![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Applied and Environmental Microbiology, June 2000, p. 2627-2630, Vol. 66, No. 6
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
One Species on the
Basis of Genetic Evidence
The Biotechnology Centre of Oslo, University of Oslo,1 and Department of Microbiology, Institute of Pharmacy,2 Blindern, 0349 Oslo, and Department of Bacteriology, National Institute of Public Health, Torshov, 0403 Oslo,3 Norway, and Toxines et Pathogenie Bacteriennes, URA 2172 CNRS, Institut Pasteur, 75724 Paris Cedex 15, France4
Received 28 December 1999/Accepted 19 March 2000
 |
ABSTRACT |
|---|
Top
Abstract
Text
References
|
|---|
Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis are members of the Bacillus cereus group of bacteria, demonstrating widely different phenotypes and pathological effects. B. anthracis causes the acute fatal disease anthrax and is a potential biological weapon due to its high toxicity. B. thuringiensis produces intracellular protein crystals toxic to a wide number of insect larvae and is the most commonly used biological pesticide worldwide. B. cereus is a probably ubiquitous soil bacterium and an opportunistic pathogen that is a common cause of food poisoning. In contrast to the differences in phenotypes, we show by multilocus enzyme electrophoresis and by sequence analysis of nine chromosomal genes that B. anthracis should be considered a lineage of B. cereus. This determination is not only a formal matter of taxonomy but may also have consequences with respect to virulence and the potential of horizontal gene transfer within the B. cereus group.
| |
TEXT |
|---|
|
Top
Abstract
Text
References
|
|---|
The spore-forming bacterium Bacillus anthracis is the cause of the acute and often lethal disease anthrax. It is therefore of concern as a possible agent in biological warfare. Virulent forms of B. anthracis harbor two plasmids, pXO1 of 181 kb and pXO2 of 93.5 kb (22), which recently have been completely sequenced (14). A sequencing project aimed at determining the total genome of a plasmid-cured strain of B. anthracis is also under way. The closest relatives of B. anthracis are the two species B. thuringiensis and B. cereus. B. thuringiensis is a very useful source of insecticidal toxins, often in the form of spore-containing preparations of crystal protein toxins that are spread from airplanes over fields. B. cereus is a ubiquitous soil bacterium and an opportunistic human pathogen, causing contamination problems in the dairy industry and paper mills. The only established difference between B. cereus and B. thuringiensis strains is the presence of genes coding for the insecticidal toxins, usually present on plasmids. If these plasmids are lost, B. thuringiensis can no longer be distinguished from B. cereus (22).
Multilocus enzyme electrophoresis (MEE) comparing the allozyme patterns of 10 to 20 housekeeping genes has for decades been used extensively in phylogenetic investigations of bacterial populations (20). We have previously employed MEE analysis to establish the relationships between 36 strains of B. cereus and B. thuringiensis, mostly from reference strain collections, and shown that the strains appear to belong to the same species (4). Analysis of B. cereus and B. thuringiensis strains isolated from soil demonstrated a very high diversity in multilocus genotypes, indicating that B. cereus and B. thuringiensis exhibit a low degree of clonality and that exchange of genetic material occurs frequently in their natural environment (9).
We present here evidence for a close similarity of the genomes of B. anthracis strains to those of B. thuringiensis and B. cereus strains, demonstrating that they should be considered as belonging to one and the same species. What distinguishes them functionally are mostly genes carried on plasmids. In view of their natural competence, horizontal spreading of plasmids may take place and has in fact been demonstrated for B. thuringiensis and B. cereus (6, 7, 19, 23). What may seem to be a minor problem of taxonomy may therefore have serious implications for virulence and pathogenicity.
Protein extracts of the isolates were electrophoresed on starch-gel, and selective enzyme staining was performed as described by Selander and coworkers (20). The 13 enzymes were assayed as previously described (9).
Oligonucleotide primers were selected on the basis of previously determined gene sequences from B. cereus ATCC 10987 (15) using Primer3 (S. Rozen and H. J. Skaletsky [http://www.genome.wi.mit.edu/genome_software/other/primer3.html]) and synthesized at the DNA Synthesis Laboratory, Biotechnology Centre of Oslo, Oslo, Norway. PCR was run for 40 cycles in a 50-µl volume using 0.8 mM each deoxynucleoside triphosphate, 0.4 µM each primer, 50 ng of genomic DNA, and 1 U of Dynazyme (Finnzymes Oy, Espoo, Finland). The appropriate annealing temperature was determined for each primer set.
PCR products were purified using a QIAquick purification kit (Qiagen, Hilden, Germany), after Seakem GTG (FMC) agarose gel electrophoresis (1× Tris-acetate-EDTA or 1× Tris-borate-EDTA running buffer), when necessary. Sequencing reactions were performed on an ALF sequencer (Pharmacia, Uppsala, Sweden) using fluorescein isothiocyanate-end-labeled oligonucleotide primers corresponding to the primers used in PCR, employing a Thermo Sequenase Cycle Sequencing kit (Vistra Systems, Amersham, Buckinghamshire, United Kingdom). DNA sequences were analyzed and assembled using GeneSkipper software (European Molecular Biology Laboratory, Heidelberg, Germany).
Preliminary sequence data of B. anthracis were obtained from The Institute for Genomic Research website (http://www.tigr.org).
In the present study we have analyzed 13 B. anthracis
strains using MEE by comparing the allozyme patterns of 13 enzyme loci to those of 227 B. cereus and B. thuringiensis
strains. The multilocus genotypes of all but one of the B. anthracis strains were identical and were, except in one locus for
which no enzymatic activity was detected, indistinguishable from the
genotype of the clone of B. cereus most frequently isolated
from patients (Fig. 1). The remaining B. anthracis
strain (Davis TE 702) differed from the other strains by presenting
distinct alleles at two enzyme loci and clustered at a genetic distance
of 0.23. B. thuringiensis subsp. thuringiensis
(HD2) from the Bacillus Genetic Stock Center, previously shown to be
closely related to B. cereus strains (4), was
also closely related to the B. anthracis cluster (Fig. 1). Ten B. cereus-like strains isolated from sites of anthrax
outbreaks were positive for the chromosomal marker Ba813
(17) but lacked the two plasmids which are necessary for
full virulence of B. anthracis (17). These
strains exhibited multilocus genotypes located within or near the
B. anthracis cluster (Fig. 1). Previous studies using other
techniques to analyze the relationships between B. anthracis
strains have all stated that B. anthracis is very homogenous
and perhaps the most monomorphic species so far identified, with the
relationship to B. cereus and B. thuringiensis
being more remote (2, 3, 8, 12). Our results confirm the genetic homogeneity of B. anthracis but demonstrate that its
apparent relatedness to B. cereus and B. thuringiensis is highly dependent on the choice of strains
studied.
|
We have further analyzed DNA sequences from nine genes to investigate
the genetic relationship between a more narrow selection of members of
the B. cereus group. A collection of gene loci were amplified by PCR and analyzed by direct DNA sequencing. Dendrograms were subsequently constructed using cluster analysis, based on pairwise
similarities of strains. The nine genes were selected from 86 previously sequenced genes from the B. cereus ATCC 10987 genome (15), and the genes were scattered on the chromosome (Fig. 2a). Four additional strains were
selected for the analysis: B. anthracis 7700 (5),
the B. cereus type strain ATCC 14579, B. thuringiensis subsp. kurstaki, which is widely used for
the preparation of biopesticides, and a B. cereus strain
isolated from a patient with periodontitis (10). Pairwise
similarities between the PCR-amplified nucleotide sequences were used
to construct distance matrices for phylogenetic analysis, based on
percentages of divergence between the sequences. By separate
examination of each gene locus, the DNA sequences were highly conserved
among the five strains, exhibiting between 92.2 and 99.6% pairwise
identity (Fig. 2b). The protein sequences were similarly conserved,
with only 25 differences among a total of 1,128 amino acid positions in
the nine deduced sequences and with 14 of the substitutions being
conservative (Table 1). The analysis
further showed that evolutionary relationships estimated on the basis
of the DNA sequence data correlated well with the results from the MEE
analysis, with B. anthracis 7700 grouping together with the
periodontal B. cereus isolate, and that the B. cereus type strain ATCC 14579 was most similar to B. thuringiensis subsp. kurstaki (Fig. 2b). B. subtilis 168, which exhibits an isoenzyme pattern too divergent
from that of the B. cereus group to be included in the
analysis of the MEE data (Fig. 1) (4), also formed the
outgroup in the sequence analysis (Fig. 2c). Similarly, the 86 putative
genes previously identified from B. cereus ATCC 10987 (15) were used to search the nonannotated DNA sequence set
representing a triple coverage of the B. anthracis genome,
available at The Institute for Genome Research website
(http://www.tigr.org/cgi-bin/BlastSearch/blast.cgi).
|
|
Putative orthologs were detected for 69 of the genes, while 17 genes were either not present in the B. anthracis strain or missed due to physical or sequence gaps in the preliminary data set. The sequence identities between the B. cereus ATCC 10987 and B. anthracis orthologs were high, averaging 96.5% at the amino acid level. DNA sequences were equally similar.
The results presented in this study clearly reveal that B. anthracis appears to be genetically indistinguishable from members of the B. cereus-B. thuringiensis group. The results
are in agreement with earlier results from DNA-DNA hybridization
analysis showing high identity among B. anthracis, B. cereus, and B. thuringiensis strains (11,
18). Furthermore, our results are in agreement with the view of
B. cereus as the more ancestral species, with many of the
strains belonging to the variants B. anthracis and B. thuringiensis encoding their most characteristic phenotypic properties from extrachromosomal DNA. Other characteristics that have
been used to differentiate B. anthracis from B. cereus and that may be chromosomally encoded, such as sensitivity
to 
-lactam antibiotics and lack of motility and hemolytic activity,
may be caused by differences in a single gene(s). For instance, 3 to 5% of B. anthracis strains are penicillin resistant
(16), which dismisses this as a characteristic feature of
the bacterium. Interestingly, PlcR, a transcriptional regulator of
putative extracellular virulence factors in B. cereus and
B. thuringiensis, is mutated and nonfunctional in B. anthracis strains (1). These mutations may thus be at least partly responsible for some of the features often associated with
B. anthracis, like the lack of lecithinase and hemolytic activity.
We have demonstrated that B. anthracis is genetically very closely related to some B. cereus and B. thuringiensis strains usually regarded as rather harmless and even beneficial. Horizontal transfer of plasmids may dramatically alter their phenotypes. It is, however, possible that for receiving and retaining the virulence plasmids of B. anthracis, additional genetic features of the chromosome are needed. Such factors remain to be elucidated.
| |
ACKNOWLEDGMENTS |
|---|
The work was supported by grants to A.-B.K. from The Norwegian Research Council and an EMBO short-term fellowship to E.H.
Preliminary sequence data of B. anthracis was obtained from The Institute for Genomic Research website at http://www.tigr.org. Sequencing of B. anthracis was accomplished with support from the Office of Naval Research. We thank J. Vaissaire (AFSSA, Maisons-Alfort, France) for providing Ba813-positive B. cereus strains.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: The Biotechnology Centre of Oslo, University of Oslo, P.O. Box 1125, Blindern, 0349 Oslo, Norway. Phone: 47-229-58460. Fax: 47-2269-4130. E-mail: annebko{at}biotek.uio.no.
| |
REFERENCES |
|---|
|
Top
Abstract
Text
References
|
|---|
| 1. | Agaisse, H., M. Gominet, O. Andreas, O. Kstad, A. B. Kolsto, and D. Lereclus. 1999. PlcR is a pleiotropic regulator of extracellular virulence factor gene expression in Bacillus thuringiensis. Mol. Microbiol. 32:1043-1053[CrossRef][Medline]. |
| 2. | Ash, C., J. A. Farrow, M. Dorsch, E. Stackebrandt, and M. D. Collins. 1991. Comparative analysis of Bacillus anthracis, Bacillus cereus, and related species on the basis of reverse transcriptase sequencing of 16S rRNA. Int. J. Syst. Bacteriol. 41:343-346[CrossRef][Medline]. |
| 3. | Bourque, S. N., J. R. Valero, M. C. Lavoie, and R. C. Levesque. 1995. Comparative analysis of the 16S to 23S ribosomal intergenic spacer sequences of Bacillus thuringiensis strains and subspecies and of closely related species. Appl. Environ. Microbiol. 61:1623-1626[Abstract]. (Erratum, 61:2811.) |
| 4. |
Carlson, C. R.,
D. Caugant, and A.-B. Kolstø.
1994.
Genotypic diversity among Bacillus cereus and Bacillus thuringiensis strains.
Appl. Environ. Microbiol.
60:1719-1725 |
| 5. | Cataldi, A., E. Labruyere, and M. Mock. 1990. Construction and characterization of a protective antigen-deficient Bacillus anthracis strain. Mol. Microbiol. 4:1111-1117[Medline]. |
| 6. | Felkner, I. C., and O. Wyss. 1964. A substance produced by competent Bacillus cereus 569 cells that affects transformability. Biochem. Biophys. Res. Commun. 16:94-99[CrossRef][Medline]. |
| 7. |
Gonzales, J. M. J.,
B. S. Brown, and B. C. Carlton.
1982.
Transfer of Bacillus thuringiensis plasmids coding for  -endotoxin among strains of Bacillus thuringiensis and Bacillus cereus.
Proc. Natl. Acad. Sci. USA
79:6951-6955 |
| 8. | Harrell, L. J., G. L. Andersen, and K. H. Wilson. 1995. Genetic variability of Bacillus anthracis and related species. J. Clin. Microbiol. 33:1847-1850[Abstract]. |
| 9. | Helgason, E., D. A. Caugant, M. M. Lecadet, Y. Chen, J. Mahillon, A. Lövgren, I. Hegna, K. Kvaloy, and A. B. Kolstø. 1998. Genetic diversity of Bacillus cereus/Bacillus thuringiensis isolates from natural sources. Curr. Microbiol. 37:80-87[CrossRef][Medline]. |
| 10. |
Helgason, E.,
D. A. Caugant,
I. Olsen, and A. B. Kolstø.
2000.
Genetic structure of population of Bacillus cereus and B. thuringiensis isolates associated with periodontitis and other human infections.
J. Clin. Microbiol.
38:1615-1622 |
| 11. | Kaneko, T., R. Nozaki, and K. Aizawa. 1978. Deoxyribonucleic acid relatedness between Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis. Microbiol. Immunol. 22:639-641[Medline]. |
| 12. |
Keim, P.,
A. Kalif,
J. Schupp,
K. Hill,
S. E. Travis,
K. Richmond,
D. M. Adair,
M. Hugh-Jones,
C. R. Kuske, and P. Jackson.
1997.
Molecular evolution and diversity in Bacillus anthracis as detected by amplified fragment length polymorphism markers.
J. Bacteriol.
179:818-824 |
| 13. |
Kumar, S.,
K. Tamura, and M. Nei.
1994.
MEGA: Molecular Evolutionary Genetics Analysis software for microcomputers.
Comput. Appl. Biosci.
10:189-191 |
| 14. | Okinaka, R., K. Cloud, O. Hampton, A. Hoffmaster, K. Hill, P. Keim, T. Koehler, G. Lamke, S. Kumano, D. Manter, Y. Martinez, D. Ricke, R. Svensson, and P. Jackson. 1999. Sequence, assembly and analysis of pX01 and pX02. J. Appl. Microbiol. 87:261-262[CrossRef][Medline]. |
| 15. | Økstad, O. A., I. Hegna, T. Lindbäck, A. L. Rishovd, and A. B. Kolstø. 1999. Genome organization is not conserved between Bacillus cereus and Bacillus subtilis. Microbiology 145:621-631[Abstract]. |
| 16. | Patra, G., P. Sylvestre, V. Ramisse, J. Therasse, and J. L. Guesdon. 1996. Isolation of a specific chromosomic DNA sequence of Bacillus anthracis and its possible use in diagnosis. FEMS Immunol. Med. Microbiol. 15:223-231[CrossRef][Medline]. |
| 17. |
Patra, G.,
J. Vaissaire,
M. Weber-Levy,
C. Le Doujet, and M. Mock.
1998.
Molecular characterization of Bacillus strains involved in outbreaks of anthrax in France in 1997.
J. Clin. Microbiol.
36:3412-3414 |
| 18. | Priest, F. G., D. A. Kaji, Y. B. Rosato, and V. P. Canhos. 1994. Characterization of Bacillus thuringiensis and related bacteria by ribosomal RNA gene restriction fragment length polymorphisms. Microbiology 140:1015-1022[Abstract]. |
| 19. | Sabelnikov, A. G., and L. V. Ulyashova. 1990. Plasmid transformation of Bacillus cereus on cellophane membranes. FEMS Microbiol. Lett. 72:123-126[CrossRef]. |
| 20. |
Selander, R. K.,
D. A. Caugant,
H. Ochman,
J. M. Musser,
M. N. Gilmour, and T. S. Whittam.
1986.
Methods of multilocus enzyme electrophoresis for bacterial population genetics and systematics.
Appl. Environ. Microbiol.
51:873-884 |
| 21. | Sneath, P. H. A., and R. R. Sokal. 1973. Numerical taxonomy: the principles of numerical classification. W. H. Freeman & Co., San Francisco, Calif. |
| 22. | Thorne, C. B. 1993. Bacillus anthracis, p. 113-124. In A. L. Sonenshein, J. A. Hoch, and R. Losick (ed.), Bacillus subtilis and other gram-positive bacteria. American Society for Microbiology, Washington, D.C. |
| 23. | Wilcks, A., N. Jayaswal, D. Lereclus, and L. Andrup. 1998. Characterization of plasmid pAW63, a second self-transmissible plasmid in Bacillus thuringiensis subsp. kurstaki HD73. Microbiology 144:1263-1270[Abstract]. |
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| J. Bacteriol. | Microbiol. Mol. Biol. Rev. | Eukaryot. Cell | All ASM Journals |
|---|
