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1 Vakgroep BFM WE10V, Laboratorium voor Microbiologie, Universiteit Gent, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium
2 School of Biological and Biomedical Sciences, Glasgow Caledonian University, Cowcaddens Road, Glasgow G4 0BA, UK
3 Institut für Bakteriologie, Mykologie und Hygiene, Veterinärmedizinische Universität, A-1210 Wien, Austria
4 Institut für Mikrobiologie und Genetik, Universität Wien, A-1030 Wien, Austria
5 BCCM/LMG Bacteria Collection, Universiteit Gent, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium
Correspondence
Jeroen Heyrman
Jeroen.Heyrman{at}rug.ac.be
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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The EMBL accession numbers for the 16S rRNA gene sequences of V. carmonensis LMG 20964T, V. necropolis LMG 19488T and V. picturae LMG 19492T are respectively AJ316302, AJ315056 and AJ315060.
Photomicrographs of sporangia and vegetative cells of the type strains of the three novel species are available as supplementary material in IJSEM Online (http://ijs.sgmjournals.org).
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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), the reliable characterization of bacteria associated with such damage is too often lacking (Heyrman & Swings, 2001). To obtain a better insight into the microbial community involved in biodeterioration of mural paintings, three sites (including the necropolis at Carmona, Spain, and the Saint-Catherine chapel of the castle at Herberstein, Austria) were sampled and investigated as part of a European research project. In a previous study (Heyrman et al., 1999), 385 strains, isolated from the different mural painting sites and samples, were analysed by fatty acid methyl ester (FAME) GC. This resulted in a grouping of the strains based on their fatty acid profiles. Representatives of the different clusters of strains with similar profiles were studied further by 16S rDNA sequencing. Several of these representative strains showed highest sequence similarity to members of the genera Salibacillus (Wainø et al., 1999; Arahal et al., 2000) and Virgibacillus (Heyndrickx et al., 1998, 1999). These two genera comprise halotolerant (Virgibacillus) or halophilic (Salibacillus) species. Among the strains of the clusters that were attributed to these genera by study of representative strains, both halotolerant and halophilic isolates were present. This paper presents the further description of 13 strains that represent three novel species. In a clustering based on the 16S rDNA sequence data, these species have positions intermediate between the genera Virgibacillus and Salibacillus. In addition, the species show intermediate G+C contents and share phenotypic properties with both genera. Therefore, we propose to combine Virgibacillus and Salibacillus in a single genus, Virgibacillus, comprising Virgibacillus pantothenticus, Virgibacillus proomii, Virgibacillus salexigens comb. nov. and Virgibacillus marismortui comb. nov., as well as the three novel species Virgibacillus carmonensis sp. nov., Virgibacillus necropolis sp. nov. and Virgibacillus picturae sp. nov. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). All novel isolates presented in this study were obtained from media with 10 % salt added and were further subcultured on Marine agar (MA, Difco) at 28 °C. Strains were checked for purity, cell morphology and spore formation.
DNA preparation.
Total genomic DNA was purified for 16S rDNA sequencing and rep-PCR using a slight modification of the method of Pitcher et al. (1989), as described by Heyndrickx et al. (1996). For determination of the G+C content and DNA–DNA hybridization, approximately 1 g biomass was harvested from agar plates. DNA was purified by a combination of the protocols of Marmur (1961) and Pitcher et al. (1989), as described previously by Logan et al. (2000).
Rep-PCR genomic fingerprinting.
PCR was performed with the (GTG)5 primer (Versalovic et al., 1994) using the PCR conditions described previously by Rademaker & de Bruijn (1997). For each strain, 6 µl PCR product mixed with 2 µl loading buffer (Rademaker & de Bruijn, 1997) was electrophoresed in a 1·5 (w/v) agarose gel and TAE buffer (1·21 g Tris base l-1, 0·2 ml 0·5 M EDTA l-1, pH 8) for 15 h at a constant 55 V and 4 °C. The first and every sixth lane were loaded with 6 µl of the molecular ruler [45·5 % (v/v) 100 bp ruler (Bio-Rad), 36·5 % (v/v) 500 bp ruler (Bio-Rad) and 18 % (v/v) loading buffer]. After staining with ethidium bromide (0·5 µg ml-1), the patterns were digitalized and a Pearson correlation of the resulting band patterns was performed using the BioNumerics 2.0 software (Applied Maths).
16S rDNA sequencing and phylogenetic analysis.
Sequence analysis was performed as described previously by Heyrman & Swings (2001). For partial sequencing, two primers were used (reverse 358–339 and reverse 536–519; Heyrman & Swings, 2001) to obtain the first 400–500 bp of the 16S rRNA gene, which has been described as the hypervariable region for the genus Bacillus (Goto et al., 2000). A phylogenetic tree was constructed using BioNumerics 2.0 by applying the neighbour-joining method of Saitou & Nei (1987) on a multiple alignment similarity matrix. The stability of relationships was assessed by a bootstrap analysis of 1000 datasets.
G+C content and relative DNA–DNA binding.
The G+C content of DNA was determined by HPLC (Mesbah et al., 1989) using the further specifications given by Logan et al. (2000). DNA–DNA hybridization was performed using a modification of the microplate method of Ezaki et al. (1989), as described by Willems et al. (2001). A hybridization temperature of 40 °C was used.
Chemotaxonomic characterization.
GC analysis of FAMEs was performed starting from strains grown on MA for 24 h at 28 °C. A quantitative analysis of cellular fatty acid compositions was further performed by the GLC procedure as described previously (Mergaert et al., 1993). Computer analysis of the resulting profiles was performed as described by Heyrman et al. (1999). Isoprenoid quinones were extracted by the method of Tindall (1990) and analysed by HPLC as described by Altenburger et al. (1996). Extraction and analysis of polar lipids by two-dimensional TLC were performed according to Ventosa et al. (1993).
Phenotypic characterization.
Strains were grown and maintained on MA, on which colony morphology and temperature range were observed. Anaerobic growth (in an anaerobic chamber on MA) and catalase activity were recorded. The ability to grow at different added salt concentrations [0·5, 5, 10 and 25 % (w/v) NaCl] was tested with trypticase soy broth (BBL) as basal medium. Utilization of different sugars as sole carbon sources was analysed using Marine broth as a base and replacing the peptone component by 1 % (w/v) of each sugar tested. Growth was measured spectrophotometrically after 72 h of incubation at 28 °C and scored weakly positive or positive if the change in OD550 was greater then 0·2 or 0·4, respectively. Testing of salt tolerance and utilization of carbon sources was also performed for V. pantothenticus LMG 7129T, V. proomii LMG 12370T, Salibacillus salexigens LMG 21520T and Salibacillus marismortui LMG 18992T. Phenotypic characterization using the API 20E and 50CHB systems and the API Biotype 100 system followed the methods of Logan & Berkeley (1984) and Heyndrickx et al. (1997), respectively; all suspension media were supplemented with 7 % (w/v) NaCl. Skim-milk agar, used when testing for casein hydrolysis, was also supplemented with 7 % NaCl. Basal media for 5 % (w/v) horse-blood agar were prepared both with and without 7 % NaCl.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). This resulted in one group of four strains with very similar patterns (further denoted as V. carmonensis sp. nov.), an ungrouped strain (V. necropolis sp. nov. LMG 19488T) and a group of eight profiles with moderate similarities (V. picturae sp. nov.), of which strain LMG 19416 showed the most dissimilar pattern. The grouping as described is in good accordance with observations of colony morphology (on MA) and the growth characteristics of the strains in relation to added salt. V. carmonensis contains salt-dependent strains with pink colonies, whereas V. necropolis (strain LMG 19488T) and V. picturae have cream-coloured colonies that also grow on media without salt. The grouping only partly reflects the origin of the strains. Indeed, while V. carmonensis contains only strains from the sample taken at Carmona, V. picturae contains strains from both murals. In this latter taxon, the two strains originating from Carmona (LMG 19492T and LMG 20963) showed very similar (GTG)5-PCR patterns, while those originating from Herberstein had very diverse rep-PCR patterns.
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), have been sequenced, except for LMG 20967 and LMG 20968, which have not been subjected to sequence analysis because they share an almost identical (GTG)5-PCR pattern with LMG 20964T. The topology of the phylogenetic tree (Fig. 2) based on neighbour-joining, which also includes the closest related taxa as retrieved from the EMBL database by FASTA search (Pearson & Lipman, 1988), supports the grouping of the mural strains as obtained by (GTG)5-PCR. The V. carmonensis strains have highly similar 16S rDNA sequences (
99·8 %) and LMG 20964T, which is representative of this taxon, shows 99·4 % similarity to LMG 19488T (V. necropolis). As sequence similarities of V. carmonensis and V. necropolis to other taxa are below the 97 % species level (Stackebrandt & Goebel, 1994) (Table 1), the strains belonging to these taxa can be attributed to at least one novel species. All sequences of the V. picturae strains were also highly similar (99·9 %), except for strain LMG 19416, which also gave a somewhat aberrant pattern in rep-PCR. LMG 19416 has a mean sequence similarity of 99·3 % towards LMG 19492T and LMG 20959. Also, the V. picturae strains have less than 97 % similarity in rDNA sequence when compared with the closest neighbour taxa. At the supra-species level, both groups of strains obtained from mural paintings share the highest sequence similarity with species of Virgibacillus and Salibacillus. They both have the highest sequence similarity to the type strain of S. marismortui and then, in descending order, to those of V. pantothenticus, V. proomii and S. salexigens. In the tree, however, they are positioned at approximately equal distances from Virgibacillus and Salibacillus. Thus, the phylogenetic tree derived from 16S rDNA sequence analysis shows two groups with high sequence similarity that are distant enough from all other described species to represent at least two novel species and that form a monophyletic group together with the species of Virgibacillus and Salibacillus. Moreover, although S. salexigens and S. marismortui group together (Fig. 2), S. marismortui shows higher pairwise similarity to the Virgibacillus species (Table 1), which indicates that the two genera are not clearly separated on the basis of 16S rDNA sequence analysis. An additional strain in the sequence tree corresponds to Bacillus sp. 2-9-3, isolated from the inside of a halite crystal that has not recrystallized since being deposited at least 250 million years ago (Vreeland et al., 2000). This strain shows very high sequence similarity to S. marismortui (99·7 %) and DNA–DNA hybridization should be performed to ascertain its species status.
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), which corresponds with the low 16S rDNA sequence similarities found.
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compared V. pantothenticus and S. salexigens, the only species described at that time, and decided that the chemotaxonomic and phenotypic data were sufficiently different to assign these species to different genera. Later, Arahal et al. (2000) reclassified Bacillus marismortui as S. marismortui and distinguished the genus Salibacillus from Virgibacillus on the basis of G+C content, salt tolerance and other characteristics. Below, we compare the data available for the four existing Virgibacillus and Salibacillus species with those for the three novel taxa and evaluate the present classification at the generic level.
DNA base composition
When the G+C contents of the novel isolates (Table 2
) are compared with data in the literature for the type strains of V. pantothenticus (36·9 mol%; Fahmy et al., 1985; 38·3 mol%; Heyndrickx et al., 1998), V. proomii (37·0 mol%; Heyndrickx et al., 1999), S. salexigens (39·5 mol%; Garabito et al., 1997) and S. marismortui (40·7 mol%; Arahal et al., 1999), one can conclude that the range of G+C contents for these taxa is below 5 mol%. Furthermore, if other strains are included, there is even a clear overlap between Virgibacillus and Salibacillus, since Garabito et al. (1997) record a range of G+C contents of 36·3–39·5 mol% for different S. salexigens strains. The argument of Arahal et al. (2000) that the G+C content discriminates between Virgibacillus and Salibacillus is therefore overruled. Representatives of the novel species proposed here all fit well (G+C contents from 37·5 to 40·0 mol%; Table 2) within the range of G+C contents of Virgibacillus and Salibacillus.
Chemotaxonomic characteristics
On the basis of fatty acid analysis, the three novel species can only be distinguished from each other on the basis of their percentages of the major fatty acid, anteiso-C15 : 0, with mean percentages of 65·5, 71·5 and 59·0 % for V. carmonensis, V. necropolis and V. picturae, respectively (Table 3). V. picturae can be further distinguished from the other two taxa by larger amounts of iso-C14 : 0 and iso-C16 : 0, while V. necropolis has somewhat less C16 : 1
7c alcohol than the other two species. Further, the taxa can be distinguished from the Virgibacillus and Salibacillus species subjected to fatty acid analysis (Heyndrickx et al., 1999; Wainø et al., 1999) by smaller amounts of iso-C15 : 0. However, it must be emphasized that, while fatty acid analysis may be helpful in a polyphasic approach, it is not reliable on its own for identification of most taxa in the Bacillus sensu lato group (Kämpfer, 1994).
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studied the polar lipid pattern of V. pantothenticus DSM 26T and S. salexigens DSM 11483T and found that both species contain major amounts of diphosphatidyl glycerol and phosphatidyl glycerol. The major differences in their polar lipid profiles are the presence of moderate amounts of phosphatidyl ethanolamine and an unknown glycolipid in V. pantothenticus DSM 26T and the presence of moderate amounts of two phospholipids of unknown structure in S. salexigens DMS 11483T. The results of our polar lipid analyses of V. pantothenticus LMG 7129T and S. salexigens LMG 21520T were in good agreement with those reported by Wainø et al. (1999). The major difference observed in the polar lipid profiles was the lack of a glycolipid in V. pantothenticus LMG 7129T. This might be explained by experimental differences. While we used 
-naphthol for detection of glycolipids, Wainø et al. (1999) employed anisaldehyde/sulfuric acid, and the glycolipid might not be detectable with -naphthol. However, when the polar lipid patterns of all type strains of the genera Virgibacillus and Salibacillus and the most dissimilar strains of V. proomii (LMG 17369) and V. picturae (LMG 19416) are analysed and compared (Table 4), the above-described differences can be attributed to variation between species but not between genera. For example, phosphatidyl ethanolamine, absent in V. picturae and V. carmonensis and only visible as traces after ninhydrin treatment in the patterns of S. salexigens LMG 21520T and V. necropolis LMG 19488T, was present in moderate amounts in the patterns of V. pantothenticus LMG 7129T and S. marismortui LMG 18992T and in minor amounts in the pattern of V. proomii. The unknown polar lipid L2 was present in minor amounts in V. proomii, S. salexigens LMG 21520T and V. picturae, but absent in V. pantothenticus LMG 7129T, S. marismortui LMG 18992T, V. carmonensis LMG 20964T and V. necropolis LMG 19488T. These results indicate that, in this phylogenetic lineage, polar lipid profiles may be considered to be species- or strain-specific. Strains of V. proomii displayed identical profiles, whereas the two strains of V. picturae each displayed distinct profiles (Table 4). Species-specific polar lipid profiles were also reported recently for members of the genus Sphingomonas (Busse et al., 1999). The presence of diphosphatidyl glycerol and phosphatidyl glycerol as major polar lipids are common characteristics of the species investigated in this study, but they are not useful for differentiation from closely related genera including Halobacillus and Gracilibacillus, which are reported to share these characteristics. In order to evaluate the importance of polar lipid profiles for genus discrimination, more species of related bacilli must be analysed.
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). Minor compounds were MK-6 and MK-8. These results are in good agreement with those of the majority of other aerobic spore-forming taxa.
Phenotypic characterization
One of the characteristics that distinguishes between the genera Virgibacillus and Salibacillus is that strains attributed to the latter genus are not able to grow on media without added salt. Therefore, the salt-tolerance of the 13 novel isolates was tested, together with the type strains of the known Virgibacillus and Salibacillus species. The results demonstrate that, while strains may differ in their abilities to grow without added salt, they have similar salt concentrations for optimal growth. V. pantothenticus LMG 7129T, V. proomii LMG 12370T, V. necropolis and V. picturae all grew weakly in trypticase soy broth without added salt, while S. salexigens LMG 21520T, S. marismortui LMG 18992T and V. carmonensis did not grow without added salt. For all strains, growth was optimal at salt concentrations of 5–10 %. In addition to growth without salt, some phenotypic traits that distinguish V. pantothenticus and V. proomii from S. salexigens and S. marismortui (Wainø et al., 1999; Arahal et al., 2000) remain, e.g. anaerobic growth, hydrogen sulphide production (though a weak reaction was recorded for the type strain of V. pantothenticus by Heyndrickx et al., 1999), acid production from D-trehalose, galactose (though contradictory results were obtained in this study) and D-xylose (though no data are available for V. proomii) and hydrolysis of starch and Tween 80 (though no data are available for V. proomii). Certain of these characteristics were also tested for the three novel taxa (Table 4) and for some, they showed reactions that would attribute them to Virgibacillus, for others to Salibacillus. Overall, these phenotypic traits are not convincing enough to maintain two separate genera and combined with the genotypic and chemotaxonomic analyses discussed above, the unification of the two genera can be justified. Additional characteristics of the three novel taxa and the other species of the genus Virgibacillus and species of the genus Salibacillus are given in Table 5 and in the descriptions below. In the API 50CH tests, the wall-painting isolates gave very weak reactions, even after protracted incubation, and only the strains allocated to V. picturae showed profiles that were largely consistent and potentially useful diagnostically (Table 5); strains of V. carmonensis and the strain of V. necropolis were essentially unreactive in this kit. All of the wall-painting isolates grew in the API Biotype 100 tests, but they showed inconsistent results, and this kit could not be used for taxonomic or diagnostic purposes. Strains of V. carmonensis and V. necropolis did not grow on 5 % horse-blood agar supplemented with 7 % NaCl, but they did grow on the unsupplemented medium.
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), the validly published species of Salibacillus should thus be transferred to Virgibacillus as V. salexigens comb. nov. and V. marismortui comb. nov. In addition, three novel Virgibacillus species are described, all originating from damaged mural paintings: V. carmonensis sp. nov., V. necropolis sp. nov. and V. picturae sp. nov. The description of V. necropolis is based on data obtained for a single strain, LMG 19488T. We acknowledge that this is not ideal and that a better understanding of the diversity of this species must await the isolation of further strains.
Description of Virgibacillus carmonensis sp. nov.
Virgibacillus carmonensis (car.mo.nen'sis. N.L. adj. carmonensis of Carmona, referring to the mural paintings of the necropolis at Carmona, Spain, from where the strains were isolated).
Cells are motile, Gram-positive rods (0·5–0·7x2–7 µm), which mostly occur singly, sometimes in pairs and short chains. They bear ellipsoidal, sometimes nearly spherical, endospores that lie in subterminal positions in swollen sporangia (see supplementary material available in IJSEM Online at http://ijs.sgmjournals.org). After 24 h on MA, colonies are 0·5–1·0 mm in diameter, low-convex, circular with slightly irregular margins, smooth and transparent with larger colonies having a pink tint. After 2 days, the colonies turn bright pink and opaque. Strains do not grow in an anaerobic chamber at 37 °C and are catalase-positive. The temperature range for growth is 10–40 °C with optimal growth at 25–30 °C. No growth without added salt and optimal growth at NaCl concentrations of 5 and 10 %. In the API 20E kit, strains gave positive results for nitrate reduction and negative results for ONPG, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, citrate utilization, hydrogen sulphide production, urease, tryptophan deaminase, indole, Voges–Proskauer and gelatinase. Casein hydrolysis positive. No haemolysis on 5 % horse-blood agar and no growth on this medium when supplemented with 7 % NaCl. With the exception of very weak reactions for aesculin hydrolysis and acid production from 5-keto-D-gluconate, strains are unreactive in the API 50CHB gallery, even when the CHB suspension medium is supplemented with 7 % NaCl. The following sugars can be used as sole carbon sources: cellobiose (weak growth), D-melibiose, raffinose, sucrose and D-trehalose. No growth on D-arabinose, D-fructose, D-glucose, DL-lactose or D-xylose. The major cellular fatty acid is anteiso-C15 : 0, present at about 65 % of the total fatty acids, while anteiso-C17 : 0 accounts for about 10 % of the total fatty acids. The following fatty acids are present at 1 %: iso-C14 : 0, iso-C15 : 0, C16 : 17c alcohol, iso-C16 : 0 and summed feature iso-C17 : 1/anteiso-C17 : 1. The polar lipid pattern of the type strain contains predominant amounts of diphosphatidyl glycerol and phosphatidyl glycerol, minor amounts of three phospholipids and one polar lipid of unknown structure and trace amounts of two additional unknown phospholipids. The main menaquinone type is MK-7. The G+C content is 38·9 mol% for the type strain, strain LMG 20964T (=DSM 14868T).
Description of Virgibacillus necropolis sp. nov.
Virgibacillus necropolis (ne.cro'po.lis. L. adj. necropolis of the necropolis, referring to the mural paintings of the necropolis of Carmona, Spain, from where the type strain was isolated).
Cells are motile, Gram-positive rods (0·5–0·7x2–5 µm) and coccoid rods, which occur singly, in pairs or short chains (with the different cells positioned at an angle). They bear ellipsoidal endospores that lie in terminal or subterminal positions or centrally in coccoid cells, in swollen sporangia (see supplementary material available in IJSEM Online at http://ijs.sgmjournals.org). After 24 h on MA, colonies are 0·2–0·5 mm in diameter, low-convex, circular with entire margins, smooth, cream-coloured and slightly transparent (opaque after 2 days growth). Does not grow in an anaerobic chamber at 37 °C and is catalase-positive. The temperature range for growth is 10–40 °C with optimal growth at 25–35 °C. Weak growth without added salt and optimal growth at NaCl concentrations of 5 and 10 %. In the API 20E kit, gives a positive result for nitrate reduction, a very weak reaction for gelatinase and negative results for ONPG, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, citrate utilization, hydrogen sulphide production, urease, tryptophan deaminase, indole and Voges–Proskauer. Casein hydrolysis is positive. Partial haemolysis on 5 % horse-blood agar, but no growth on this medium when supplemented with 7 % NaCl. Generally unreactive in the API 50CHB gallery, using CHB suspension medium supplemented with 7 % NaCl, but very weak reactions that do not qualify as positive results are seen in the following tests: glycerol, ribose, D-glucose, D-fructose, D-mannose, N-acetylglucosamine, D-trehalose, D-tagatose and 5-keto-D-gluconate. The following sugars can be used as sole carbon sources: cellobiose, D-glucose, DL-lactose (weak growth), D-melibiose, raffinose, sucrose and D-trehalose. No growth on D-arabinose, D-fructose or D-xylose. The major cellular fatty acid is anteiso-C15 : 0, present at about 72 % of the total fatty acids, while anteiso-C17 : 0 accounts for about 10 % of the total fatty acids. The following fatty acids are present at 1 %: iso-C14 : 0, iso-C15 : 0, C16 : 17c alcohol, iso-C16 : 0 and summed feature iso-C17 : 1/anteiso-C17 : 1. The polar lipid pattern contains predominant amounts of diphosphatidyl glycerol and phosphatidyl glycerol, minor amounts of two phospholipids and one polar lipid of unknown structure, traces of three additional unknown phospholipids and traces of phosphatidyl ethanolamine after ninhydrin treatment. The main menaquinone type is MK-7. The G+C content is 37·3 mol% for the type strain, strain LMG 19488T (=DSM 14866T).
Description of Virgibacillus picturae sp. nov.
Virgibacillus picturae (pic.tu'rae. L. gen. n. picturae pertaining or belonging to a painting).
Cells are motile, Gram-positive rods (0·5–0·7x2–6 µm) that occur singly or in pairs. They bear ellipsoidal, sometimes nearly spherical, endospores that lie in terminal positions in swollen sporangia (see supplementary material available in IJSEM Online at http://ijs.sgmjournals.org). After 24 h on MA, colonies are 0·5–1 mm in diameter, low-convex, circular or spread out with entire margins, smooth, cream-coloured and slightly transparent at the edges (opaque after 2 days growth). Strains do not grow in an anaerobic chamber at 37 °C and are catalase-positive. The temperature range for growth is 5–40 °C with optimal growth at 25–35 °C. Weak growth without added salt and optimal growth at NaCl concentrations of 5 and 10 %. In the API 20E kit, gives positive results for ONPG and nitrate reduction, very weak or negative reaction for gelatinase and negative results for arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, citrate utilization, hydrogen sulphide production, urease, tryptophan deaminase, indole and Voges–Proskauer. Casein hydrolysis is weakly positive. Partial haemolysis on 5 % horse-blood agar and on this medium supplemented with 7 % NaCl, with faster growth on the latter medium. In the API 50CHB gallery, using CHB suspension medium supplemented with 7 % NaCl, gives weakly positive (sometimes moderately positive) results for aesculin hydrolysis and for acid production from N-acetylglucosamine, galactose, D-glucose, D-fructose, glycerol, D-mannose, mannitol and D-melibiose. Weak acid production varies between strains for the following substrates: D-cellobiose, maltose, lactose, D-trehalose, starch, glycogen, xylitol, gentiobiose and turanose. Growth on different sugars as sole carbon source only results in weakly positive results: raffinose can be used as a sole carbon source by all strains; DL-lactose, D-melibiose and D-trehalose can usually be used as sole carbon sources; use of sucrose varies between strains; D-glucose is not usually used as a sole carbon source; D-arabinose, cellobiose, D-fructose and D-xylose are not used. The major cellular fatty acid is anteiso-C15 : 0, present at about 60 % of the total fatty acids, while anteiso-C17 : 0 and iso-C14 : 0 respectively account for about 12 and 11 % of the total fatty acids. The following fatty acids are present at 1 %: iso-C15 : 0, C16 : 17c alcohol, iso-C16 : 0 and summed feature iso-C17 : 1/anteiso-C17 : 1. The polar lipid pattern, determined for the type strain, LMG 19492T (= DSM 14867T), and the most dissimilar strain, LMG 19416, contains predominant amounts of diphosphatidyl glycerol, moderate amounts of phosphatidyl glycerol, trace to moderate amounts of five phospholipids of unknown structure and minor amounts of two unknown polar lipids. Presence of three other phospholipids is variable. The main menaquinone type is MK-7. The G+C content is 39·5 mol% for the type strain.
Of the variable characters listed above, the type strain is positive for growth on D-glucose as a sole carbon source and negative for growth on D-melibiose and sucrose, negative for gelatinase in the API 20E kit and negative for acid production from D-cellobiose, lactose, D-trehalose, starch, glycogen, xylitol, gentiobiose and turanose.
Description of Virgibacillus salexigens (Garabito et al. 1997) Heyrman, Logan, Busse, Balcaen, Lebbe, Rodriguez-Diaz, Swings and De Vos comb. nov.
Basonyms: Bacillus salexigens Garabito et al. 1997; Salibacillus salexigens (Garabito et al. 1997) Wainø et al. 1999.
The description matches the original description given by Garabito et al. (1997) and emended by Wainø et al. (1999). The type strain is strain C-20MoT (=ATCC 700290T =DSM 11483T =CCM 4646T =LMG 21520T).
Description of Virgibacillus marismortui (Arahal et al. 1999) Heyrman, Logan, Busse, Balcaen, Lebbe, Rodriguez-Diaz, Swings and De Vos comb. nov.
Basonyms: Bacillus marismortui Arahal et al. 1999; Salibacillus marismortui (Arahal et al. 1999) Arahal et al. 2000.
The description matches that given by Arahal et al. (1999). Additional chemical characters found in this study are as follows. In the polar lipid profile, diphosphatidyl glycerol is the predominant compound. Phosphatidyl glycerol and phosphatidyl ethanolamine are present in moderate amounts and five phospholipids, one aminophospholipid and one polar lipid of unknown structure are present in minor amounts or traces. The main menaquinone type is MK-7. The type strain is strain 123T (=DSM 12325T =ATCC 700626T =CIP 105609T =CECT 5066T =LMG 18992T).
Emended description of the genus Virgibacillus Heyndrickx et al. 1998
Virgibacillus (Vir.gi.ba.cil'lus. L. n. virga a green twig, transf., a branch in a family tree; L. dim. n. bacillus from Bacillus, a genus of aerobic endospore-forming bacteria; N.L. n. Virgibacillus a branch of the genus Bacillus).
Cells are motile, Gram-positive rods (0·3–0·7x2–6 µm) that occur singly, in pairs or short chains or filaments. They bear oval to ellipsoidal endospores that lie in swollen sporangia. Colonies are small, circular, low-convex and slightly transparent to opaque. Members of the genus are catalase-positive. In the API 20E strip and in conventional tests, the Voges–Proskauer reaction is negative, indole is not produced, citrate is usually not used and nitrate reduction to nitrite is variable. Urease and hydrogen sulphide are usually not produced. Gelatin, aesculin and casein are usually hydrolysed. Growth is stimulated by 4–10 % NaCl. Growth may occur between 5 and 50 °C, with an optimum of about 28 or 37 °C. D-Raffinose and D-melibiose can be used as sole carbon sources; no growth on D-arabinose, D-fructose or D-xylose. The different members of the genus show a wide range of activities in routine phenotypic tests, and this may reflect undiscovered requirements for growth factors and/or special environmental conditions. The major fatty acid is anteiso-C15 : 0. The major polar lipids are diphosphatidyl glycerol and phosphatidyl glycerol. Five phospholipids and one polar lipid of unknown structure are present in all species of the genus. Presence of phosphatidyl ethanolamine and other lipids is variable. The main menaquinone type is MK-7, with minor to trace amounts of MK-6 and MK-8. In the species tested, the cell wall contains peptidoglycan of the meso-diaminopimelic acid type (Claus & Berkeley, 1986; Arahal et al., 1999). The G+C content is in the range 36–43 mol%. The type species is Virgibacillus pantothenticus.
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