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Applied and Environmental Microbiology, October 2001, p. 4891-4895, Vol. 67, No. 10
Institute of Microbiology and Genetics,
University of Vienna, A-1030 Vienna, Austria1;
Institute of Microbiology, Technical University of Denmark,
Lyngby, Denmark2; and Genalysis GmbH,
D-14943 Luckenwalde, Germany3
Received 6 April 2001/Accepted 16 July 2001
Several moderately halophilic gram-positive, spore-forming bacteria
have been isolated by conventional enrichment cultures from damaged
medieval wall paintings and building materials. Enrichment and
isolation were monitored by denaturing gradient gel electrophoresis and
fluorescent in situ hybridization. 16S ribosomal DNA analysis showed
that the bacteria are most closely related to Halobacillus litoralis. DNA-DNA reassociation experiments identified the
isolates as a population of hitherto unknown
Halobacillus species.
Recently, a number of
investigations based on molecular techniques (9, 22, 24,
25) as well as on cultivation techniques (10, 16, 17,
27) have demonstrated that objects of art and building materials
may be habitats for extremely salt tolerant and moderately halophilic
bacteria. In fact, this group of microorganisms has been detected by
molecular means on the mural paintings from the 14th century located in
the Catherine chapel of the castle of Herberstein (Styria, Austria)
(24, 25), the subject of this study. The aim of this work
was the selective isolation of halophilic bacteria from wall paintings
and building materials.
Two samples were collected from different areas delimited in squares of
around 20 cm2 from the wall paintings of the
chapel of the castle. Sample H1 was taken below the chancel's east
wall window, where a brownish biofilm was observed; sample H6 was taken
from the chancel's north wall from a zone of the painting with an
intense rosy discoloration. An additional sample (m7) was taken on the
outside of the chapel from a lime wall also showing an intense rosy
discoloration. All samples were collected with a sterile scalpel by
scraping off surface material and plaster to a depth of 1 to 3 mm.
Small amounts of samples were used immediately for cultivation.
Enrichment cultures were set up in 300-ml Erlenmeyer flasks containing
30 ml of M2 medium (20% [wt/vol] NaCl) (34) and
maintenance medium (MM) medium (10% [wt/vol] NaCl)
(31). To avoid fungal growth, media were supplemented with
50 µg of cycloheximide (Sigma) ml Genomic DNA from enrichment cultures was extracted according to the
protocol described by Ausubel et al. (1). Amplification of
16S ribosomal DNA (rDNA) using specific archaeal primers
(22) did not give any positive results, indicating that no
Archaea were present in the enrichment cultures. However,
16S rDNA amplification using specific eubacterial primers
(29) gave positive results. For DGGE analysis, 200-bp
fragments of the 16S rDNA were amplified using the forward primer 341f
GC (19) and the reverse primer 518r (19). PCR
and DGGE were performed as described by Schabereiter-Gurtner et al.
(29) and Muyzer et al. (19), respectively.
DGGE profiles revealed only one band, demonstrating the low microbial
diversity present in the enrichments (Fig.
1).
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.10.4891-4895.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Detection of Indigenous Halobacillus
Populations in Damaged Ancient Wall Paintings and Building
Materials: Molecular Monitoring and Cultivation
ABSTRACT
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1. All
enrichments were incubated aerobically at room temperature (22 ± 3°C) and at 37oC in a water bath using magnetic
stirring. Aliquots of the same enrichment cultures were used for
denaturing gradient gel electrophoresis (DGGE) analysis, whole-cell
hybridization, and isolation of bacteria by plating as described below.
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FIG. 1.
Enrichments of wall painting sample H6 monitored by DGGE
analysis using eubacterium-specific primers. Lanes: 1, enrichment from
sample H6 on M2 medium incubated at 30°C; 2, enrichment from sample
H6 on M2 medium incubated at 37°C; 3, H. litoralis DSM
10405T; M, marker (to allow gel-to-gel comparison of
different samples). The marker containing 16S rDNA PCR products derived
from 10 different bacterial species was designed as previously
described by Schabereiter-Gurtner et al. (29).
Enrichment samples were fixed and hybridized with the eubacterial probe
EUB338 (32) as described before (23). All
samples showed a high green autofluorescence probably due to
organic-inorganic autofluorescent particles or to cellular
interference, as has been reported by other authors (20).
However, this problem was overcome by hybridizing bacteria with the
probe EUB338 labeled with Cy5 (EUB338-Cy5), which fluoresces in
the far red range. No signal was detected without the use of the
specific dye, indicating no false results due to autofluorescence using
this fluorochrome (data not shown). Figure
2 shows the whole-cell hybridization experiment performed with enrichment cultures of sample H6 in M2 and MM
media. A change in cellular morphology of the enrichment cultures was
observed at different salt concentrations. In both enrichment media
long filaments giving specific hybridization with the eubacterial probe
EUB338 were observed. However, the average length of the filaments was
greater in 20% NaCl (Fig. 2A) than in 10% NaCl (Fig. 2B and C). Cell
discrimination directly on the original sample, H6, using the
eubacterial probe EUB338-Cy5 was attempted. However, it was not
possible to discriminate the cells in situ due to the high red
autofluorescence of the material. Additionally, it could be possible
that due to a slow growth activity of the cells in the sample material
their ribosomal contents are too low to allow detection using
fluorescent in situ hybridization (FISH).
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DGGE and FISH analyses are known to be useful tools for monitoring the microbial composition in enrichment cultures as well as for isolation of pure cultures from cocultures (12, 15, 28, 33).
For the isolation of detected microorganisms, 100-µl aliquots of enrichment cultures were plated onto solid versions of M2 and MM medium as well as on M2A medium (6). All media were incubated aerobically at room temperature and 37°C. After 3 weeks, four brick red-pigmented colonies (K3, K5, U5, and U6) were isolated from enrichment cultures of sample H6. Two different colony types were observed: small, circular colonies with an intense coloration in the center and larger colonies which were less intensely pigmented. The two colony types were separated, and pure cultures were designated K3-1, K3-2, K5-1, K5-2, U5-1, U5-2, U6-1, and U6-2. In addition, we isolated from samples H1 and m7 two yellow-to-brown-pigmented bacterial strains, I3 and I7, respectively. DGGE analysis was performed with all isolates, and the migrations of the DGGE bands were compared. The positions of the DGGE bands were identical for all isolates to the positions of DGGE bands obtained from the same enrichment cultures shown in Fig. 1 (data not shown). These results indicate that the isolated strains are phylogenetically related despite their different colony morphologies.
All isolated strains were able to grow in M2A or MM medium in the presence of 5 to 20% (wt/vol) NaCl. Optimum growth occurred in media containing 5 to 10% NaCl; no growth occurred in the absence of NaCl. In the standard bacteriological tests (30) all of the strains were catalase and oxidase positive and did not grow anaerobically, as tested by using an atmospheric generation system (Anaerogen; Oxoid). Cells were gram positive and rod shaped, and in stationary cultures single endospores were observed in the cells by phase-contrast microscopy (Leitz Diaplan). One strain of the eight red isolates (strain K3-1) was selected from the H6 enrichment culture, as well as strains I3 and I7 from H1 and m7 enrichment cultures, respectively, for further phylogenetic investigations.
For sequence analysis, 16S rDNA fragments were amplified according to
the method of Lane (18). Individual 16S rDNA fragments amplified from isolates K3-1, I3, and I7 (1,425 bp, 1,326 bp, and 1,322 bp, respectively) were purified and sequenced as previously described
(29). Sequences were compared with sequences of known microorganisms using the EMBL nucleotide sequence database. The FASTA
search option (21) for the EMBL database was used to
search for close evolutionary relatives. Comparison of 16S rDNA
sequences showed that the new strains are most closely related to the
genus Halobacillus (97.1 to 98.4% similarity). To verify
their phylogenetic positions, evolutionary distances were calculated
and a phylogenetic tree was constructed (Fig.
3). The sequence alignment was corrected manually and end gaps on the 5' and 3' ends, as well as positions of
ambiguous base pairs, were omitted from the analysis. Therefore, a
total of 1,298 bases were compared. Pairwise evolutionary similarities and distances (14) were computed by using the DNADIST
program in the phylogeny inference package PHYLIP (version 3.57c,
University of Washington, Seattle). The phylogenetic tree was
constructed by using the neighbor-joining method of Saitou and Nei
(26). To determine the statistical significance of
branching patterns, a bootstrap analysis (1,000 replicates) was
performed using the SEQBOOT program; subsequently a consensus tree was
produced by using the CONSENSE program.
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The high sequence similarity clearly indicates that the isolates are closely related phylogenetically but does not allow single-species differentiation (8). Therefore, a DNA-DNA hybridization experiment was performed by the Identification Service of the Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany. Genomic DNA from strains K3-1, I3, I7, and Halobacillus litoralis DSM 10405T was isolated by hydroxyapatite chromatography using the procedure of Cashion et al. (3). DNA-DNA hybridizations were determined spectrophotometrically, as described by De Ley et al. (5) with modifications (7, 11), using a Gilford System model 2600 spectrophotometer equipped with a Gilford model 2527-R thermoprogrammer and plotter. Renaturation rates were computed with the TRANSFER.BAS program (13). The levels of DNA relatedness were 23.5% between K3-1 and DSM 10405T, 23.7% between I3 and DSMT, and 25.0% between I7 and DSM 10405T. The DNA homology levels between the wall paintings and building material isolates were 42.7% between K3-1 and I3, 29.7% between I3 and I7, and 83.4% between K3-1 and I7. The level of DNA relatedness found between strains K3-1 and I7 clearly suggests that the two organisms are highly related genotypically. DNA reassociation values obtained with Halobacillus litoralis DSM10405T were otherwise clearly below the usual standard criterion of approximately 70%, which supports the idea that the new isolates represent two distinct taxa (species) within the genus Halobacillus.
Halobacillus species have been previously isolated from hypersaline sediments, Antarctic sea ice, and fermented foods (2, 4, 31). To our knowledge this is the first report on the isolation of Halobacillus species from historic buildings and monuments.
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ACKNOWLEDGMENTS |
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This work is part of the European Union-funded project "Novel molecular tools for the analysis of unknown microbial communities of mural paintings and their implementation into the conservation/restoration practise" (EU-ENV4-CT98-0705). The work of G.P. was supported by a European Community Marie Curie Training Grant (Contract BIO4-CT98-5057).
We thank Søren Molin for permission to use the facilities at the Technical University of Denmark (Lyngby).
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FOOTNOTES |
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* Corresponding author. Mailing address: Institute of Microbiology and Genetics, University of Vienna, Dr. Bohr-Gasse 9, A-1030 Vienna, Austria. Phone: 0043-1-4277 54675 or 0043-1-4277 54632. Fax: 0043-1-4277 54674. E-mail: upe{at}gem.univie.ac.at.
Present address: Department of Genetics, Faculty of Sciences,
University of Málaga, 29071-Málaga, Spain.
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REFERENCES |
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Abstract
Text
References
|
|---|
| 1. | Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.). 1991. Current protocols in molecular biology. John Wiley and Sons, Inc., New York, N.Y. |
| 2. | Bowman, J. P., S. A. McCammon, M. V. Brown, D. S. Nichols, and T. A. McMeekin. 1997. Diversity and association of psychrophilic bacteria in Antarctic sea ice. Appl. Environ. Microbiol. 63:3068-3078[Abstract]. |
| 3. | Cashion, P., M. A. Hodler-Franklin, J. McCully, and M. Franklin. 1977. A rapid method for base ratio determination of bacterial DNA. Anal. Biochem. 81:461-466[CrossRef][Medline]. |
| 4. | Chaiyanan, S., S. Chaiyanan, T. Maugel, A. Huq, F. T. Robb, and R. R. Colwell. 1999. Polyphasic taxonomy of a novel Halobacillus, Halobacillus thailandesis sp. nov. isolated from fish sauce. Syst. Appl. Microbiol. 22:360-365[Medline]. |
| 5. | De Ley, J., H. Cattoir, and A. Reynaerts. 1970. The quantitative measurement of DNA hybridization from renaturation rates. Eur. J. Biochem. 12:133-142[Medline]. |
| 6. |
Denner, E. B. M.,
T. J. McGenity,
H.-J. Busse,
W. D. Grant,
G. Wanner, and H. Stan-Lotter.
1994.
Halococcus salifodine sp. nov., an archaeal isolate from an Austrian salt mine.
Int. J. Syst. Bacteriol.
44:774-780 |
| 7. | Escara, J. F., and J. R. Hutton. 1980. Thermal stability and renaturation of DNA in dimethyl sulfoxide solutions: acceleration of renaturation rate. Biopolymers 19:1315-1327[CrossRef][Medline]. |
| 8. |
Fox, G. C.,
J. D. Wisotzkey, and P. Jurtshuk, Jr.
1992.
How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identity.
Int. J. Syst. Bacteriol.
42:166-170 |
| 9. | Gurtner, C., J. Heyrman, G. Piñar, W. Lubitz, J. Swings, and S. Rölleke. 2000. Comparative analyses of the bacterial diversity on two different biodeteriorated wall paintings by DGGE and 16S rDNA sequence analysis. Int. Biodeterior. Biodegradation 46:229-239. |
| 10. | Heyrman, J., J. Mergaert, R. Denys, and J. Swings. 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]. |
| 11. | Huss, V. A. R., H. Festl, and K. H. Schleifer. 1983. Studies on the spectrometric determination of DNA hybridization from renaturation rates. Syst. Appl. Microbiol. 4:184-192. |
| 12. |
Jackson, C. R.,
E. E. Roden, and P. F. Churchill.
1998.
Changes in bacterial species composition in enrichment cultures with various dilutions of inoculum as monitored by denaturing gradient gel electrophoresis.
Appl. Environ. Microbiol.
64:5046-5048 |
| 13. | Jahnke, K.-D. 1992. Basic computer program for evaluation of spectroscopic DNA renaturation data from Gilford System 2600 spectrometer on a PC/XT/AT type personal computer. J. Microbiol. Methods 15:61-73. |
| 14. | Jukes, T. H., and C. R. Cantor. 1969. Evolution of protein molecules, p. 21-132. In H. N. Munro (ed.), Mammalian protein metabolism. Academic Press, Inc., New York, N.Y. |
| 15. |
Kane, M. D.,
L. K. Poulsen, and D. A. Stahl.
1993.
Monitoring the enrichment and isolation of sulfate-reducing bacteria using oligonucleotide hybridization probes designed from environmentally derived 16S rRNA sequences.
Appl. Environ. Microbiol.
59:682-686 |
| 16. | Krumbein, W. E., V. Schostak, and K. Petersen. 1991. On novel halophilic and extremely halotolerant bacteria from environments near the North Sea coast and the Steinhuder Meer. Kiel. Meeresforsch. Sonderh. 8:173-177. |
| 17. | Laiz, L., D. Recio, B. Hermosin, and C. Saiz-Jimenez. 2000. Microbial communities in salt efflorescences, p. 77-88. In O. Ciferri, P. Tiano, and G. Mastromei (ed.), On microbes and art. Proceedings of an International Conference on Microbiology and Conservation (ICMC). Kluwer Academic/Plenum Publishers, New York, N.Y. |
| 18. | Lane, D. J. 1991. 16S/23S rRNA sequencing, p. 115-175. In E. Stackebrandt, and M. Goodfellow (ed.), Nucleic acid techniques in bacterial systematics. John Wiley & Sons, Chichester, United Kingdom. |
| 19. |
Muyzer, G.,
E. C. de Waal, and A. G. Uitterlinden.
1993.
Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA.
Appl. Environ. Microbiol.
59:695-700 |
| 20. | Muyzer, G., and N. B. Ramsing. 1995. Molecular methods to study the organization of microbial communities. Water Sci. Technol. 32:1-9. |
| 21. | Pearson, W. R. 1994. Rapid and sensitive sequence comparison with FAST and FASTA. Methods Enzymol. 183:63-98. |
| 22. | Piñar, G., C. Gurtner, W. Lubitz, and S. Rölleke. 2001. Identification of Archaea in objects of art by DGGE analysis and shotgun cloning. Methods Enzymol. 336:356-366[Medline]. |
| 23. |
Poulsen, L. K.,
G. Ballard, and D. A. Stahl.
1993.
Use of rRNA fluorescence in situ hybridization for measuring the activity of single cells in young and established biofilms.
Appl. Environ. Microbiol.
59:1354-1360 |
| 24. | Rölleke, S., G. Muyzer, C. Wawer, G. Wanner, and W. Lubitz. 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]. |
| 25. |
Rölleke, S.,
A. Witte,
G. Wanner, and W. Lubitz.
1998.
Medieval wall painting a habitat for archaea: identification of archaea by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified gene fragments coding 16S rRNA in a medieval wall painting.
Int. Biodeterior. Biodegradation
41:85-92.
|
| 26. | Saitou, N., and M. Nei. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406-425[Abstract]. |
| 27. | Saiz-Jimenez, C., and L. Laiz. 2000. Occurrence of halotolerant/halophilic bacterial communities in deteriorated monuments. Int. Biodeterior. Biodegradation 46:319-326[CrossRef]. |
| 28. | Santegoeds, C. M., S. C. Nold, and D. M. Ward. 1996. Denaturing gradient gel electrophoresis used to monitor the enrichment culture of aerobic chemoorganotrophic bacteria from a hot spring cyanobacterial mat. Appl. Environ. Microbiol. 62:3922-3928[Abstract]. |
| 29. | Schabereiter-Gurtner, C., G. Piñar, W. Lubitz, and S. Rölleke. 2001. An advanced molecular strategy to identify bacterial communities on art objects. J. Microbiol. Methods 45:77-87[CrossRef][Medline]. |
| 30. | Smibert, R. M., and N. R. Krieg. 1994. Phenotypic characterization, p. 607-654. In P. Gerhardt, R. G. E. Murray, W. A. Woods, and N. R. Krieg (ed.), Methods for general and molecular bacteriology. American Society for Microbiology, Washington, D.C. |
| 31. |
Spring, S.,
W. Ludwig,
M. C. Marquez,
A. Ventosa, and K.-H. Schleifer.
1996.
Halobacillus gen. nov., with descriptions of Halobacillus litoralis sp. nov. and Halobacillus trueperi sp. nov., and transfer of Sporosarcina halophila to Halobacillus halophilus comb. nov.
Int. J. Syst. Bacteriol.
46:492-496 |
| 32. | Stahl, D. A., R. Devereux, R. I. Amann, B. Flesher, C. Lin, and J. Stromley. 1989. Ribosomal RNA based studies of natural microbial diversity and ecology, p. 669-673. In T. Hattori, Y. Ishida, Y. Maruyama, R. Morita, and A. Uchida (ed.), Recent advances in microbial ecology. Japan Scientific Societies Press, Tokyo, Japan. |
| 33. | Teske, A., P. Sigalevich, Y. Cohen, and G. Muyzer. 1996. Molecular identification of bacteria from a coculture by denaturing gradient gel electrophoresis of 16S ribosomal DNA fragments as a tool for isolation in pure cultures. Appl. Environ. Microbiol. 62:4210-4215[Abstract]. |
| 34. | Tomlinson, G. A., and L. I. Hochstein. 1976. Halobacterium saccharovorum sp. nov., a carbohydrate-metabolizing, extremely halophilic bacterium. Can. J. Microbiol. 22:587-591[Medline]. |
Applied and Environmental Microbiology, October 2001, p. 4891-4895, Vol. 67, No. 10
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.10.4891-4895.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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