| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Note |
1 Vakgroep BFM WE10V, Laboratorium voor Microbiologie, Universiteit Gent, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium
2 BCCM/LMG Bacteria Collection, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium
3 School of Biological and Biomedical Sciences, Glasgow Caledonian University, Cowcaddens Road, Glasgow G4 0BA, UK
Correspondence
Jeroen Heyrman
Jeroen.Heyrman{at}rug.ac.be
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
The EMBL accession numbers for the 16S rRNA gene sequences of Bacillus decolorationis strains LMG 19507T (almost complete) and LMG 21001 (partial) are AJ315075 and AJ316304.
A 16S rDNA-based phylogenetic tree showing the position of B. decolorationis sp. nov. in relation to closely related species, B. subtilis and the type species of related genera is available as supplementary material in IJSEM Online (http://ijs.sgmjournals.org).
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
). In cultivation studies of deteriorated mural and rock paintings, bacterial isolates have frequently been attributed to the genus Bacillus (Weirich, 1989; Karpovich-Tate & Rebrikova, 1990; Gonzalez et al., 1999; Heyrman et al., 1999; Saiz-Jimenez & Laiz, 2000). Other studies that have included DNA-based techniques to characterize the bacterial community involved in biodeterioration have shown that a substantial part of the bacterial population is made up of so far unknown species (Altenburger et al., 1996; Rölleke et al., 1996; Gurtner et al., 2000; Heyrman & Swings, 2001). In this paper, we report the characterization of a group of ten strains isolated from different damaged parts of two mural painting sites, the Servilia tomb of the Roman necropolis at Carmona (Spain) and the Saint-Catherine chapel of Castle Herberstein (Austria). The strains are attributed to a novel species in the genus Bacillus, Bacillus decolorationis sp. nov.
Strain designations and their origins are listed in Table 1. The isolation procedure followed was described previously by Heyrman et al. (1999). Isolates were grown on trypticase soy agar (TSA; BBL) at 28 °C for examination of cell morphology and spore formation. The temperature range for growth was tested on the same medium. Analysis of the fatty acid content of the cell wall was performed as described previously (Heyrman et al., 1999), starting from cells grown on TSA for 24 h at 28 °C. Purification of total genomic DNA for 16S rDNA sequencing and rep-PCR genomic fingerprinting was performed 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 and DNA was purified as in Logan et al. (2000). Rep-PCR genomic fingerprinting was performed with the (GTG)5 primer (Versalovic et al., 1994) applying the PCR conditions described by Rademaker & de Bruijn (1997). The resulting band patterns were digitized and grouped using Pearson's correlation coefficient with the BioNumerics 2.0 software (Applied Maths). Sequence analysis was performed as described previously by Heyrman & Swings (2001). The G+C content of DNA was determined by HPLC (Mesbah et al., 1989) using further specifications given by Logan et al. (2000). DNA–DNA hybridization was performed using a modification of the microplate method described by Ezaki et al. (1989), as described by Willems et al. (2001). A hybridization temperature of 37 °C was used. 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. Suspension media for API 20E and API 50CH were supplemented with 7 % (w/v) NaCl. Skim milk agar, used when testing for casein hydrolysis, was not supplemented with 7 % (w/v) NaCl.
|
). The cut-off level of the group was at 85 % Canberra metric similarity. The mean contents of the fatty acids of the ten strains as percentages of total fatty acid content (fatty acids representing less then 1 % of the total amount are omitted and standard deviations are given in parentheses) were: iso-C14 : 0, 2·95 (0·52); C14 : 0, 1·17 (0·24); iso-C15 : 0, 2·71 (1·05); anteiso-C15 : 0, 67·67 (3·70); C16 : 1
7c alcohol, 1·09 (0·29); iso-C16 : 0, 4·53 (0·48); summed feature C16 : 17c/iso-C15 : 0 2OH, 1·45 (0·17); C16 : 0, 1·92 (1·40); anteisoA-C17 : 1, 3·74 (1·08); anteiso-C17 : 0, 10·70 (2·87). All fatty acids occurred in all strains. The individual profiles were compared with the TSBA4.0 database using the Microbial Identification System (Microbial ID) and they resembled the profiles of Micrococcus varians and Arthrobacter protophormiae/ramosus most closely, though with low, and thus not taxonomically relevant, respective mean similarities of 0·28 and 0·26. Cell morphology studies contradicted a close relationship between the mural isolates and these taxa.
The rep-PCR fingerprinting of the isolates allowed determination of their genotypic diversity. Studies that have compared rep-PCR genomic fingerprint analysis with DNA–DNA relatedness studies have shown that the two techniques yield results that are in close agreement (Nick et al., 1999; Rademaker et al., 2000). These studies therefore suggest that rep-PCR fingerprinting can be used as a genomic screening method to differentiate at the species level and to select representatives for DNA–DNA reassociation experiments. The (GTG)5-PCR patterns revealed two clusters based on Pearson's correlation coefficient, comprising eight and two isolates (Fig. 1). Within the clusters, the (GTG)5 patterns were nearly identical and it can be stated that the isolates of the same clusters are genotypically closely related and probably belong to the same species. Remarkably, the clustering did not correspond to the geographical divergence of the isolates. Indeed, isolates from different mural painting sites or samples were found to have identical (GTG)5 patterns, while isolates from the same sample were also attributed to different (GTG)5 groups. One strain from each (GTG)5 cluster (LMG 19507T and LMG 21001) was selected for 16S rDNA sequencing and DNA–DNA hybridization.
|
), was sequenced (467 bp). The two sequences show 99·9 % similarity in the most variable part of the gene (they only differ in one base; a T in the sequence of LMG 19507T is C or T in the sequence of LMG 21001). A FASTA search (Pearson, 1994) indicated that the highest similarity for the LMG 19507T sequence is 95·4 %, to Bacillus pseudofirmus DSM 8715T (X76439), and sequence similarity to other species of the genus Bacillus was below 95 %. Although the 16S rDNA sequence similarities were fairly low, strains LMG 19507T and LMG 21001 clustered tightly within the genus Bacillus (a phylogenetic tree with the closest related Bacillus species, Bacillus subtilis and the type species of related genera is available as supplementary material in IJSEM Online at http://ijs.sgmjournals.org). These observations support a separate species rank for the isolates within this genus.
Since sequence similarities to entries in the EMBL database were well below 97 %, the level below which strains are generally attributed to separate taxa (Stackebrandt & Goebel, 1994), DNA–DNA relatedness needed to be verified only between the representatives of each (GTG)5 cluster. The reciprocal DNA–DNA relatedness values of 87·6 and 82·6 % between strains LMG 19507T and LMG 21001 assign the two strains and thus the two clusters to a single species. The G+C contents of strains LMG 19507T and LMG 21001 were respectively 39·8 and 40·0 mol%.
All of the 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. In the API 50CH tests, the wall-painting isolates gave weak reactions, even after protracted incubation. In the API 20E strip, with or without added salt, strains showed nitrate reduction; however, gelatin hydrolysis needed generous inocula for consistent results. Despite the inconclusive results in the API systems, characteristics were found that distinguish the mural painting isolates from the phylogenetically closest related Bacillus species, as determined by a FASTA search (Table 2). For example, the isolates do not show growth at pH 8 or 9, whereas the closest related Bacillus species are all alkaliphilic (Nielsen et al., 1995), they give a positive reaction for nitrate reduction and are able to utilize 5-keto-D-gluconate.
|
Description of Bacillus decolorationis sp. nov.
Bacillus decolorationis (de.co.lo.ra.ti.on'is. L. gen. n. decolorationis of discoloration).
Cells are motile, coccoid rods, 0·5–0·8 µm wide and 1·0–4·0 µm long, that occur singly, in pairs or short chains (Fig. 2) and show a variable Gram-stain reaction. Spores are produced slowly and in small numbers in culture; they are ellipsoidal, sometimes nearly spherical, central to subterminal and swell the sporangia slightly. Colonies on TSA are cream-coloured to beige, circular with a smooth to slightly irregular margin, low-convex with a glistening and rough surface. No growth in an anaerobic chamber. Oxidase- and catalase-positive. The temperature range for growth is 5–40 °C with optimal growth at 25–37 °C. The NaCl concentration for growth is 0–10 % (w/v), with an optimum of 4–7 % (w/v). Casein hydrolysis is positive within 4 days incubation. In the API 20E strip, conversion of nitrates to nitrite and dinitrogen is positive and gelatin hydrolysis occurs with or without added salt, but only with generous inocula. Reactions are negative for arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, citrate utilization, hydrogen sulphide production, urease, tryptophan deaminase, indole production and Voges–Proskauer. The ONPG reaction is negative without added NaCl and variable when supplemented with 7 % NaCl. Acid is produced weakly and without gas from the following carbohydrates in the API 50 CH gallery using the CHB suspension medium supplemented with 7 % NaCl: cellobiose, D-fructose, gentiobiose, D-glucose, 5-keto-D-gluconate, maltose, D-mannose, N-acetylglucosamine, ribose, salicin, sucrose and trehalose. Aesculin hydrolysis is positive with or without added NaCl. Results are variable amongst strains for acid production from arbutin, galactose, glycerol, lactose and D-mannitol. Acid is not produced from the following carbohydrates: adonitol, amygdalin, D- or L-arabinose, D- or L-arabitol, dulcitol, erythritol, D- or L-fucose, gluconate, glycogen, meso-inositol, inulin, 2-keto-D-gluconate, D-lyxose, melibiose, methyl 
-D-glucoside, methyl -D-mannoside, methyl xyloside, D-raffinose, rhamnose, sorbitol, L-sorbose, D- or L-xylose, D-melezitose, starch, D-tagatose, D-turanose or xylitol. The major fatty acid is anteiso-C15 : 0, present at about 68 %; anteiso-C17 : 0 accounts for about 11 % of the total. Other fatty acids present at at least 1 % are listed above. Isolated from mural paintings, discoloured by microbial growths. The G+C content is 39·8 mol% for the type strain, strain LMG 19507T (=DSM 14890T). In the variable characters listed above, the type strain is positive for ONPG, arbutin, glycerol and D-mannitol.
|
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Ciferri, O. (1999). Microbial degradation of paintings. Appl Environ Microbiol 65, 879–885.
Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.
Gonzalez, I., Laiz, L., Hermosin, B., Caballero, B., Incerti, C. & Saiz-Jimenez, C. (1999). Bacteria isolated from rock art paintings: the case of Atlanterra shelter (south Spain). J Microbiol Methods 36, 123–127.[CrossRef][Medline]
Goto, K., Omura, T., Hara, Y. & Sadaie, Y. (2000). Application of the partial 16S rDNA sequence as an index for rapid identification of species in the genus Bacillus. J Gen Appl Microbiol 46, 1–8.
Gurtner, C., Heyrman, J., Piñar, G., Lubitz, W., Swings, J. & Rölleke, S. (2000). Comparative analyses of the bacterial diversity on two different biodeteriorated wall paintings by DGGE and 16S rDNA sequence analysis. Int Biodeterior Biodegrad 46, 229–239.
Heyndrickx, M., Vauterin, L., Vandamme, P., Kersters, K. & De Vos, P. (1996). Applicability of combined amplified ribosomal DNA restriction analysis (ARDRA) patterns in bacterial phylogeny and taxonomy. J Microbiol Methods 26, 247–259.
Heyndrickx, M., Lebbe, L., Vancanneyt, M. & 7 other authors (1997). A polyphasic reassessment of the genus Aneurinibacillus, reclassification of Bacillus thermoaerophilus (Meier-Stauffer et al. 1996) as Aneurinibacillus thermoaerophilus comb. nov., and emended descriptions of A. aneurinilyticus corrig., A. migulanus, and A. thermoaerophilus. Int J Syst Bacteriol 47, 808–817.
Heyrman, J. & Swings, J. (2001). 16S rDNA sequence analysis of bacterial isolates from biodeteriorated mural paintings in the Servilia tomb (necropolis of Carmona, Seville, Spain). Syst Appl Microbiol 24, 417–422.[CrossRef][Medline]
Heyrman, J., Mergaert, J., Denys, R. & Swings, J. (1999). The use of fatty acid methyl ester analysis (FAME) for the identification of heterotrophic bacteria present on three mural paintings showing severe damage by microorganisms. FEMS Microbiol Lett 181, 55–62.[CrossRef][Medline]
Karpovich-Tate, N. & Rebrikova, N. L. (1990). Microbial communities on damaged frescoes and building materials in the cathedral of the nativity of the virgin in the Pafnutii-Borovskii monastery, Russia. Int Biodeterior 27, 281–296.
Logan, N. A. & Berkeley, R. C. W. (1984). Identification of Bacillus strains using the API system. J Gen Microbiol 130, 1871–1882.
Logan, N. A., Lebbe, L., Hoste, B. & 7 other authors (2000). Aerobic endospore-forming bacteria from geothermal environments in northern Victoria Land, Antarctica, and Candlemas Island, South Sandwich archipelago, with the proposal of Bacillus fumarioli sp. nov. Int J Syst Evol Microbiol 50, 1741–1753.
Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39, 159–167.
Nick, G., Jussila, M., Hoste, B., Niemi, R. M., Kaijalainen, S., de Lajudie, P., Gillis, M., de Bruijn, F. J. & Lindström., K. (1999). Rhizobia isolated from root nodules of tropical leguminous trees characterized using DNA-DNA dot-blot hybridisation and rep-PCR genomic fingerprinting. Syst Appl Microbiol 22, 287–299.
Nielsen, P., Fritze, D. & Priest, F. G. (1995). Phenetic diversity of alkaliphilic Bacillus strains: proposal for nine new species. Microbiology 141, 1745–1761.
Pearson, W. R. (1994). Rapid and sensitive sequence comparison with FAST and FASTA. Methods Enzymol 193, 63–98.
Rademaker, J. L. W. & de Bruijn, F. J. (1997). Characterization and classification of microbes by rep-PCR genomic fingerprinting and computer assisted pattern analysis. In DNA Markers: Protocols, Applications and Overviews, pp. 151–171. Edited by G. Gaetano-Anollés & P. M. Gresshoff. New York: Wiley.
Rademaker, J. L. W., Hoste, B., Louws, F. J., Kersters, K., Swings, J., Vauterin, L., Vauterin, P. & de Bruijn, F. J. (2000). Comparison of AFLP and rep-PCR genomic fingerprinting with DNA–DNA homology studies: Xanthomonas as a model system. Int J Syst Evol Microbiol 50, 665–677.[Abstract]
Rölleke, S., Muyzer, G., Wawer, C., Wanner, G. & Lubitz, W. (1996). Identification of bacteria in a biodegraded wall painting by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA. Appl Environ Microbiol 62, 2059–2065.[Abstract]
Saiz-Jimenez, C. & Laiz, L. (2000). Occurrence of halotolerant/halophilic bacterial communities in deteriorated monuments. Int Biodeterior Biodegrad 46, 319–326.[CrossRef]
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846–849.
Versalovic, J., Schneider, M., de Bruijn, F. J. & Lupksi, J. R. (1994). Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction. Methods Mol Cell Biol 5, 25–40.
Weirich, G. (1989). Untersuchungen über Mikroorganismen von Wandmalereien. Mater Org 24, 139–159.
Willems, A., Doignon-Bourcier, F., Goris, J., Coopman, R., de Lajudie, P., De Vos, P. & Gillis, M. (2001). DNA–DNA hybridization study of Bradyrhizobium strains. Int J Syst Evol Microbiol 51, 1315–1322.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  M. S. Borchert, P. Nielsen, I. Graeber, I. Kaesler, U. Szewzyk, T. Pape, G. Antranikian, and T. Schafer Bacillus plakortidis sp. nov. and Bacillus murimartini sp. nov., novel alkalitolerant members of rRNA group 6 Int J Syst Evol Microbiol, December 1, 2007; 57(12): 2888 - 2893. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. L. Brodie, T. Z. DeSantis, J. P. M. Parker, I. X. Zubietta, Y. M. Piceno, and G. L. Andersen Urban aerosols harbor diverse and dynamic bacterial populations PNAS, January 2, 2007; 104(1): 299 - 304. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-M. Lim, C. O. Jeon, and C.-J. Kim Bacillus taeanensis sp. nov., a halophilic Gram-positive bacterium from a solar saltern in Korea Int J Syst Evol Microbiol, December 1, 2006; 56(12): 2903 - 2908. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-M. Lim, C. O. Jeon, S.-M. Lee, J.-C. Lee, L.-H. Xu, C.-L. Jiang, and C.-J. Kim Bacillus salarius sp. nov., a halophilic, spore-forming bacterium isolated from a salt lake in China Int J Syst Evol Microbiol, February 1, 2006; 56(2): 373 - 377. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Wieser, H. Worliczek, P. Kampfer, and H.-J. Busse Bacillus herbersteinensis sp. nov. Int J Syst Evol Microbiol, September 1, 2005; 55(5): 2119 - 2123. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. N. Allan, L. Lebbe, J. Heyrman, P. De Vos, C. J. Buchanan, and N. A. Logan Brevibacillus levickii sp. nov. and Aneurinibacillus terranovensis sp. nov., two novel thermoacidophiles isolated from geothermal soils of northern Victoria Land, Antarctica Int J Syst Evol Microbiol, May 1, 2005; 55(3): 1039 - 1050. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-H. Yoon, I.-G. Kim, K. H. Kang, T.-K. Oh, and Y.-H. Park Bacillus hwajinpoensis sp. nov. and an unnamed Bacillus genomospecies, novel members of Bacillus rRNA group 6 isolated from sea water of the East Sea and the Yellow Sea in Korea Int J Syst Evol Microbiol, May 1, 2004; 54(3): 803 - 808. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
85 % of strains positive; V, 16–84 % positive; -, 
15 % positive; ND, not determined. All species grow in the presence of 5 % NaCl.