Institut Pasteur de Bruxelles, rue Engeland 642, B-1180 Brussels, Belgium1
Université Libre de Bruxelles, Unité de Vectorologie et Oncologie Expérimentale, Hopital Erasme, Route de Lennik 808, B-1070 Brussels, Belgium2
Author for correspondence: Jean Content. Tel: +32 2 3733416. Fax: +32 2 3733291. e-mail: jcontent{at}pasteur.be
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ABSTRACT |
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Keywords: homologous recombination, genomic duplication, merodiploidy, alcohol dehydrogenase C
Abbreviations: ADHC, alcohol dehydrogenase C; BCG-ADHC, M. bovis BCG ADHC; DCO, double cross-over; Ms-ADHC, M. smegmatis mc2155 ADHC; SCO, single cross-over
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INTRODUCTION |
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Alcohol dehydrogenase C (ADHC) has been suggested to be a potential target for the development of new antibiotics. This protein, initially purified and biochemically characterized from Mycobacterium bovis BCG, was suggested to contribute to the hydrophobic content of the mycobacterial envelope through its involvement in the biosynthesis of the free lipids required for envelope formation (De Bruyn et al., 1981a , b
; Wilkin et al., 1999
). The M. bovis BCG ADHC (BCG-ADHC) gene was cloned and found to be identical to the adhC gene from M. tuberculosis (Stelandre et al., 1992
; Wilkin et al., 1999
). BCG-ADHC is a dimeric zinc, NADP-dependent enzyme belonging to the long-chain alcohol/polyol dehydrogenase family, class C. To test the hypothesis that ADHC is essential in mycobacteiral envelope formation we undertook experiments to knock out the adhC gene and examine the effects of this mutation.
To avoid the difficulty of generating mutants by homologous recombination in slow-growing mycobacterial strains (McFadden, 1996 ), and to speed up the clarification of the physiological role of mycobacterial ADHCs, we first looked for such an enzyme in a fast-growing and non-pathogenic mycobacterial strain. We have previously reported the identification of M. smegmatis mc2155 ADHC (Ms-ADHC; Galamba et al., 2001
), which shares 78% identity with BCG-ADHC and M. tuberculosis ADHC. The Ms-ADHC gene was cloned and sequenced, and the protein was purified, partially characterized and compared with BCG-ADHC. The two enzymes were found to be similar and functioned as aldehyde reductases in vitro, processing alcohols far less efficiently than aliphatic and aromatic aldehydes. It was also found that Ms-ADHC shares a strong degree of amino acid sequence similarity with the ADHCs of M. avium and M. paratuberculosis (76%), and M. leprae (75%). These results suggested that M. smegmatis mc2155 could be used as a model to study the physiological role of the alcohol dehydrogenases in pathogenic mycobacteria. Thus, we used the cloned adhC gene to generate an ADHC knockout mutant of M. smegmatis mc2155, by homologous recombination with a double cross-over (DCO) event. The resulting adhC-disrupted mutant should allow us to test the potential role of ADHCs in the mycobacterial cell envelope and to evaluate their in vivo significance as a target for anti-tuberculosis drugs.
In this paper we describe how the experiments undertaken to disrupt the adhC chromosomal allele (with a kanamycin-resistance gene) uncovered a large duplication in the M. smegmatis mc2155 genome. In addition, the AdhC+/- and AdhC-/- mutants were characterized and compared to the M. smegmatis wild-type with respect to growth and the utilization of some aldehydes in vitro.
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METHODS |
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Analysis of the transformants.
To distinguish between the single cross-over (SCO) and DCO homologous recombinants, the KanR clones were plated out onto both sucrose/kanamycin SNBA [10% (w/v) sucrose; 20 µg kanamycin ml-1] and hygromycin SNBA (100 µg hygromycin ml-1) plates. The KanR HygS SucR DCOs were further analysed by Southern blotting and PCR. The DNA from the M. smegmatis mutants was extracted as previously described (Galamba et al., 2001 ). Southern blots of the DNA from each mutant were prepared after the DNA had been digested with BamHI, ApaI, SfiI or BglII. The restriction fragments were run out on 1% agarose gels and transferred to Hybond-N nylon membranes. Hybridizations were performed at 65 °C as previously described (Galamba et al., 2001
); the probes used for the hybridizations were Padh, Pkan, PsacB and Pdws (see Fig. 1
and Table 1
). All of the probes were labelled by random priming with [
-32P]dCTP using a random priming kit (Amersham). PCR analysis was carried out either on extracted DNA or on individual colonies from the SCO and DCO recombinant strains. To carry out PCR from individual colonies, a colony from each transformant was resuspended in 1 ml water, boiled for 25 min to liberate chromosomal DNA and cooled on ice for 10 min. The liberated DNA was then used as a template for PCR amplification. PCR was carried out in 50 µl reaction volumes containing 15 µl of colony suspension and Taq DNA polymerase (Promega) under the following conditions: 1 min at 95 °C; 30 cycles of 1 min at 95 °C, 1 min at 60 °C, 1·5 min at 72 °C; and, a final 10 min extension period at 72 °C. Primers R59, 5'-TTCGTCGACTCCTGCCGAGA-3', and G345, 5'-GTCGATGACGAAGCGGTAGCGCAC-3', were used for the PCR amplification. These primers were designed to amplify a 760 bp fragment from the wild-type strain, and a 1500 bp fragment from mutant strains that contained the aphA-3 cassette insertion.
PFGE and Southern blot hybridization of M. smegmatis wild-type and mutant strains.
Chromosomal DNA embedded in agarose plugs was obtained from M. smegmatis mc2155, M. smegmatis AdhC+/- and M. smegmatis AdhC-/- grown in 5 ml modified Middlebrook 7H9 broth (Difco) containing 0·5 M sucrose, 0·05% (w/v) Tween 80, 0·2% (w/v) D-glucose and 10% (v/v) oleic acidalbumin complex (Beckman Dickson), for 24 h at 37 °C. Four hundred microlitres of a solution containing 0·2 M glycine, 60 µg D-cycloserine ml1, 20 mM LiCl, 200 µg lysozyme ml1 and 5 mM EDTA were then added to the cultures; the cultures were incubated for an additional 16 h and were then centrifuged at 1000 g for 20 min at 4 °C. The bacteria were recovered and resuspended in TS buffer (50 mM Tris, 0·5 M sucrose, pH 7·6) (1/50 culture vol.). The cell suspensions were transferred to microcentrifuge tubes, immediately frozen in dry icemethanol and then thawed on wet ice. The cell suspensions were then mixed with an equal volume of 1% low-melting-point agarose at 50 °C (Incert agarose; FMC Bioproducts) in TEN buffer (50 mM Tris, 250 mM EDTA, 200 mM NaCl, pH 7·6) and cast in plugs. The plugs were left to set for 20 min, and were incubated overnight at 37 °C in TE buffer (10 mM Tris, 1 mM EDTA, pH 8) containing 1 mg lysozyme ml-1 to lyse the cells. The plugs were then transferred to TE buffer containing 1% SDS and 1 mg proteinase K ml-1 and incubated for 48 h, 55 °C. The plugs were transferred into fresh TE buffer and washed for 30 min, then transeferred to TE buffer containing 0·04 mg PMSF ml-1 incubated at 55 °C for 30 min. They were then washed three times, using only fresh TE buffer for each wash. The plugs were stored in 0·2 M EDTA at 4 °C. Before restriction digestion the plugs were washed twice in TE/Triton X-100 (0·1%) (v/v) at 4 °C for 1 h, and twice in restriction enzyme buffer/Triton X-100 (0·1%) (v/v) for 1 h at room temperature; the plugs were then incubated overnight at 37 °C in the presence of DraI (20 U). The restriction fragments were separated on a 1% agarose gel (Seakem GTG, FMC) in 0·5 x TBE buffer (0·025 M Tris, 0·5 mM EDTA and 0·025 M boric acid) containing 50 µM thiourea, using a CHEF Mapper system (Bio-Rad) at 14 °C and 200 V. For DraI-digested DNA from the wild-type and from clone 31, pulse times were ramped linearly from 140 s for 22 h; pulse times for DraI-digested DNA from clone 18 were ramped 520 s for 20 h. Bacteriophage concatemers (48·5 kb) were used as DNA standards. Gels were stained with ethidium bromide, and examined and photographed under UV light on a transilluminator. The agarose gels were then treated with 0·25 M HCl for 30 min before being treated following the standard Southern method and transferred by capillarity to a Hybond-N nylon membrane. Southern blot hybridizations were performed using probes P600 and Pups (Table 1
). P600 was obtained by PCR amplification of pAGA5 with the primers G344, 5'-GCGCCGCTGTTGTGCGCGGGC-3', and G345.
SDS-PAGE and Western blot analysis.
SDS-PAGE was carried out as described by Laemmli (1970) with 12% polyacrylamide gels on a Miniprotean II system (Bio-Rad). Proteins were transferred onto nitrocellulose filters with an LKB Multiphor II electrophoresis unit by semidry electroblotting. ADH was detected by incubation with a murine monoclonal antibody raised against the BCG-ADHC (4B5) (Stelandre et al., 1992
) and developed with the Protoblot Western blot alkaline phosphatase system (Promega), according to the manufacturers instructions.
Biochemical characterization of the adhC knockout mutants.
M. smegmatis mc2155, AdhC+/- and AdhC-/- mutants were grown on Sauton medium as surface pellicles for 3 d at 37 °C. The pellicles were recovered by filtration, sonicated with a probe tip sonicator (Vibra Cell) at 50% duty cycle for 10 min and then centrifuged (11000 g, 15 min). The protein concentration in the cell lysates was determined by the Coomassie brillant blue method (Spector, 1978 ) with BSA as the standard. ADH activity was assayed by monitoring the oxidation of NADPH by using a spectrophotometer (DU640B; Beckman Coulter) at A340 (A366 for cinnamaldehyde). This assay was carried out at room temperature in a 1 ml reaction mixture [(0·02 M KH2PO4/Na2HPO4 buffer, pH 7·3; 0·25 mM NADPH; 25 µg total protein) plus the aldehyde substrate (either 100 µM octanal, 200 µM benzaldehyde, 200 µM cinnamaldehyde or 1 mM butyraldehyde)].
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RESULTS |
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The KanR colonies obtained were initially screened by plating them onto plates with 10% sucrose and plates containing hygromycin, to distinguish between integration events (SCOs or DCOs) and spontaneous KanR arising after electroporation. Since the delivery vector we used is unable to replicate in mycobacteria, HygR SucS clones were assumed to have integrated the plasmid into the chromosome by a SCO event. Analysis of the 150 transformants obtained showed that 51 (34%) were straightforward DCOs and all the others were the result of SCOs. However, Southern blot analysis of eight of the DCO clones (KanR HygS SucR) with Padh after BamHI digestion did not confirm the expected genotype (Fig. 2a): hybridization resulted in two bands for all eight of the DCO candidate mutants. Although the
4000 bp band that was consistent with a DCO event was present in each clone, we could also detect a
3300 bp band that corresponded to the wild-type adhC. When BamHI-digested DNA from these DCO clones was probed with Pkan, only the
4000 bp band was detected (Fig. 2b
). On the other hand, when the blot was probed with PsacB no hybridization could be detected, confirming that the delivery vector had not been integrated into the chromosome of these clones, again suggesting a double recombination event (Fig. 2b
).
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Analysis of some of the SCO clones showed that they resulted from a SCO event in the adhC coding region. Southern blot hybridization of eight SCO clones (KanR HygR SucS) digested with BamHI (Fig. 2c), ApaI and BglII (data not shown) and probed with Padh (Fig. 2c
) revealed two types of SCO mutant, and it was possible to determine that they corresponded to SCO events that took place either upstream or downstream of the adhC gene (Fig. 2d
). In the latter case an unexpected band was found to hybridize to Padh. It is possible that a wild-type adhC was also present in the clones in which the SCO event occurred upstream of adhC; however, since the band generated was the same size as the one generated by the BamHI fragment containing the wild-type adhC (
3300 bp), only one band, corresponding to two DNA fragments, could be seen. These results argue against the existence of sequences other than adhC that are homologous to the plasmid in the M. smegmatis genome, and provide preliminary evidence that adhC could be duplicated within the M. smegmatis genome.
To confirm the duplication hypothesis, we repeated the electroporation of M. smegmatis competent cells with the delivery vector, using a two-step screening strategy. This enabled the selection of DCOs resulting from a second SCO event in a homologous SCO clone. If adhC was not an essential gene, a second cross-over event should have occurred in some of the SCO clones to generate a DCO clone in which adhC had been replaced by a disrupted copy of the gene. The presence of a non-disrupted copy of adhC together with a disrupted copy would confirm the hypothesis.
One of the SCO mutants selected on plates containing kanamycin and hygromycin, after having been characterized by Southern hybridization as a homologous SCO (data not shown), was picked and streaked out onto plates lacking antibiotics. Following cultivation, a loopful of cells was resuspended in liquid medium and serial dilutions were plated onto a sucrose-enriched medium to select for clones that had lost the integrated plasmid through a second SCO. SucR colonies were streaked onto kanamycin plates with and without hygromycin, and scored for growth to distinguish DCOs from SCOs which had acquired spontaneous resistance to sucrose. One hundred KanR HygS SucR colonies were analysed by PCR (Fig. 3), but again both the 760 and 1500 bp bands were detected in each DCO mutant. Some of these clones were further analysed by Southern blotting, using the probes Padh and Pkan, which confirmed the results obtained by PCR (data not shown). These results, together with the Southern blotting and PCR results for the first set of DCO mutants, strongly suggested that there were two copies of adhC in the M. smegmatis genome and that the DCO clones obtained with both screening strategies have the phenotype AdhC+/-.
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The definitive proof of the adhC duplication in the M. smegmatis genome was obtained when a clone in which two DCO events had occurred was isolated. Competent cells from clone 31, one of the first DCO mutants, were prepared and electroporated with the suicide delivery vector pAGA6KM. A two-step strategy was followed to screen DCO mutants, as the cells were already KanR and it was not possible to use kanamycin as the only selectable marker. SCO events were first selected on plates containing kanamycin and hygromycin; a second SCO was subsequently screened for by using the sacB and hyg genes. Five SCO mutants were picked and streaked out onto plates lacking antibiotics. Following growth, a loopful of cells was resuspended in liquid medium and serial dilutions were plated onto a sucrose-enriched medium. Seventy SucR colonies were streaked onto kanamycin plates with and without hygromycin to select for DCO mutants. Forty-nine KanR HygS SucR clones were selected and 27 were analysed by PCR. Among the tested clones only one (clone 18) showed the expected genotype for a double DCO event. The genomic DNA from this clone was extracted and analysed by Southern blotting (Fig. 4a). The probe Padh was used and was found to hybridize only to one BamHI DNA fragment of
4000 bp, which confirmed that the
3300 bp band has been removed to become
4000 bp, as expected from disruption of the second adhC gene. These results also demonstrated that adhC is not an essential gene for M. smegmatis.
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PFGE to determine the position of the two adhC genes
To determine the localization of the two adhC genes and obtain information about the size of this duplication in the M. smegmatis genome, agarose plugs containing DNA from M. smegmatis wild-type, clone 31 and clone 18, were digested with DraI and resolved by PFGE. By comparative Southern blot hybridization analysis, and thanks to the DraI restriction site in the kanamycin cassette used to disrupt the adhC, it was possible to obtain some information about the duplication within the M. smegmatis genome. Southern blot hybridization with P600, a 760 bp fragment of the ADHC ORF downstream of the site where the kanamycin cassette containing the DraI had been inserted, revealed that each adhC of M. smegmatis wild-type was within a 250 kb DraI DNA fragment. On the other hand,
250 kb and
20 kb fragments from clone 31, and a
20 kb fragment from clone 18, were found to hybridize with P600 (Fig. 5a
, b
). The hybridization of the DraI-digested DNA from clone 18 with Pups, a 343 bp fragment upstream of adhC, revealed a
230 kb fragment (Fig. 5b
). Though our results do not allow us to determine the exact size of this duplication, it may be at least
250 kb. Fig. 5(c)
shows our proposed interpretation of the results obtained by PFGE. We propose the existence of a tandem duplication as the most probable explanation; however, even if the duplication is not in tandem this does not affect the predicted size of the duplication found within the M. smegmatis genome. The relative position of both of the copies of adhC in the DraI fragments is shown in Fig. 5(c)
. The
250 kb band obtained with P600 on M. smegmatis wild-type DNA, the
20 kb band obtained with P600 on clone 18 DNA, and the
230 kb band obtained with Pups on clone 18 should correspond to two DraI DNA fragments each.
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Biochemical characterization of the M. smegmatis AdhC+/- and AdhC-/- mutants
Lysates from 3 d pellicles of wild-type M. smegmatis and both recombinant strains were obtained, and ADH specific activity was determined by measuring the rate of oxidation of 0·25 mM NADPH at A340 in the presence of concentrations of octanal and benzaldehyde well above the Km that had been previously determined for the Ms-ADHC in vitro (Galamba et al., 2001 ). These two substrates had previously been shown to be, in vitro, the best available substrates for this enzyme. As can be seen in Fig. 7(a
, b
) the M. smegmatis AdhC-/- could not reduce either of these two aldehydes, while M. smegmatis AdhC+/- showed intermediate activities for both benzaldehyde and octanal, when compared with the M. smegmatis wild-type and M. smegmatis AdhC-/-. We also tested M. smegmatis AdhC-/- with two other aldehydes, butyraldehyde and cinnamaldehyde, but neither of them was reduced by the M. smegmatis AdhC-/- lysates (Fig. 7c
, d
). The absence of activity in the double-knockout mutant could be explained either by the fact that ADHC is the only M. smegmatis ADH which uses NADPH as a cofactor, or that the other putative ADHs which can be deduced from the M. smegmatis genome sequence available (http://www.tigr.org/tdb/mdb/mdbinprogress.html) are either not expressed or are inactive in our crude bacterial extracts.
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DISCUSSION |
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The initial attempts to select an ADHC knockout mutant were unsuccessful, though a large number of DCO mutants were generated. The one-step strategy initially used can be useful to generate marked mutations, but in some cases the required mutants will not be obtained, e.g. if the gene is essential or the recombination frequency at the locus is low. In these cases, especially for slow-growing mycobacteria, where gene replacement has not proved to be straightforward (McFadden, 1996 ), a two-step strategy has been usually employed, whereby SCO events were first selected, and then screening for the second cross-over was carried out to yield the mutant strains. We thus decided to switch to a two-step strategy that should ensure that the DCO clones selected are homologous, and also help determine whether adhC is duplicated. DCO mutants were obtained from SCO clones where another recombination event had taken place. These DCO mutants were characterized and found to have the same genotype as the DCO mutants previously obtained with the one-step strategy. They were further characterized by Western blotting (data not shown) and seemed to have less ADHC present than the wild-type M. smegmatis. Altogether, our results provided strong evidence that there are two copies of the adhC in M. smegmatis mc2155.
This hypothesis was confirmed when competent cells of one DCO mutant were prepared and another DCO event was selected from this strain, using a two-step strategy. This led us to isolate one clone which, by both PCR and Southern blot hybridization analysis, was shown to have the two copies of adhC replaced by two disrupted copies. Western blot hybridization results confirmed the results obtained by Southern blot hybridizations. Thus, a defective mutant unable to express ADHC was only obtained when two functional native adhC copies were disrupted by homologous recombination.
These results showed that adhC is not an essential gene for the in vitro growth of M. smegmatis. Surprisingly, we observed a difference in the growth and morphology for both M. smegmatis AdhC+/- and AdhC-/- when compared with the wild-type AdhC+/+ strain. In the absence of further information about the physiological role of the ADHC enzyme these observations are difficult to explain.
The preliminary biochemical characterization of the M. smegmatis AdhC+/- and AdhC-/- mutants did not help us to find out which aldehydes are closest to the ones used in vivo by this mycobacterial ADH, as the AdhC-/- knockout mutant does not use any of the structurally different substrates tested. Preliminary analysis of the lipid and aldehyde contents of the mutant strains compared to the wild-type M. smegmatis strain has not yet shown any significant differences.
M. smegmatis mc2155 genome duplication
Evidence accumulated during the generation of the M. smegmatis mc2155 adhC knockout mutants uncovered a large duplication in its genome. PFGE, which enables the separation of large DNAs and can be used to investigate genome organization, was used to obtain information about the duplication extension and localization. Although PFGE is routinely used in many mycobacterial species, we were not successful in analysing M. smegmatis DNA by PFGE when using the classical protocols for mycobacteria (Levy-Frebault et al., 1989 ; Philipp et al., 1996
). Similar difficulties had been previously described for the analysis of Streptomyces lividans DNA (Ray et al., 1992
), and were associated with Tris-dependent site-specific cleavage of the DNA. Tris-derived nucleolytic species have been shown to react with thiourea, which could thus protect the DNA from strand cleavage (Evans & Dyson, 1993
). We achieved non-degradative PFGE of the M. smegmatis DNA only when 50 µM thiourea was added to the running buffer. Southern blot analysis of DraI-digested genomic DNA from M. smegmatis mc2155, clone 31 and clone 18 resolved by PFGE demonstrated that M. smegmatis mc2155 harbours a large chromosomal duplication whose size may be at least
250 kb. Our results did not prove that the whole
250 kb region was duplicated; however, DraI sites are relatively rare in mycobacteria and it seems quite unlikely that two DraI sites would exist in the M. smegmatis genome at the same distance from the two adhC genes, even if they did not belong to a duplicated region. The chances of the existence of two identical (same size and sequence) fragments running together in the gel is much greater than the existence of two fragments of the same size, but with different sequences. Thus, the size of the duplication found in the M. smegmatis genome could be at least
250 kb.
Chromosomal duplication and resultant gene duplication are ubiquitous features of genome evolution and have been viewed as the predominant mechanisms for the evolution of new gene functions and adaptive responses (Lupski et al., 1996 ). Chromosomal duplications provide a means for increasing gene dosage and for generating novel functions from potential gene fusion events at duplication end points, and represent a source of redundant DNA for divergence. Over half of the proteins present in the tubercle bacillus have arisen from ancient gene duplication and adaptation events (Cole et al., 1998
; Tekaia et al., 1999
).
The generally accepted model for the formation of chromosomal duplication in bacteria is that, after chromosomal replication, misaligned repeated sequences (e.g. rrn operons, IS elements, transposons) (Lupski et al., 1996 ) or short DNA homologies (Edlund & Normark, 1981
) act as substrates for homologous recombination, leading to duplication or deletion of the specific region between the repeated sequences. The duplicated regions are usually flanked by repeated sequences in a directed orientation, and duplication formation is generally found to be highly dependent on the RecA function. Certain duplications have been observed which confer a growth advantage under specific selective conditions and play a role in the adaptation of micro-organisms as a method for gene amplification. When conditions arise which cannot be compensated for by an alteration in gene expression, either in the laboratory or in nature, selection may favour an increase in the copy number of a gene or a group of genes. In general these duplications are rather large, up to one-third of the chromosome, but they are highly unstable as they are typically tandem duplications and are lost by homologous recombination when environmental conditions change, thus representing a readily reversible source of genomic variation (Anderson & Roth, 1977
). Spontaneous tandem chromosomal duplications are common in populations of E. coli and Salmonella typhimurium (Haack & Roth, 1995
).
The existence of two tandem chromosomal duplications of the genome of M. bovis BCG Pasteur, DU1 (29668 bp) and DU2 (36161 bp) (Brosch et al., 2000 ) was recently reported and showed that these genomic regions of the BCG genome are still dynamic. This study concluded that BCG Pasteur is diploid for at least 58 genes and that at a certain point in its evolution contained duplicate copies of a further 60 genes which were lost when a deletion internal to DU2 arose.
The presence of a large (>250 kb) cis duplication in the M. smegmatis genome is described here for the first time. An amplification has already been described in M. smegmatis, in the gene pstB, encoding the putative ATPase subunit of the phosphate-specific transporter system, in a fluoroquinolone-resistant M. smegmatis strain (Bhatt et al., 2000 ). However, this is one of those cases where selection for increased gene dosage resulted in spontaneous amplification of one gene and there was no evidence that other flanking genes were duplicated or amplified.
We cannot exclude the possibility that the duplication we describe here had originated as the result of specific environmental conditions, but this is certainly a large chromosomal duplication that includes many genes. We propose that the duplication we found is a tandem duplication because non-tandem or inverted duplications appear to be very rare compared with the tandem ones. In fact, most of the duplications that have been analysed in detail in bacteria appear to be tandem duplications (Anderson & Roth, 1977 , 1981
; Brosch et al., 2000
; Heath, 1992
). The restriction analysis of M. smegmatis DNA with several restriction enzymes showed that this M. smegmatis chromosomal duplication is probably a recent one in which divergence of gene sequences has not yet occurred. The mechanism responsible for this duplication is still obscure. Since the assembled complete genome sequence of M. smegmatis is not yet available, we do not know which sequences could have been involved in the recombination event that originally generated this duplication.
A preliminary study of the sequencing and assembly of the complete M. smegmatis genome indicates that this, at nearly 7 Mbp, is approximately 50% larger than that of its slow-growing relatives (Merkel et al., 2001 ). Since the genome sequence of M. smegmatis was based upon sequencing shotgun clones which do not detect possible genomic duplications or other genomic rearrangements, the previewed size for the genome of this mycobacterial species might have been underestimated. Bacterial artificial chromosome (BAC) libraries, representing the complete genome of M. smegmatis, could be used for detection and localization of other possible duplications. Quantitative DNADNA hybridization has also been reported as a method which allows accurate determination of the size of merodiploid chromosomes and the identification of genes present in two copies per chromosome (Hauser & Karamata, 1994
).
Future strategies for disruption of M. smegmatis genes should consider the possibility of having to mutate more than one gene copy. Availability of the assembled complete genome sequence from M. smegmatis will enable the determination of which genes are included within the 250 kb duplicated region, but additional techniques (such as BACs or heteroduplex mapping) will be needed to determine its exact size, orientation and origin, and to detect other duplications and other possible complex genomic rearrangements within the M. smegmatis genome.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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---|
Anderson, P. & Roth, J. (1981). Spontaneous tandem genetic duplications in Salmonella typhimurium arise by unequal recombination between rRNA (rrn) cistrons. Proc Natl Acad Sci USA 78, 3113-3117.[Abstract]
Baulard, A., Gurdyal, S., Brennan, B. & Brennan, P. J. (1999). The cell-wall core of Mycobacterium: structure, biogenesis and genetics. In Mycobacteria. Molecular Biology and Virulence , pp. 240-259. Edited by C. Ratledge & J. Dale. London:Blackwell Science.
Besra, G. S., Morehouse, C. B., Rittner, C. M., Waechter, C. J. & Brennan, P. J. (1997). Biosynthesis of mycobacterial lipoarabinomannan. J Biol Chem 272, 18460-18466.
Bhatt, K., Banerjee, S. K. & Chakraborty, P. K. (2000). Evidence that phosphate specific transporter is amplified in a fluoroquinolone resistant Mycobacterium smegmatis. Eur J Biochem 267, 4028-4032.
Brosch, R., Gordon, S. V., Buchrieser, C., Pym, A. S., Garnier, T. & Cole, S. T. (2000). Comparative genomics uncovers large tandem chromosomal duplications in Mycobacterium bovis BCG Pasteur. Yeast 17, 111-123.[Medline]
Cole, S. T., Brosch, R., Parkhill, J. & 38 other authors (1998). Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537544.[Medline]
De Bruyn, J., Johannes, A., Weckx, M. & Beumer-Jochmans, M. P. (1981a). Partial purification and characterization of an alcohol dehydrogenase of Mycobacterium tuberculosis var. bovis (BCG). J Gen Microbiol 124, 359-363.[Medline]
De Bruyn, J., Weckx, M. & Beumer-Jochmans, M. P. (1981b). Effect of zinc deficiency on Mycobacterium tuberculosis var. bovis (BCG). J Gen Microbiol 124, 353-357.[Medline]
Dye, C., Scheele, S., Dolin, P., Pathania, V. & Raviglione, M. C. (1999). Consensus statement. Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country. WHO global surveillance and monitoring project. J Am Med Assoc 282, 677-686.
Edlund, T. & Normark, S. (1981). Recombination between short DNA homologies causes tandem duplication. Nature 292, 269-271.[Medline]
Evans, M. & Dyson, P. (1993). Pulsed-field gel electrophoresis of Streptomyces lividans DNA. Trends Genet 9, 72.[Medline]
Galamba, A., Soetaert, K., Buyssens, P., Monnaie, D., Jacobs, P. & Content, J. (2001). Molecular and biochemical characterisation of Mycobacterium smegmatis alcohol dehydrogenase C. FEMS Microbiol Lett 196, 51-56.[Medline]
Haack, K. R. & Roth, J. R. (1995). Recombination between chromosomal IS200 elements supports frequent duplication formation in Salmonella typhimurium. Genetics 141, 1245-1252.
Hauser, P. M. & Karamata, D. (1994). Characterization of the chromosomes of Bacillus subtilis merodiploid strains by quantitative DNADNA hybridization. Microbiology 140, 1605-1611.[Abstract]
Heath, J. D. (1992). Control of chromosomal rearrangements in Escherichia coli. PhD thesis, University of Texas, Health Science Center.
Hinds, J., Mahenthiralingam, E., Kempsell, K. E., Duncan, K., Stokes, R. W., Parish, T. & Stoker, N. G. (1999). Enhanced gene replacement in mycobacteria. Microbiology 145, 519-527.[Abstract]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head bacteriophage T4. Nature 227, 680-685.[Medline]
Lee, R. E., Brennan, P. J. & Besra, G. S. (1996). Mycobacterium tuberculosis cell envelope. Curr Top Microbiol Immunol 215, 1-27.[Medline]
Levy-Frebault, V. V., Thorel, M. F., Varnerot, A. & Gicquel, B. (1989). DNA polymorphism in Mycobacterium paratuberculosis, wood pigeon mycobacteria, and related mycobacteria analyzed by field inversion gel electrophoresis. J Clin Microbiol 27, 2823-2826.[Medline]
Lupski, J. R., Roth, J. R. & Weinstock, G. M. (1996). Chromosomal duplications in bacteria, fruit flies, and humans. Am J Hum Genet 58, 21-27.[Medline]
McFadden, J. (1996). Recombination in mycobacteria. Mol Microbiol 21, 205-211.[Medline]
Menard, R., Sansonetti, P. J. & Parsot, C. (1993). Nonpolar mutagenesis of the ipa genes defines IpaB, IpaC, and IpaD as effectors of Shigella flexneri entry into epithelial cells. J Bacteriol 175, 5899-5906.[Abstract]
Merkel, J., Feldblyum, T., Young, B., Kriakov, J., Jacobs, W. R.Jr & Fleishmann, R. D. (2001). Sequencing and assembly of the Mycobacterium smegmatis genome. In Keystone Symposium B1: Molecular and Cellular Aspects of Tuberculosis Research in the Post Genome Era , pp. 62. Edited by G. Kaplan, S. T. Cole & B. Brennan. Taos,New Mexico.
Mikusova, K., Mikus, M., Besra, G. S., Hancock, I. & Brennan, P. J. (1996). Biosynthesis of the linkage region of the mycobacterial cell wall. J Biol Chem 271, 7820-7828.
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, 525-534.[Medline]
Parish, T. & Stoker, N. G. (2000). Use of a flexible cassette method to generate a double unmarked Mycobacterium tuberculosis tlyA plcABC mutant by gene replacement. Microbiology 146, 1969-1975.
Parish, T., Gordhan, B. G., McAdam, R. A., Duncan, K., Mizrahi, V. & Stoker, N. G. (1999). Production of mutants in amino acid biosynthesis genes of Mycobacterium tuberculosis by homologous recombination. Microbiology 145, 3497-3503.
Pavelka, M. S.Jr & Jacobs, W. R.Jr (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, 4780-4789.
Pelicic, V., Reyrat, J. M. & Gicquel, B. (1996a). Expression of the Bacillus subtilis sacB gene confers sucrose sensitivity on mycobacteria. J Bacteriol 178, 1197-1199.[Abstract]
Pelicic, V., Reyrat, J. M. & Gicquel, B. (1996b). Generation of unmarked directed mutations in mycobacteria using sucrose counter-selectable suicide vectors. Mol Microbiol 20, 919-925.[Medline]
Philipp, W. J., Poulet, S., Eiglmeier, K. & 7 other authors (1996). An integrated map of the genome of the tubercle bacillus, Mycobacterium tuberculosis H37Rv, and comparison with Mycobacterium leprae. Proc Natl Acad Sci USA 93, 31323137.
Ray, T., Weaden, J. & Dyson, P. (1992). Tris-dependent site-specific cleavage of Streptomyces lividans DNA. FEMS Microbiol Lett 96, 247-252.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Spector, T. (1978). Refinement of the Coomassie blue method of protein quantitation. A simple and linear spectrophotometric assay for less than or equal to 0·5 to 5·0 microgram of protein. Anal Biochem 86, 142-146.[Medline]
Stelandre, M., Bosseloir, Y., De Bruyn, J., Maes, P. & Content, J. (1992). Cloning and sequence analysis of the gene encoding an NADP-dependent alcohol dehydrogenase in Mycobacterium bovis BCG. Gene 121, 79-86.[Medline]
Tekaia, F., Gordon, S. V., Garnier, T., Brosch, R., Barrell, B. G. & Cole, S. T. (1999). Analysis of the proteome of Mycobacterium tuberculosis in silico. Tuber Lung Dis 79, 329-342.[Medline]
Wilkin, J. M., Soetaert, K., Stelandre, M. & 7 other authors (1999). Overexpression, purification and characterization of Mycobacterium bovis BCG alcohol dehydrogenase. Eur J Biochem 262, 299307.
Received 15 June 2001;
revised 30 July 2001;
accepted 14 August 2001.