University of New Mexico School of Medicine, Dept of Internal Medicine, 915 Camino de Salud, Albuquerque, NM 87131, USA1
Department of Medicine, Albuquerque Veterans Affairs Medical Center, 1501 San Pedro SE, Albuquerque, NM 87108, USA2
UNM Health Science Center Cancer Research and Treatment Center, 900 Camino de Salud, Albuquerque, NM 87131, USA3
Author for correspondence: C. Richard Lyons. Tel: +1 505 272 4450. Fax: +1 505 272 9912. e-mail: clyons{at}salud.unm.edu
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: rapidly growing mycobacteria, insertion element, horizontal transfer, genetic variation
Abbreviations: DR, direct repeat; IR, inverted repeat; IS, insertion sequence; RGM, rapidly growing mycobacteria
b The GenBank accession number for the sequence reported in this paper is AF513500.
a Present address: Center for Pulmonary and Infectious Disease Control, The University of Texas Health Center at Tyler, Tyler, TX 75708-3154, USA.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Several species of RGM, particularly Mycobacterium abscessus, Mycobacterium chelonae and Mycobacterium fortuitum, are opportunistic pathogens and can cause infections ranging from localized abscesses to pulmonary and disseminated disease (Griffith et al., 1993 ; Howard & Byrd, 2000
; Wright & Wallace, 1995
). RGM-associated disease tends to be sporadic and usually associated with injury or surgical procedures (Wright & Wallace, 1995
). However, large outbreaks of post-operative wound infections (Chadha et al., 1998
) and post-injection abscesses (Galil et al., 1999
; Villaneuva et al., 1997
) have been attributed to contamination of medical reagents or instruments with M. abscessus. M. abscessus is also the leading cause of pulmonary disease due to RGM (Griffith et al., 1993
).
M. abscessus was originally classified as a subspecies of M. chelonae but has been reclassified as a distinct species on the basis of DNADNA hybridization and sequence analysis of the 16S rRNA gene (Kusunoki & Ezaki, 1992 ; Pitulle et al., 1992
). Some phylogenetic analyses place M. abscessus close to M. chelonae (Brown et al., 1999
; Domenech et al., 1997
), whereas others place it on an older branch of the phylogenetic tree (Pitulle et al., 1992
; Shojaei et al., 1997
). Epidemiological investigations utilizing RFLP analysis, PFGE or random amplified PCR (Villaneuva et al., 1997
; Wallace et al., 1993
; Zhang et al., 1997
) have indicated that there is genetic variation within M. abscessus but specific polymorphic regions have not been identified. Molecular characterization has focused primarily on genes that allow rapid speciation of isolates (Kim et al., 1999
; Ringuet et al., 1999
) and those with a role in drug resistance (Ainsa et al., 1998
; Guillemin et al., 1995
; Prammananan et al., 1998
). Available genomic data on M. abscessus are primarily limited to partial sequences of housekeeping genes in the GenBank database.
We have been characterizing strain 390R, which is a clinical isolate of M. abscessus, and its mutant derivative, 390S (Byrd & Lyons, 1999 ). During the course of our investigation, we identified a large polymorphic region in M. abscessus and discovered a novel insertion sequence (IS) with significant nucleotide identity to IS elements from both slowly growing and rapidly growing environmental mycobacteria.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Extraction of mycobacterial genomic DNA.
For routine extraction of DNA, bacterial cells were lysed by bead-beating using 0·1 mm silwica/zirconium beads in a BioSpec Mini-beadbeater and DNA was extracted using a Puregene DNA isolation kit (Gentra Systems) followed by phenol/chloroform extraction and precipitation with 2-propanol. For the genomic subtraction procedure, DNA was extracted from bead-beaten cells using the Qiagen genomic DNA maxiprep extraction kit with minor modifications.
Genomic subtraction.
The protocol of Straus & Ausubel (1990) was followed with some modifications.
(i) Preparation of DNA.
DNA from M. abscessus 390S and 390R was used as driver and test DNA, respectively. 390S DNA was sheared into random fragments using a Branson Sonifier 450 cup horn sonicator at 70% output for 2x25 s. Fragment sizes ranged from 0·5 to 12 kb, but the majority of fragments were 14 kb in size. Sheared DNA was biotinylated using the Clontech photoactivatable biotin-labelling kit, according to the manufacturers instructions. After butanol extraction and ethanol/ammonium acetate precipitation, biotinylated DNA was resuspended at a concentration of 1 µg µl-1 in 1x EE buffer [10 mM N-(2-hydroxyethyl)piperazine-N'-(3-propanesulfonic acid), pH 8·0/1 mM EDTA] (Straus & Ausubel, 1990 ). 390R DNA was partially digested with the restriction enzyme Sau3AI, extracted with phenol/chloroform and ethanol-precipitated. Fragments ranged up to 12 kb in size but the majority of fragments were <600 bp.
(ii) Subtraction.
Five micrograms of biotinylated 390S DNA, 0·5 µg Sau3AI-digested 390R DNA and 40 µg yeast tRNA in a final volume of 20 µl 2x EE were denatured, lyophilized and resuspended in 4 µl 2·5x EE. After the addition of 1 µl 5 M NaCl, samples were annealed overnight at 65 °C in a Perkin-Elmer Gene Amp 9600 Thermocycler with heated lid. Ninety-five microlitres of EEN (1x EE/500 mM NaCl) was added and the total mixture was added to MagneSphere streptavidin-coated paramagnetic particles (Promega) prewashed once in 0·5x SSC and three times in EEN. Samples were incubated with paramagnetic particles for 20 min at room temperature with occasional mixing, the particles were captured, and the supernatant was incubated with a fresh aliquot of prewashed particles. After particle capture, supernatants were pooled, mixed with 20 µg tRNA and precipitated with ethanol. Pellets were washed with 70% ethanol, dried and resuspended in 10 µl EE. After removal of 1 µl for analysis, the sample was mixed with 1 µl EE, 5 µl biotinylated 390S DNA, 1 µl tRNA (20 µg) and 3 µl 10x EE. The subtraction procedure was then repeated for another four cycles beginning with the denaturation step.
(iii) Addition of adapters and PCR.
For amplification of the remaining fragments of 390R DNA, the protocol of Straus & Ausubel (1990 ) was used. Sau3AI adapters were prepared and ligated to the pool of fragments followed by amplification using a primer that annealed to the adapters. Electrophoresis of the PCR products on an agarose gel showed a range of amplicons <800 bp in size for each sample. Several discrete bands of 300500 bp were visible beginning with the third round of subtraction.
(iv) Extraction of fragments for hybridization and cloning.
From subtraction round five, amplicons between 350 and 500 bp were excised from an agarose gel, purified and re-amplified by PCR. Amplicons between 350 and 500 bp were again purified and then radiolabelled for use as a probe or cloned into the TA cloning vector pCR2.1 (Invitrogen) for sequence analysis.
Southern blotting.
For Southern blots, 2 µg genomic DNA was digested with 20 U restriction enzyme and electrophoresed on 0·8% agarose gels. Following alkali treatment and neutralization (Sambrook et al., 1989 ), gels were blotted onto GeneScreen Plus hybridization transfer membranes (NEN) by capillary transfer. Membranes were prehybridized in Clontech Express Hybridization solution and then hybridized overnight with [
-32P]dCTP-labelled DNA probes. Washed membranes were exposed to autoradiographic film.
Cloning of genomic restriction fragments.
Restriction fragments of genomic DNA in the desired size range were eluted from agarose gels, ligated into pGEM3zf+ (Promega) and transformed into E. coli. Colony blots were prepared using standard procedures (Sambrook et al., 1989 ) and clones were identified by hybridization with overlapping probes. Positive colonies were amplified and the identity of the clones was confirmed by Southern blot analysis of extracted plasmids.
Cosmid library construction.
390R DNA was partially digested with Sau3AI, and restriction fragments of 3040 kb were excised from a 0·8% agarose gel and purified using GeneClean (Bio101) according to the manufacturers instructions for large DNA fragments. Purified fragments were ligated into BclI-digested pYUB178 (Pascopella et al., 1994 ) and ligations were packaged into lambda particles using the Gigapack III XL packaging extract (Stratagene) according to the manufacturers instructions. The packaged library was transduced into the XL-1 Blue MR strain of E. coli (Stratagene) and plated on LB agar containing 25 µg kanamycin ml-1. Colony blots were prepared from the cosmid library and hybridized with a probe to the deletion region. Restriction digests and Southern blot analysis of cos10 showed that it had an insert of approximately 35 kb and that it contained the full deletion and flanking regions (data not shown).
Sequence analysis and contig assembly.
Sequence analysis was performed on an ABI Prism 377 DNA Sequencer using the ABI BigDye Terminator Cycle Sequencing kit (PE Applied Biosystems). The manufacturers modified protocol was used with cosmid DNA. Sequence editing was performed using ABI Editview software. GeneTool 1.0 (BioTools) was used for sequence assembly and for detection of ORFs using a minimum cutoff size of 70 aa. The BLAST programs (Altschul et al., 1997 ) at the NCBI website were used for sequence comparisons. Additional comparisons were conducted against completed and partial genome sequences at www.tigr.org using BLASTN 2.0 and TBLASTN (W. Gish, http://blast.wustl.edu). The M. smegmatis IS-like sequences IS-A and IS-B are located in contigs 3311 and 3312, respectively, of the April 8 2002 update of the TIGR database. Putative functions for some M. tuberculosis genes were verified using the website at http://genolist.pasteur.fr/TubercuList/. The designation ISMab1 was provided by P. Siguier from the IS database registry (www-is.biotoul.fr).
PC.
Primers S4M13RY (GAT GAC GTT GAC CAA GTG) and Ma-ISD' (GTC GCT GAG CCA CAA CAT G) which amplify a portion of ISMab1 ORFB were used to detect the IS element. Primers S10T7S (CAT GCA GCA ATT TCA GG) and S4T7A (GAT CGG AGA CAG CTA CG) were used to amplify the 5'-end insertion site.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Examination of the sequence structure flanking the deletion breakpoints (Fig. 2) revealed an imperfect palindrome of 43 bases overlapping the left side of the deletion (left and right refer to the orientation of the region as depicted in Fig. 1
). The left breakpoint is near the axis of symmetry of the palindrome (vertical arrow, Fig. 2
); the A at the axis of symmetry is the first base of the predicted start codon (TTG) of ORF R5c which is transcribed in the opposite direction. Internal palindromes and a direct repeat (DR) are contained within the larger palindrome.
|
There is no obvious similarity between the breakpoints to suggest that the deletion occurred through homologous recombination; however, the features surrounding the breakpoints may have promoted regional instability. DRs and IRs including palindromes can be unstable in eukaryotes and prokaryotes and may lead to deletion of intervening or overlapping sequences (Glickman & Ripley, 1984 ; Goodchild et al., 1985
; Henderson & Petes, 1993
; Nasar et al., 2000
). Furthermore, coincident palindromes and DRs appear to be favoured deletion sites (Glickman & Ripley, 1984
).
R/S-D1 occurs within a polymorphic region
Several clinical strains of M. abscessus with different colony morphologies were examined but no direct correlation between R/S-D1 and colony morphology was found. Hybridization analysis showed that two rough strains, 1056 and 1475, were missing fragment R-sst3 (Fig. 3a), which lies within the deletion (Fig. 1
). Further analysis (data not shown) revealed that strains 1056 and 1475 were also missing fragments R-sst10.5 and R-bam9 (Fig. 1
), indicating both that this region is not required for a rough phenotype and, furthermore, that it exhibits significant polymorphism. In addition, the deletion fragment R-sst3 was present in the smooth strain 6639 as well as in two strains with an intermediate phenotype (8243 and 8988) (Fig. 3a
). These three strains as well as the type strain ATCC 19977, which has a smooth phenotype, produced the same hybridization patterns as 390R using R-sst10.5 and R-bam9 as probes (data not shown). The single exception was strain 8243, which was missing about 2·2 kb from a 7·4 kb PvuII fragment that overlaps the R-sst10.5 probe. The reduction in size of this fragment appears to be due to the absence of the IS element ISMab1 from this strain as indicated below.
|
Comparison of the M. abscessus ORF sequences with GenBank and completed bacterial genome sequences provided by TIGR databases (www.tigr.com) revealed strongest similarities to the actinomycetes M. tuberculosis (Cole et al., 1998 ) and Streptomyces coelicolor (Redenbach et al., 1996
) and to the Gram-negative environmental organisms Pseudomonas aeruginosa (Stover et al., 2000
) and Caulobacter crescentus (Nierman et al., 2001
) (Table 1
). Table 1
also includes the results of TBLASTN searches conducted against the unannotated TIGR databases for M. avium and M. smegmatis. All of the species listed in Table 1
have GC-rich genomes, and therefore to some degree similarities may reflect preferences for amino acids with GC-rich codons as has been described for M. tuberculosis (Cole et al., 1998
). However, some ORFs show much stronger similarities to genes from particular species and this may indicate closer functional relationships. R15, for example, has 62% identity and 75% similarity to the P. aeruginosa acyl-CoA dehydrogenase PA4435.
The slowly growing species M. avium showed strongest similarity to M. abscessus ORFs in this region while the rapid-grower M. smegmatis had the least similarity to M. abscessus among the mycobacteria listed. Most matches to M. leprae were to short sequences from pseudogenes (data not shown). M. abscessus ORFs within the same protein families were also compared using pairwise BLAST analysis (Tatusova & Madden, 1999 ). Identities ranged from 26% for the putative transcriptional regulators R3c and R5c to 3038% among the monooxygenases (R8, R9, R11) and acyl-CoA dehydrogenases (R15, R19). These M. abscessus ORFs were therefore generally less similar to each other than to related genes from other species, again suggesting that interspecies matches may reflect functional similarities.
Predicted functions for most of the homologous genes suggest that much of this region is involved in fatty acid metabolism. Acyl-CoA dehydrogenases such as R15 and R19 have a putative role in ß-oxidative metabolism and are related to families of M. tuberculosis proteins proposed to be involved in the degradation of host cell lipids for energy and metabolic precursors (Cole et al., 1998 ). It has been noted for P. aeruginosa that dehydrogenase genes are often clustered with genes encoding enzymes with related functions such as oxidoreductases and monooxygenases (Stover et al., 2000
), and similar clustering is evident within this region of M. abscessus (Fig. 1
).
Given the change in colony morphology of 390S (Byrd & Lyons, 1999 ), we had expected to find genes related to the synthesis of glycopeptidolipids (GPLs). Altered colony morphology in M. avium has been linked to changes in GPL structure (Belisle et al., 1993a
, b
) and M. abscessus is known to possess GPLs (Lopez-Marin et al., 1994
), but none of the missing genes resembles those reported to be involved in GPL synthesis in other mycobacteria (Belisle et al., 1993a
, b
; Recht et al., 2000
). Together with the data showing the absence of this region from some rough strains and its presence in some smooth strains (Fig. 3
), this suggests that this region is not directly associated with colony phenotype.
Structure of ISMab1
ISMab1 is a novel IS element which was discovered at the 3'-end of R19 (Fig. 1). The proposed structure of ISMab1 is shown in Fig. 4
; including the predicted 7 bp terminal IR (Fig. 4
, IR 1), the element is 1767 bp in length. Comparative sequence analysis with IS-like elements from M. avium and M. smegmatis indicates that ISMab1 is composed of two major sections, each of which is highly related to different mycobacterial IS elements (Fig. 4
).
|
At the nucleotide level, this first section has 82% identity to the region of M. avium IS1601 containing ORF4 (Eckstein et al., 2000 ) and 84% identity to M. avium IS999 including the 28 bp IR and 3 bp DR described for that element (J.-P. Laurent & G. Cangelosi, GenBank accession no. AF232829) (Fig. 4
and data not shown). Nucleotide comparisons indicate that IS999 is 8083% identical to the region of IS1601 containing ORFs 3 and 4 and that the 28 bp IR of IS999 is also present in IS1601 and overlaps the 15 bp IR 2 originally described for that element (Eckstein et al., 2000
). Interestingly, a shorter related sequence, designated IS-A in Fig. 4
, is also found in M. smegmatis and it includes one copy of the IS999 IR.
The IS1601-related sequence in the first section ends abruptly at the left copy of DR 1, suggesting that it was disrupted by the insertion of the section containing ORFs B and C (Fig. 4). This 1·27 kb section may be an independent transposable element: it has 83% nucleotide identity over its entire length to an uncharacterized IS-like sequence from M. smegmatis (designated IS-B in Fig. 4
), and in both M. smegmatis and M. abscessus it is flanked by IR 3 (Fig. 4
and data not shown). In addition to flanking the entire section, IR 3 is an inverted copy of the DR 1 and IR 2 sequences which flank ORFB. The IS-B sequence from M. smegmatis appears to be part of a larger 1·7 kb IS-like element which is less related to M. abscessus at the 5'-end (data not shown). TBLASTN comparisons showed that ORFs B and C have 88% identity (92% similarity) and 86% identity (91% similarity), respectively, to IS-B ORFs.
Both ORFs B and C of ISMab1 show homology to transposases. GenBank searches show that ISMab1 ORFB has 38% aa identity (59% similarity) and 36% aa identity (54% similarity) to, respectively, the tranposase of IS6110 (McAdam et al., 1990 ; Thierry et al., 1990
) and a putative transposase from Saccharopolyspora (GenBank accession no. AF045021); it is also of similar length to these ORFs. ORFC has 3235% aa identity to a large group of transposases including the IS3 putative transposase of E. coli and M. avium IS1601 ORF4.
Distribution of ISMab1
Examination of the clinical isolates for the presence of ISMab1 showed that strains 1056 and 1475, which were missing R/S-D1 and the upstream region, are also missing this element (Fig. 3b). There was also no specific hybridization to strain 8243, which is consistent with the shorter PvuII fragment identified by the R-sst10.5 probe (see above). The ISMab1 probe detected single bands in 390R, 390S, and in strains 6639 and 8988. As SstI cuts once within the IS element, the single bands in the SstI digests indicate that there is only one complete copy of the element in these strains. PCR analysis confirmed the distribution of ISMab1 and also indicated that it was present in the type strain ATCC 19977 (data not shown). In addition, sequence analysis of the PCR fragments for 8988 and the type strain showed that ISMab1 was located in the same position in these strains as in 390R.
Conclusions
ISMab1 is the first IS element to be identified in M. abscessus. It is not known, though, whether it functions as a single mobile element. IR 1 is shorter than most IRs described for IS elements (Chandler, 1998 ) and no DRs were found. Also, only single copies of the element have been detected. ISMab1 is remarkable, though, for its close relationship to IS elements from other environmental mycobacteria. It has been suggested that horizontal transfer of genetic elements may occur between mycobacteria and related species (Gordon et al., 1999
). For example, the transposase of IS1552 from M. tuberculosis shares 80% aa identity to a transposase from Rhodococcus (Gordon et al., 1999
), and ORFs in M. avium IS1601 show high amino acid similarity to transposases from other slowly growing mycobacteria (Eckstein et al., 2000
). Horizontal transfer is further supported by the high degree of nucleotide identity between ISMab1 and elements from M. avium and M. smegmatis.
ISMab1 is located within a region that showed unexpected polymorphism in clinical isolates. Polymorphism associated with large deletions has also been observed in clinical isolates of M. tuberculosis (Ho et al., 2000 ; Vera-Cabrera et al., 1997
) but, as with our M. abscessus findings, the relationship to biological properties is unknown. Although some of the altered phenotypes of M. abscessus 390S (Byrd & Lyons, 1999
) may be linked to the loss of this region, this remains to be determined as we have been unable to reintroduce this region into M. abscessus 390S using cosmids. We expect that other genetic changes will be found in 390S, but genomic subtraction was useful for detecting a major genetic difference. This method, or variations of it, has been used to identify bacterial virulence factors and other unique sequences (Emmerth et al., 1999
; Mahairas et al., 1996
; Morrow et al., 1999
; Reckseidler et al., 2001
; Sawada et al., 1999
; Schmidt et al., 1999
; Zhang et al., 2000
) and in some cases has been successfully used to compare less closely related bacterial strains (Emmerth et al., 1999
; Mahairas et al., 1996
; Morrow et al., 1999
; Zhang et al., 2000
). It may be a suitable approach for identifying genetic differences in other uncharacterized mycobacterial genomes.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alland, D., Steyn, A. J., Weisbrod, T., Aldrich, K. & Jacobs, W. R.Jr (2000). Characterization of the Mycobacterium tuberculosis iniBAC promoter, a promoter that responds to cell wall biosynthesis inhibition. J Bacteriol 182, 1802-1811.
Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 3389-3402.
Belisle, J. T., McNeil, M. R., Chatterjee, D., Inamine, J. M. & Brennan, P. J. (1993a). Expression of the core lipopeptide of the glycopeptidolipid surface antigens in rough mutants of Mycobacterium avium. J Biol Chem 268, 10510-10516.
Belisle, J. T., Klaczkiewicz, K., Brennan, P. J., Jacobs, W. R. & Inamine, J. M. (1993b). Rough morphological variants of Mycobacterium avium. J Biol Chem 268, 10517-10523.
Brown, B. A., Springer, B., Steingrube, V. A. & 10 other authors (1999). Mycobacterium wolinskyi sp. nov. and Mycobacterium goodii sp. nov., two new rapidly growing species related to Mycobacterium smegmatis and associated with human wound infections: a cooperative study from the International Working Group on Mycobacterial Taxonomy. Int J Syst Bacteriol 49, 14931511.[Abstract]
Byrd, T. F. & Lyons, C. R. (1999). Preliminary characterization of a Mycobacterium abscessus mutant in human and murine models of infection. Infect Immun 67, 4700-4707.
Chadha, R., Grover, M., Sharma, A., Lakshmy, A., Deb, M., Kumar, A. & Mehta, G. (1998). An outbreak of post-surgical wound infections due to Mycobacterium abscessus. Pediatr Surg Int 13, 406-410.[Medline]
Chandler, M. S. (1998). Insertion sequences and transposons. In Bacterial Genomes, Physical Structure and Analysis , pp. 30-37. Edited by F. J. de Bruijn, J. R. Lupski & G. M. Weinstock. New York:International Thomson Publishing.
Cole, S. T., Brosch, R., Parkhill, J. & 39 other authors (1998). Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537544.[Medline]
Cole, S. T., Eiglmeier, K., Parkhill, J. & 41 other authors (2001). Massive gene decay in the leprosy bacillus. Nature 409, 10071011.[Medline]
Domenech, P., Jimenez, M. S., Menendez, M. C., Bull, T. J., Samper, S., Manrique, A. & Garcia, M. J. (1997). Mycobacterium mageritense sp. nov. Int J Syst Bacteriol 47, 535-540.
Dussurget, O., Timm, J., Gomez, M., Gold, B., Yu, S., Sabol, S. Z., Holmes, R. K., Jacobs, W. R.Jr & Smith, I. (1999). Transcriptional control of the iron-responsive fxbA gene by the mycobacterial regulator IdeR. J Bacteriol 181, 3402-3408.
Eckstein, T. M., Inamine, J. M., Lambert, M. L. & Belisle, J. T. (2000). A genetic mechanism for deletion of the ser2 gene cluster and formation of rough morphological variants of Mycobacterium avium. J Bacteriol 182, 6177-6182.
Emmerth, M., Goebel, W., Miller, S. I. & Hueck, C. J. (1999). Genomic subtraction identifies Salmonella typhimurium prophages, F-related plasmid sequences, and a novel fimbrial operon, stf, which are absent in Salmonella typhi. J Bacteriol 181, 5652-5661.
Falkinham, J. O.III (1996). Epidemiology of infection by nontuberculous mycobacteria. Clin Microbiol Rev 9, 177-215.
Fujii, S., Akiyama, M., Aoki, K. & 10 other authors (1999). DNA replication errors produced by the replicative apparatus of Escherichia coli. J Mol Biol 18, 835850.
Galil, K., Miller, L. A., Yakrus, M. A., Wallace, R. J.Jr, Mosley, D. G., England, B., Huitt, G., McNeil, M. M. & Perkins, B. A. (1999). Abscesses due to Mycobacterium abscessus linked to injection of unapproved alternative medication. Emerg Infect Dis 5, 681-687.[Medline]
Glickman, B. W. & Ripley, L. S. (1984). Structural intermediates of deletion mutagenesis: a role for palindromic DNA. Proc Natl Acad Sci USA 81, 512-516.[Abstract]
Goodchild, J., Michniewicz, J., Seto-Young, D. & Narang, S. (1985). A novel deletion found during cloning of a synthetic palindromic DNA. Gene 33, 367-371.[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, 881-892.[Abstract]
Griffith, D. E., Girard, W. M. & Wallace, R. J.Jr (1993). Clinical features of pulmonary disease caused by rapidly growing mycobacteria. Am Rev Respir Dis 147, 1271-1278.[Medline]
Guillemin, I., Cambau, E. & Jarlier, V. (1995). Sequences of conserved region in the A subunit of DNA gyrase from nine species of the genus Mycobacterium: phylogenetic analysis and implication for intrinsic susceptibility to quinolones. Antimicrob Agents Chemother 39, 2145-2149.[Abstract]
Henderson, S. T. & Petes, T. D. (1993). Instability of a plasmid-borne inverted repeat in Saccharomyces cerevisiae. Genetics 133, 57-62.
Ho, T. B., Robertson, B. D., Taylor, G. M., Shaw, R. J. & Young, D. B. (2000). Comparison of Mycobacterium tuberculosis genomes reveals frequent deletions in a 20 kb variable region in clinical isolates. Yeast 17, 272-282.[Medline]
Howard, S. T. & Byrd, T. F. (2000). The rapidly-growing mycobacteria: saprophytes and parasites. Microbes Infect 11, 1845-1853.
Kim, B. J., Lee, S. H., Lyu, M. A., Kim, S. J., Bai, G. H., Chae, G. T., Kim, E. C., Cha, C. Y. & Kook, Y. H. (1999). Identification of mycobacterial species by comparative sequence analysis of the RNA polymerase gene (rpoB). J Clin Microbiol 37, 1714-1720.
Kusunoki, S. & Ezaki, T. (1992). Proposal of Mycobacterium peregrinum sp. nov., nom. rev., and elevation of Mycobacterium chelonae subsp. abscessus (Kubica et al.) to species status: Mycobacterium abscessus comb. nov. Int J Syst Bacteriol 42, 240-245.[Abstract]
Lee, J. H. & Holmes, R. K. (2000). Characterization of specific nucleotide substitutions in DtxR-specific operators of Corynebacterium diphtheriae that dramatically affect DtxR binding, operator function, and promoter strength. J Bacteriol 182, 432-438.
López-Marín, L. M., Gautier, N., Lanéelle, M. A., Silve, G. & Daffé, M. (1994). Structures of the glycopeptidolipid antigens of Mycobacterium abscessus and Mycobacterium chelonae and possible chemical basis of the serological cross-reactions in the Mycobacterium fortuitum complex. Microbiology 140, 1109-1118.[Abstract]
Mahairas, G. G., Sabo, P. J., Hickey, M. J., Singh, D. C. & Stover, C. K. (1996). Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis. J Bacteriol 178, 1274-1282.[Abstract]
McAdam, R. A., Hermans, P. W., van Soolingen, D., Zainuddin, Z. F., Catty, D., van Embden, J. D. & Dale, J. W. (1990). Characterization of a Mycobacterium tuberculosis insertion sequence belonging to the IS3 family. Mol Microbiol 4, 1607-1613.[Medline]
Morrow, B. J., Graham, J. E. & Curtiss, R.III (1999). Genomic subtractive hybridization and selective capture of transcribed sequences identify a novel Salmonella typhimurium fimbrial operon and putative transcriptional regulator that are absent from the Salmonella typhi genome. Infect Immun 67, 5106-5116.
Nasar, F., Jankowski, C. & Nag, D. K. (2000). Long palindromic sequences induce double-strand breaks during meiosis in yeast. Mol Cell Biol 20, 3449-3458.
Nierman, W. C., Feldblyum, T. V., Laub, M. T. & 34 other authors (2001). Complete genome sequence of Caulobacter crescentus. Proc Natl Acad Sci USA 98, 41364141.
Pascopella, L., Collins, F. M., Martin, J. M., Lee, M. H., Hatfull, G. F., Stover, C. K., Bloom, B. R. & Jacobs, W. R. (1994). Use of in vivo complementation in Mycobacterium tuberculosis to identify a genomic fragment associated with virulence. Infect Immun 62, 1313-1319.[Abstract]
Pitulle, C., Dorsch, M., Kazda, J., Wolters, J. & Stackebrandt, E. (1992). Phylogeny of rapidly growing members of the genus Mycobacterium. Int J Syst Bacteriol 42, 337-343.[Abstract]
Prammananan, T., Sander, P., Brown, B. A., Frischkorn, K., Onyi, G. O., Zhang, Y., Bottger, E. C. & Wallace, R. J.Jr (1998). A single 16S ribosomal RNA substitution is responsible for resistance to amikacin and other 2-deoxystreptamine aminoglycosides in Mycobacterium abscessus and Mycobacterium chelonae. J Infect Dis 177, 1573-1581.[Medline]
Recht, J., Martinez, A., Torello, S. & Kolter, R. (2000). Genetic analysis of sliding motility in Mycobacterium smegmatis. J Bacteriol 182, 4348-4351.
Reckseidler, S. L., DeShazer, D., Sokol, P. A. & Woods, D. E. (2001). Detection of bacterial virulence genes by subtractive hybridization: identification of capsular polysaccharide of Burkholderia pseudomallei as a major virulence determinant. Infect Immun 69, 34-44.
Redenbach, M., Kieser, H. M., Denapaite, D., Eichner, A., Cullum, J., Kinashi, H. & Hopwood, D. A. (1996). A set of ordered cosmids and a detailed genetic and physical map for the 8 Mb Streptomyces coelicolor A3(2) chromosome. Mol Microbiol 21, 77-96.[Medline]
Ringuet, H., Akoua-Koffi, C., Honore, S., Varnerot, A., Vincent, V., Berche, P., Gaillard, J. L. & Pierre-Audigier, C. (1999). hsp65 sequencing for identification of rapidly growing mycobacteria. J Clin Microbiol 37, 852-857.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sawada, K., Kokeguchi, S., Hongyo, H., Sawada, S., Miyamoto, M., Maeda, H., Nishimura, F., Takashiba, S. & Murayama, Y. (1999). Identification by subtractive hybridization of a novel insertion sequence specific for virulent strains of Porphyromonas gingivalis. Infect Immun 67, 5621-5625.
Schmidt, K. D., Schmidt-Rose, T., Romling, U. & Tummler, B. (1999). Differential genome analysis of bacteria by genomic subtractive hybridization and pulsed field gel electrophoresis. Electrophoresis 19, 509-514.
Shinnick, T. M. & Good, R. C. (1994). Mycobacterial taxonomy. Eur J Clin Microbiol Infect Dis 13, 884-901.[Medline]
Shojaei, H., Goodfellow, M., Magee, J. G., Freeman, R., Gould, F. K. & Brignall, C. G. (1997). Mycobacterium novocastrense sp. nov., a rapidly growing photochromogenic mycobacterium. Int J Syst Bacteriol 47, 1205-1207.
Stover, C. K., Pham, X. Q., Erwin, A. L. & 23 other authors (2000). Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406, 959964.[Medline]
Straus, D. & Ausubel, F. M. (1990). Genomic subtraction for cloning DNA corresponding to deletion mutations. Proc Natl Acad Sci USA 87, 1889-1893.[Abstract]
Tatusova, T. A. & Madden, T. L. (1999). Blast 2 sequences a new tool for comparing protein and nucleotide sequences. FEMS Microbiol Lett 174, 247-250.[Medline]
Thierry, D., Cave, M. D., Eisenach, K. D., Crawford, J. T., Bates, J. H., Gicquel, B. & Guesdon, J. L. (1990). IS6110, an IS-like element of Mycobacterium tuberculosis complex. Nucleic Acids Res 18, 188.[Medline]
Timsit, Y. (1999). DNA structure and polymerase fidelity. J Mol Biol 293, 835-853.[Medline]
Vera-Cabrera, L., Howard, S. T., Laszlo, A. & Johnson, W. M. (1997). Analysis of genetic polymorphism in the phospholipase region of Mycobacterium tuberculosis. J Clin Microbiol 35, 1190-1195.[Abstract]
Villaneuva, A., Calderon, R. V., Vargas, B. A., Ruiz, F., Aguero, S., Zhang, Y., Brown, B. A. & Wallace, R. J.Jr (1997). Report of an outbreak of postinjection abscesses due to Mycobacterium abscessus, including management with surgery and clarithromycin therapy and comparison of strains by random amplified polymorphic DNA polymerase chain reaction. Clin Infect Dis 24, 1147-1153.[Medline]
Wallace, R. J.Jr, Zhang, Y., Brown, B. A., Fraser, V., Mazurek, G. H. & Maloney, S. (1993). DNA large restriction fragment patterns of sporadic and epidemic nosocomial strains of Mycobacterium chelonae and Mycobacterium abscessus. J Clin Microbiol 31, 2697-2701.[Abstract]
Wright, P. W. & Wallace, R. J.Jr (1995). Syndromes, diagnosis, and treatment of rapidly growing mycobacteria. In Tuberculosis: Clinical Management and New Challenges , pp. 373-389. Edited by M. R. R. Rossman. New York:McGraw-Hill.
Zeng, X. & Saxild, H. H. (1999). Identification and characterization of a DeoR-specific operator sequence essential for induction of dra-nupC-pdp operon expression in Bacillus subtilis. J Bacteriol 181, 1719-1727.
Zhang, Y., Rajagopalan, M., Brown, B. A. & Wallace, R. J.Jr (1997). Randomly amplified polymorphic DNA PCR for comparison of Mycobacterium abscessus strains from nosocomial outbreaks. J Clin Microbiol 35, 3132-3139.[Abstract]
Zhang, Y. L., Ong, C. T. & Leung, K. Y. (2000). Molecular analysis of genetic differences between virulent and avirulent strains of Aeromonas hydrophila isolated from diseased fish. Microbiology 146, 999-1009.
Received 21 January 2002;
revised 19 April 2002;
accepted 18 June 2002.