Department of Biological Sciences, Center for Tropical Disease Research and Training, University of Notre Dame, 130 Galvin Life Science Center, Notre Dame, IN 46556, USA
Correspondence
Jeffrey S. Schorey
schorey.1{at}nd.edu
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ABSTRACT |
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Present address: Department of Medical Microbiology, Medical University of Gdansk, Do Studzienki 38, 80-227 Gdansk, Poland.
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INTRODUCTION |
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The genus Mycobacterium contains over 70 species of human and animal pathogens as well as non-pathogenic saprophytes. The major human pathogens are Mycobacterium tuberculosis, the causative agent of tuberculosis, Mycobacterium leprae, causing leprosy, and Mycobacterium avium, responsible for opportunistic infections, particularly in AIDS patients. It is estimated that M. tuberculosis infects up to one-third of the world's population and results in 3 million deaths annually (Snider, 1994).
In Mycobacterium HGT has not been clearly demonstrated. Several existing reports suggesting this process are based on circumstantial evidence involving similarity in sequences found in distantly related bacterial or even eukaryotic species (Gamieldien et al., 2002; Kinsella et al., 2003
; Le Dantec et al., 2001
; Poelarends et al., 2000
). In addition, population genetic studies indicate that, unlike other bacteria, mycobacteria seem not to exchange genetic material between individuals. Genome-wide multilocus analyses of M. tuberculosis and Mycobacterium bovis environmental samples consistently detected highly significant linkage disequilibrium, suggesting either extreme rarity or non-existence of recombination in natural settings (Smith et al., 2003
; Supply et al., 2003
). This notion is consistent with early unsuccessful attempts at mutagenesis via allelic exchange (homologous recombination of native gene with an inactivated copy) in these slow-growing mycobacteria. It has been suggested that the unusual structure of the Mycobacterium recA gene, which encodes a key protein involved in recombination, DNA repair and regulation of the SOS response (Walker, 1984
), may be responsible for inefficient homologous recombination in the M. tuberculosis complex (McFadden, 1996
). The recA gene in these mycobacteria and in M. leprae is interrupted by an in-frame ORF encoding an intein that is removed from a precursor protein by a protein-splicing reaction (Davis et al., 1992
, 1991
). However, subsequent experiments showed that the intein does not affect RecA protein function and the frequency of double cross-over homologous recombination events (Papavinasasundaram et al., 1998
). To date, gene knock-outs using allelic exchange have been successfully achieved in both fast-growing Mycobacterium smegmatis (Husson et al., 1990
) and slow-growing M. tuberculosis (Balasubramanian et al., 1996
) and M. bovis BCG (Aldovini et al., 1993
). Pavelka & Jacobs (1999)
demonstrated that the recombination frequencies in M. smegmatis, M. tuberculosis and M. bovis were similar, suggesting that the homologous recombination machinery in fast- and slow-growing mycobacteria functions with comparable efficiency.
In another study (Krzywinska et al., 2004) we performed a phylogenetic analysis of 13 M. avium strains using as markers two DNA fragments (a 926 bp fragment of the gtfB gene and a 2150 bp region spanning rtfA-mtfC genes; cf. Fig. 1
a) lying in each other's proximity within the gene cluster responsible for biosynthesis of glycopeptidolipids (GPLs). Trees inferred from each marker had significantly incongruent topologies due to a well supported alternative placement of M. avium strain 2151. Such incongruence strongly suggests HGT involving one of the markers between the studied strains (Ragan, 2001
). Here we present the results of further analysis of the available orthologous sequences of the GPL biosynthesis gene cluster from M. avium strains 2151, 104, 724 and A5, and show unequivocal evidence for HGT and homologous recombination in Mycobacterium.
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METHODS |
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Sequence analysis.
The sequences were aligned using CLUSTALX (Thompson et al., 1997). The putative recombinant and parental sequences were identified upon inspection of variable alignment sites retrieved using MEGA 2.1 (Kumar et al., 2001
). The putative recombinant sequence was subjected to a maximum-likelihood analysis using the program LARD developed by Holmes et al. (1999)
. The likelihood of the null hypothesis, H0, that there has been no recombination event and the likelihood of the hypothesis H1, that recombination took place, were assessed from a simple unrooted tree of a putative recombinant (strain 2151) and two parental sequences (strains 724 and 104). The likelihood ratio test was performed using the joint likelihood of the two trees on either side of the putative breakpoint and the likelihood of a single tree was calculated on the entire alignment. To assess whether H1 is a significantly better fit to the data, the likelihood ratio of experimental data was compared to a null distribution obtained by LARD analysis of 500 sequence datasets generated by a Monte Carlo simulation of clonal evolution using Seq-Gen (Rambaut & Grassly, 1997
).
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RESULTS AND DISCUSSION |
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Our recent phylogenetic analysis of M. avium strains using two closely linked markers, gtfB and rtfA-mtfC from the GPL biosynthesis gene cluster, unexpectedly revealed a potential horizontal transfer of one of the marker sequences between the strains belonging to different serotypes (Krzywinska et al., 2004). The notion of transfer was based on a well-supported alternative position of one of the strains (i.e. 2151) in the trees inferred from each marker individually. Inspection of the sequence substitution patterns from all studied strains revealed that within the rtfA-mtfC marker, strain 2151 shared identical sequence with strain 724, whereas within the gtfB gene fragment it was identical to five other strains, including 104, but substantially different from strain 724.
To further explore the transfer hypothesis and identify a potential sequence breakpoint, we retrieved and analysed all M. avium genomic sequences that exist in the publicly available databases which corresponded to the GPL cluster. These included sequences from strains 104, 724, 2151 and A5. We focused our study on the highly conserved 5' region of the GPL cluster stretching between the mtfB and gtfB genes (Krzywinska & Schorey, 2003). The alignment of the four 10 624 bp sequences was unambiguous. The pattern of the variable alignment positions within that region provided clear evidence for a mosaic structure within the strain 2151 genomic sequence (Fig. 1
). Its 5' half, containing the rtfA-mtfC marker, was identical to the sequence from strain 724. A potential breakpoint was located within the intergenic spacer between mtfC and mtfD genes, at which point the subsequent sequence of strain 2151 shared identity with strain 104. Remarkably, another potential breakpoint was identified at the end of the aligned region, within the gtfB gene. Downstream from the gtfB marker sequence, the pattern of distribution of nucleotide substitutions reverted to the one observed within the 5' half of the alignment. To substantiate our hypothesis of gene transfer we performed sequence analysis using the LARD maximum-likelihood method (Holmes et al., 1999
), in which the likelihoods of the null hypothesis, H0, (i.e. there has been no recombination event at the analysed regions) and of the alternative hypothesis, H1, were calculated. This method allowed us to identify the two breakpoint regions within the alignment, which were found to be before positions 6219 and 10340. Monte Carlo simulation used to assess whether H1 is a significantly better fit to the data (i.e. that the likelihood ratio of H1 to H0 is greater than we would expect by chance) revealed that the log likelihood ratio of the real data (recombination event involving region spanning positions 621910 340) was significantly greater than for any of the simulated dataset (73·4 vs maximum of 15·09). A mosaic structure of the aligned region with blocks of sequences having unambiguously different evolutionary affiliations and identification of both breakpoints allowed us to pinpoint with confidence an entire region horizontally transferred into strain 2151. The maintenance of sequence continuity (lack of indels) and the absence of mobile elements within both breakpoints clearly points to homologous recombination as a mechanism of the foreign DNA integration into the strain 2151 genome.
It is worth noting that within the recombination region an insertion sequence IS1245 was embedded, although the significance of this finding remains unknown. Insertion sequence (IS) elements are known to modify a bacterial genome by mediating mobility of DNA fragments within an individual. The widespread occurrence and high degree of similarity between IS elements in different Mycobacterium species suggest that they spread across members of the genus through horizontal transfer (Gordon et al., 1999; Howard et al., 2002
). However, evidence from the analysis of the M. tuberculosis H37Rv genome shows no apparent link between potential IS transfer and introduction of novel genes into the bacillus (Gordon et al., 1999
).
Correct identification of ancient HGT events is difficult and is associated with a high degree of uncertainty. Many of the reports claiming such ancient transfer based on sequence similarity or phylogenetic distribution of sequences are likely to be false positives, and at least one such case apparently concerns mycobacteria. It has been proposed that 19 genes found in the M. tuberculosis genome, and implicated to have a role in mycobacterial pathogenesis, are of eukaryotic origin (Gamieldien et al., 2002). However, this finding was shown to be an artifact of insufficient taxon sampling, when the genes were reanalysed in the context of a larger dataset (Kinsella & McInerney, 2003
). Recently Kinsella et al. (2003)
suggested that a close phylogenetic affiliation of genes involved in fatty acid biosynthesis in mycobacteria and proteobacteria, two distantly related groups of organisms, is a signature of HGT. Although plausible, considering a broad spectrum of taxa used for the analysis, their hypothesis leaves a tinge of suspicion, because the codon usage bias in the putatively transferred genes was typical of Mycobacterium genes. For this reason, ancient gene duplications and subsequent retention of sequences in unrelated lineages can serve as an alternative hypothesis explaining the pattern observed in that study.
Apparently, recent gene transfer has been implicated in the evolution of the catabolic pathway for the man-made chemical 1,2-dibromoethane, found in Mycobacterium sp. strain GP1 (Poelarends et al., 2000). This highly toxic compound is degraded by the haloalkane dehalogenase (dhaA) gene product. A highly conserved dhaA gene was also found in phylogenetically distant Pseudomonas pavonaceae and Rhodococcus rhodochrous (Kulakova et al., 1997
), organisms isolated, like Mycobacterium sp. strain GP1, from different strongly contaminated environmental samples. Remarkably, in Rhodococcus rhodochrous the dhA region was located on the autotransmissible plasmid pRTL1 (Kulakova et al., 1997
). Another case of potentially recent HGT was suggested by the sequence analysis of the linear plasmid pCLP from Mycobacterium celatum, which revealed loci with high nucleotide sequence identity to loci on the M. tuberculosis chromosome (Le Dantec et al., 2001
). Moreover, the acquisition of a plasmid encoding mycolactone, a key toxin responsible for skin tissue destruction, was likely to be a recent event, resulting in the emergence of Mycobacterium ulcerans as a causative agent of Buruli ulcer (Stinear et al., 2004
). Whereas the mycolactone gene cluster in M. ulcerans remained on a plasmid, the other two examples of presumably recent HGT implied incorporation of the transferred genes into the genome by either excisive-integrative recombination or integrase-dependent acquisition. Our study is the first to report naturally occurring transfer and incorporation by homologous recombination of the transferred DNA into the Mycobacterium chromosome. The transfer most likely occurred very recently, since the recombination event has not been obscured by subsequent nucleotide substitutions within the replaced region, as shown by its comparison with the parental sequence. Recent successful allelic exchange experiments confirmed the feasibility of homologous recombination in M. avium (Maslow et al., 2003
; Otero et al., 2003
; Krzywinska
& others, unpublished). In two of those studies a gene knock-out has been achieved in the rtfA and mtfD genes located within the GPL cluster (Maslow et al., 2003
; Krzywinska
& others, unpublished).
Several mechanisms have been proposed for the exchange of chromosomal DNA between bacteria by homologous recombination as a consequence of conjugation, transduction and transformation. However, there is little information regarding the significance of these mechanisms in Mycobacterium. It has been shown that mycobacterial plasmids can replicate within most Mycobacterium species, so they can theoretically be spread horizontally, promoting gene transfer between mycobacteria (Kirby et al., 2002; Le Dantec et al., 2001
). Several plasmids were found in the M. avium complex (MAC) (Hellyer et al., 1991
; Meissner & Falkinham, 1986
), among them plasmid pVT2, which is thought to be conjugative (Kirby et al., 2002
), and raises the possibility for conjugative transfer in MAC a mechanism known to occur between M. smegmatis strains (Parsons et al., 1998
) and between M. smegmatis and other bacterial species (Gormley & Davies, 1991
). The widespread occurrence of related plasmids from M. avium, Mycobacterium intracellulare and Mycobacterium scrofulaceum (Jucker & Falkinham, 1990
) suggests that the plasmids have the ability to transfer between hosts in the environment, where MAC strains are ubiquitous. Transfer can also occur via a transformation process. Spontaneous plasmid transformation was discovered in M. smegmatis (Bhatt et al., 2002
) and natural competence for transformation by chromosomal DNA was reported in M. avium (Tsukamura et al., 1960
). Moreover, DNA fragments can be transferred between bacterial cells in a transduction process mediated by bacteriophages, over 250 of which have been described from Mycobacterium (Hatfull & Jacobs, 1994
). Genomic characterization of 14 mycobacteriophages revealed within their genomes over 50 genes not associated with phage growth (Pedulla et al., 2003
). Intriguingly, two of these phages contained a close homologue of a gene found in M. leprae and M. tuberculosis, which encodes the antigen Lsr2, a strong stimulator of the immune response, hinting at a possible role of phages in mycobacterial virulence. It is not clear if mycobacteriophages transmit such genes to bacterial genomes (Pedulla et al., 2003
); however, experiments in which mycobacteriophages have been used to create systems for gene delivery in mycobacteria (Bardarov et al., 1997
) show the feasibility of such a process.
Within pathogenic species, recombination may have important ramifications regarding adaptation to host, response to control or tracing of strains for epidemiological purposes (Ochman, 2001). The presence of different gene delivery mechanisms and functional homologous recombination machinery in various Mycobacterium species raises the possibility of naturally occurring transfer and recombination not only in M. avium, but also in other members of the genus. Moreover, the evidence presented in this study may contribute to the understanding of the mechanisms of drug resistance development in Mycobacterium. In M. tuberculosis all antibiotic resistance is chromosomally mediated and results from point mutations in different genes (depending on the antibiotic), rather than from acquisition of new genetic elements encoding antibiotic-altering enzymes (Musser, 1995
). The rate of spontaneous mutations in vitro is low in M. tuberculosis, relative to the range noticed in other bacteria (David & Newman, 1971
). This contrasts with a high frequency of emergence of drug resistance in tuberculosis patients (Espinal et al., 2000
). Likewise, multiple drug resistance, which occurs in 13 % of total tuberculosis isolates, is inexplicably high. It has been shown that DnaE2 polymerase, which is responsible for inducible mutagenesis in M. tuberculosis, can contribute directly to the appearance of resistance in vivo (Boshoff et al., 2003
). Homologous recombination brings a new potential explanation for the generation of multiple drug resistance. Although HGT has not been shown to be involved in the generation of drug resistance, it is possible that the mutated allele conferring resistance to a given antibiotic in one Mycobacterium can be transferred to another. If this isolate, containing a different resistance phenotype, incorporated the mutated allele into its genome through homologous recombination, one could obtain a multidrug-resistant strain. Simultaneous infection of patients by two different strains of M. tuberculosis (Braden et al., 2001
) or M. avium (von Reyn et al., 1995
) also creates the opportunity for such a DNA exchange.
In summary, we have unequivocally shown naturally occurring homologous recombination within the GPL biosynthesis cluster of M. avium 2151. At present, it is not yet known how often recombination of highly selected loci contributes to pathogenesis or the generation of multidrug resistance in mycobacteria.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Balasubramanian, V., Pavelka, M. S. J., Bardarov, S. S., Martin, J., Weisbrod, T. R., McAdam, R. A., Bloom, B. R. & Jacobs, W. R., Jr (1996). Allelic exchange in Mycobacterium tuberculosis with long linear recombination substrates. J Bacteriol 178, 273279.[Abstract]
Bardarov, S., Kriakov, J., Carriere, C. & 7 other authors (1997). Conditionally replicating mycobacteriophages: a system for transposon delivery to Mycobacterium tuberculosis. Proc Natl Acad Sci U S A 94, 1096110966.
Bhatt, A., Kieser, H. M., Melton, R. E. & Kieser, T. (2002). Plasmid transfer from Streptomyces to Mycobacterium smegmatis by spontaneous transformation. Mol Microbiol 43, 135146.[Medline]
Boshoff, H. I., Reed, M. B. & Barry, C. E. (2003). DnaE2 polymerase contributes to in vivo survival and the emergence of drug resistance in Mycobacterium tuberculosis. Cell 113, 183193.[Medline]
Braden, C. R., Morlock, G. P., Woodley, C. L. & 7 other authors (2001). Simultaneous infection with multiple strains of Mycobacterium tuberculosis. Clin Infect Dis 33, 4247.[CrossRef]
David, H. L. & Newman, C. M. (1971). Some observations on the genetics of isoniazid resistance in the tubercle bacilli. Am Rev Respir Dis 104, 508515.[Medline]
Davis, E. O., Sedgwick, S. G. & Colston, M. J. (1991). Novel structure of the recA locus of Mycobacterium tuberculosis implies processing of the gene product. J Bacteriol 173, 56535662.[Medline]
Davis, E. O., Jenner, P. J., Brooks, P. C., Colston, M. J. & Sedgwick, S. G. (1992). Protein splicing in the maturation of M. tuberculosis RecA protein: a mechanism for tolerating a novel class of intervening sequence. Cell 71, 201210.[Medline]
Espinal, M. A., Kim, S. J., Suarez, P. G. & 7 other authors (2000). Standard short-course chemotherapy for drug-resistant tuberculosis: treatment outcomes in 6 countries. JAMA 283, 25372545.
Gamieldien, J., Ptitsyn, A. & Hide, W. (2002). Eukaryotic genes in Mycobacterium tuberculosis could have a role in pathogenesis and immunomodulation. Trends Genet 18, 58.[CrossRef][Medline]
Gordon, S. V., Heym, B., Parkhill, J., Barrell, B. & Cole, S. T. (1999). New insertion sequences and a novel repeated sequence in the genome of Mycobacterium tuberculosis H37Rv. Microbiology 145, 881892.[Abstract]
Gormley, E. P. & Davies, J. (1991). Transfer of plasmid RSF1010 by conjugation from Escherichia coli to Streptomyces lividans and Mycobacterium smegmatis. J Bacteriol 173, 67056708.[Medline]
Hatfull, G. F. & Jacobs, W. R. J. (1994). Mycobacteriophages: cornerstones of mycobacterial research. In Tuberculosis: Pathogenesis, Protection and Control, pp. 165183. Edited by B. R. Bloom. Washington, DC: American Society for Microbiology.
Hellyer, T. J., Brown, I. N., Dale, J. W. & Easmon, C. S. (1991). Plasmid analysis of Mycobacterium avium-intracellulare (MAI) isolated in the United Kingdom from patients with and without AIDS. J Med Microbiol 34, 225231.[Abstract]
Holmes, E. C., Worobey, M. & Rambaut, A. (1999). Phylogenetic evidence for recombination in dengue virus. Mol Biol Evol 16, 405409.[Abstract]
Howard, S. T., Byrd, T. F. & Lyons, C. R. (2002). A polymorphic region in Mycobacterium abscessus contains a novel insertion sequence element. Microbiology 148, 29872996.
Husson, R. N., James, B. E. & Young, R. A. (1990). Gene replacement and expression of foreign DNA in mycobacteria. J Bacteriol 172, 519524.[Medline]
Jucker, M. T. & Falkinham, J. O. (1990). Epidemiology of infection by nontuberculous mycobacteria IX. Evidence for two DNA homology groups among small plasmids in Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum. Am Rev Respir Dis 142, 858862.[Medline]
Kinsella, R. J. & McInerney, J. O. (2003). Eukaryotic genes in Mycobacterium tuberculosis? Possible alternative explanations. Trends Genet 19, 687689.[CrossRef][Medline]
Kinsella, R. J., Fitzpatrick, D. A., Creevey, C. J. & McInerney, J. O. (2003). Fatty acid biosynthesis in Mycobacterium tuberculosis: lateral gene transfer, adaptive evolution, and gene duplication. Proc Natl Acad Sci U S A 100, 1032010325.
Kirby, C., Waring, A., Griffin, T. J. & Falkinham, J. O. (2002). Cryptic plasmids of Mycobacterium avium: Tn552 to the rescue. Mol Microbiol 43, 173186.[CrossRef][Medline]
Krzywinska, E. & Schorey, J. S. (2003). Characterization of genetic differences between Mycobacterium avium subsp. avium strains of diverse virulence with a focus on the glycopeptidolipid biosynthesis cluster. Vet Microbiol 91, 249264.[CrossRef][Medline]
Krzywinska, E., Krzywinski, J. & Schorey, J. S. (2004). Phylogeny of Mycobacterium avium strains inferred from glycopeptidolipid biosynthesis pathway genes. Microbiology 150, 16991706.
Kulakova, A. N., Larkin, M. J. & Kulakov, L. A. (1997). The plasmid-located haloalkane dehalogenase gene from Rhodococcus rhodochrous NCIMB13064. Microbiology 143, 109115.[Abstract]
Kumar, S., Tamura, K., Jakobsen, I. B. & Nei, M. (2001). MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17, 12441245.
Le Dantec, C., Winter, N., Gicquel, B., Vincent, V. & Picardeau, M. (2001). Genomic sequence and transcriptional analysis of a 23-kilobase mycobacterial linear plasmid: evidence for horizontal transfer and identification of plasmid maintenance systems. J Bacteriol 183, 21572164.
Maslow, J. N., Irani, V. R., Lee, S. H., Eckstein, T. M., Inamine, J. M. & Belisle, J. T. (2003). Biosynthetic specificity of the rhamnosyltransferase gene of Mycobacterium avium serovar 2 as determined by allelic exchange mutagenesis. Microbiology 149, 31933202.
McFadden, J. (1996). Recombination in mycobacteria. Mol Microbiol 21, 205211.[Medline]
Meissner, P. S. & Falkinham, J. O., 3rd (1986). Plasmid DNA profiles as epidemiological markers for clinical and environmental isolates of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum. J Infect Dis 153, 325331.[Medline]
Morschhauser, J., Kohler, G., Ziebuhr, W., Blum-Oehler, G., Dobrindt, U. & Hacker, J. (2000). Evolution of microbial pathogens. Philos Trans R Soc Lond B Biol Sci 355, 695704.[CrossRef][Medline]
Musser, J. M. (1995). Antimicrobial agent resistance in mycobacteria: molecular genetic insights. Clin Microbiol Rev 8, 496514.[Abstract]
Ochman, H. (2001). Lateral and oblique gene transfer. Curr Opin Genet Dev 11, 616619.[CrossRef][Medline]
Ochman, H., Lawrence, J. G. & Groisman, E. A. (2000). Lateral gene transfer and the nature of bacterial innovation. Nature 405, 299304.[CrossRef][Medline]
Otero, J., Jacobs, W. R. J. & Glickman, M. S. (2003). Efficient allelic exchange and transposon mutagenesis in Mycobacterium avium by specialized transduction. Appl Environ Microbiol 69, 50395044.
Papavinasasundaram, K. G., Colston, M. J. & Davis, E. O. (1998). Construction and complementation of a recA deletion mutant of Mycobacterium smegmatis reveals that the intein in Mycobacterium tuberculosis recA does not affect RecA function. Mol Microbiol 30, 525534.[CrossRef][Medline]
Parsons, L. M., Jankowski, C. S. & Derbyshire, K. M. (1998). Conjugal transfer of chromosomal DNA in Mycobacterium smegmatis. Mol Microbiol 28, 571582.[CrossRef][Medline]
Pavelka, M. S. J. & Jacobs, W. R. J. (1999). Comparison of the construction of unmarked deletion mutations in Mycobacterium smegmatis, Mycobacterium bovis bacillus Calmette-Guerin, and Mycobacterium tuberculosis H37Rv by allelic exchange. J Bacteriol 181, 47804789.
Pedulla, M. L., Ford, M. E., Houtz, J. M. & 17 other authors (2003). Origins of highly mosaic mycobacteriophage genomes. Cell 113, 171182.[Medline]
Poelarends, G. J., Kulakov, L. A., Larkin, M. J., van Hylckama Vlieg, J. E. & Janssen, D. B. (2000). Roles of horizontal gene transfer and gene integration in evolution of 1,3-dichloropropene- and 1,2-dibromoethane-degradative pathways. J Bacteriol 182, 21912199.
Ragan, M. A. (2001). Detection of lateral gene transfer among microbial genomes. Curr Opin Genet Dev 11, 620626.[CrossRef][Medline]
Rambaut, A. & Grassly, N. C. (1997). Seq-Gen: an application for the Monte Carlo simulation of DNA sequence evolution along phylogenetic trees. Comput Appl Biosci 13, 235238.[Abstract]
Smith, N. H., Dale, J., Inwald, J., Palmer, S., Gordon, S. V., Hewinson, R. G. & Smith, J. M. (2003). The population structure of Mycobacterium bovis in Great Britain: clonal expansion. Proc Natl Acad Sci U S A 100, 1527115275.
Snider, D. E. J. (1994). Global burden of tuberculosis. In Tuberculosis: Pathogenesis, Protection, and Control, pp. 359. Edited by B. R. Bloom. Washington DC: American Society for Microbiology.
Stinear, T. P., Mve-Obiang, A., Small, P. L. C. & 12 other authors (2004). Giant plasmid-encoded polyketide synthases produce the macrolide toxin of Mycobacterium ulcerans. Proc Natl Acad Sci U S A 101, 13451349.
Supply, P., Warren, R. M., Banuls, A. L. & 6 other authors (2003). Linkage disequilibrium between minisatellite loci supports clonal evolution of Mycobacterium tuberculosis in a high tuberculosis incidence area. Mol Microbiol 47, 529538.[CrossRef][Medline]
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 48764882.
Tsukamura, M., Hashimoto, H. & Noda, Y. (1960). Transformation of isoniazid and streptomycin resistance in Mycobacterium avium by desoxyribonucleate derived from isoniazid- and streptomycin-double-resistant cultures. Am Rev Respir Dis 81, 403406.[Medline]
von Reyn, C. F., Jacobs, N. J. R., Arbeit, D., Maslow, J. N. & Niemczyk, S. (1995). Polyclonal Mycobacterium avium infections in patients with AIDS: variations in antimicrobial susceptibilities of different strains of M. avium isolated from the same patient. J Clin Microbiol 33, 10081010.[Abstract]
Walker, G. C. (1984). Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli. Microbiol Rev 48, 6093.[Medline]
Weigel, L. M., Clewell, D. B., Gill, S. R. & 7 other authors (2003). Genetic analysis of a high-level vancomycin-resistant isolate of Staphylococcus aureus. Science 302, 15691571.
Received 9 February 2004;
revised 15 March 2004;
accepted 22 March 2004.
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