide and 2- and 5-keto-D-gluconate. Growth occurs seldom and weakly from the following substrates: adonitol, L-alanine, L-arabinose, L-arabitol, benzoate, fumarate, D-galacturonate, D-gluconate, -D-glucose, histamine, 3-hydroxybutyrate, D-lyxose, D-malate, methyl -galactopyranoside, methyl -D-glucopyranoside, N-acetyl D-glucosamine, phenylacetate, propionate, putrescine, D-raffinose, D-ribose, D-saccharate, L-sorbose, succinate, D-tagatose, L-tartrate, tricarballylate, trigonelline and D-xylose. The following substrates are not used as sole carbon source: cis- and trans-aconitate, D-alanine, 4-aminobutyrate, D-arabitol, L-aspartate, betaine, caprate, caprylate, D-cellobiose, citrate, m-coumarate, dulcitol, erythritol, -D-fructose, -L-fucose, D-galactose, -gentiobiose, L-glutamate, glycerol, m-hydroxybenzoate, myo-inositol, itaconate, -lactose, lactulose, L-malate, maltitol, maltose, maltotriose, D-mannitol, D-mannose, -D-melibiose, D-melezitose, methyl -galactopyranoside, 3-methyl D-glucopyranoside, methyl -D-glucopyranoside, mucate, palatinose, 3-phenylpropionate, L-proline, -L-rhamnose, sucrose, L-serine, D-sorbitol, D-tartrate, D-trehalose, tryptamine, D-turanose and xylitol.
The type strain is R-7190T (=MB 1878T=LMG 22080T=DSM 16014T). Where results are variable, the type strain uses the following substrates: aesculin, 5-aminovalerate, L-arabinose, benzoate, ethanolamine, D-galacturonate, gentisate, D-glucuronate, glutarate, DL-glycerate, histamine, p-hydroxybenzoate, 3-hydroxybutyrate, hydroxyquinoline--glucuronide, 2- and 5-keto-D-gluconate, 2-oxoglutarate, DL-lactate, D-lyxose, N-acetyl D-glucosamine, phenylacetate, protocatechuate, putrescine, quinate, D-raffinose, meso-tartrate, tricarballylate, L-tryptophan and D-xylose. The major cellular fatty acids (>5 % of total cellular fatty acids) are iso-C15 : 0, anteiso-C15 : 0, anteiso-C17 : 0, C16 : 111c and iso-C17 : 0. The DNA G+C content of the type strain is 41·9 mol%.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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