Department of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, Keppel Street, London WC1E 7HT, UK 1
Molecular Biology Unit, South African Institute for Medical Research, PO Box 1038, Johannesburg 2000, South Africa 2
Immunopathology Unit, Glaxo Wellcome Research and Development, Medicines Research Centre, Stevenage, UK3
Department of Haematology and Molecular Medicine, University of the Witwatersrand, Johannesburg, South Africa4
Author for correspondence: Neil G. Stoker. Tel: +44 20 7927 2425. Fax: +44 20 7637 4314. e-mail: neil.stoker{at}lshtm.ac.uk
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ABSTRACT |
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Keywords: auxotrophs, gene replacement, lacZ , Casamino acids, pre-treated DNA
Abbreviations: DCO, double crossover; HR, homologous recombination; IR, illegitimate recombination; hygR, hygromycin resistant; SCO, single crossover
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INTRODUCTION |
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We have previously shown that pre-treatment of the transforming DNA increases the rate of HR, which enabled us to isolate mutants in three mycobacterial species: M. smegmatis, Mycobacterium intracellulare and M. tuberculosis (Hinds et al. , 1999 ). Although we had obtained a tlyA mutant of M. tuberculosis using pre-treated DNA (Hinds et al., 1999
), it was important to look at different genes in order to assess the general applicability of this delivery system. Here we describe the use of DNA pre-treatment to test our system with a further 11 genes and present an improved procedure for simply and reliably isolating targeted mutants of M. tuberculosis. We selected genes involved in amino acid biosynthesis as our candidates for knockouts because mutants in these genes may be attenuated in vivo as has been documented for Salmonella typhimurium (OCallaghan et al., 1988
). Such mutants would provide important information regarding the host environment encountered by the bacterium and may be potentially useful in new vaccine development. The additional advantages were that mutants would have easily testable phenotypes and the genes are unlikely to be essential on rich media in vitro.
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METHODS |
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Construction of lacZ delivery vectors.
The complete Escherichia coli lacZ gene from plasmid pATB12 (Martin Everett, Glaxo Wellcome) was cloned as a Hin dIII fragment into the HindIII site of five suicide vectors (Table 2) and expression of lacZ confirmed in E. coli as demonstrated by the formation of blue colonies on X- Gal plates. The lac promoter is not itself functional in mycobacteria, so we assume that lacZ expression in mycobacteria occurs from a cryptic promoter.
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Treatment of transforming DNA.
Vector DNA was treated prior to transformation as described (Hinds et al., 1999 ). Briefly, UV irradiation of DNA was carried out in an UV Stratalinker 1800 (Stratagene) at 100 mJ cm -2. Alkali denaturation was performed in a 100 µl volume containing 0·2 M NaOH, 0·2 mM EDTA at 37 °C for 30 min, followed by ethanol precipitation.
Analysis of transformants.
Transformants were inoculated into 10 ml liquid media (plus hygromycin and supplements) and cultured standing at 37 °C for DNA preparation by the method of Belisle & Sonnenberg (1998) or Santos et al. (1992)
. Southern blotting was carried out using probes labelled by the AlkPhos Direct method (Amersham). Initial auxotrophy testing was carried out by patching transformant colonies onto 7H10/OADC plates with and without the appropriate supplements and cultured at 37 °C for 34 weeks before reading results. Auxotrophy was confirmed both by plating liquid cultures onto 7H10/OADC plates with and without individual amino acid supplements and by subculturing into unsupplemented 7H9/OADC/Tween broth and checking for lack of growth.
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RESULTS |
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Isolation of DCOs using lacZ
The large number of spontaneous hygR mutants obtained prompted us to include the lacZ gene from E. coli as a screenable marker in the suicide vectors. This would enable us to identify SCOs in the initial transformation since they would form blue colonies on plates containing X-Gal and hygromycin; white colonies should arise from DCO events (as well as spontaneous hygromycin resistance). The lacZ gene was therefore cloned into three vectors already used (containing disrupted trpD, hisD and glnE genes) and two new suicide vectors were constructed, containing disrupted proC and argF genes (Table 2 ). DNA was pre-treated with either UV or alkali and used in electroporations.
Results from several experiments are shown in Table 4
. The total number of transformants was much higher than in the previous experiments, but the numbers were still very variable between experiments. Furthermore, the number of transformants obtained (and the ability to isolate recombinants) did not appear to correlate with the electroporation efficiencies obtained using replicating plasmids. For example, with the glnE plasmid, on one occasion we obtained 200 blue and 2 white colonies (10 4 c.f.u. per µg replicating plasmid) and on another we obtained only 3 blue and 11 white colonies (105 c.f.u. per µg replicating plasmid). We do not know what factors are responsible for this variation.
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For glnE, a large number of blue colonies (500) from three experiments, but very few whites, were isolated. All of the white colonies were found to be spontaneous hygR mutants; the absence of any DCOs may indicate that this gene is essential for growth on the medium used. The use of lacZ enables one to determine the frequency at which HR is taking place to produce SCOs, thus allowing a quick preliminary screen for essential genes the absence of DCOs in such a large population of SCOs is indicative of HR, but suggests that DCOs may be lethal under the conditions used. No argF mutants were isolated; the number of blue colonies was lower than that obtained with glnE, so there was less compelling evidence that a DCO would be deleterious in this case.
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DISCUSSION |
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The metB mutant obtained was not auxotrophic, showing no growth requirement for methionine. This gene encodes a putative cystathionine -synthase, which converts O-succinyl-L- homoserine to cystathionine. One possible explanation is the possession of another gene/enzyme, that can complement or compensate for the loss of MetB activity. The complete genome sequence reveals that another gene (metZ; Rv0391) may also be a cystathionine
- synthase, so it is possible that this gene product may have an overlapping function. Alternatively, the site of the hyg gene insertion may not have inactivated the gene function, although the insertion lies near the 5' end (70 bp downstream of the predicted start codon).
Initial attempts to construct defined auxotrophs by HR were unsuccessful. Only one DCO mutant was obtained and that was prototophic. The hisD auxotroph was tested for its ability to grow on media supplemented with Casamino acids and failed to grow, confirming that they are toxic at the concentration we used (0·5%). This confirms the recent observations of Pavelka & Jacobs (1999) , and is an important consideration for other groups trying to isolate auxotrophs.
In the case of the SCOs analysed, the side on which recombination had occurred was that on which the flanking DNA was longer. The size ranged from 1·5 kb (aroK) to 3·8 kb ( glnE). For the four DCOs where HR had necessarily occurred on both sides, the range of flanking DNA was from 0·7 kb ( metB) to 3·3 kb (trpD). Therefore, the need for regions of homology >1kb on each side of the mutated gene is not an absolute requirement, but is probably desirable. However, there is a trade-off in using longer flanking DNA to increase the efficiency of recombination since the efficiency of transformation is expected to fall with increasing plasmid size.
IR and spontaneous mutants
IR was not observed in any of these experiments. Care was taken to ensure that the restriction enzymes and probes used for Southern blotting would distinguish SCOs from illegitimate recombinants. IR has been one of the barriers to routinely obtaining gene knockouts, since it masks the low frequency of HR and requires that significant numbers of transformants are analysed in order to find the HR transformants which, for M. tuberculosis, is a laborious and time-consuming process. IR has been reported in M. tuberculosis (Kalpana et al., 1991 ; Pavelka & Jacobs, 1999
), M. bovis (Wilson et al., 1997
) and M. bovis BCG (Aldovini et al., 1993
; Kalpana et al., 1991
), but now generally appears to be a nuisance rather than an overwhelming problem. The success of our method in attaining HR in 11 genes without any IR may be due to the fact that IR is not as big a problem as originally feared or that the method we use favours HR over IR, or it may be a combination of these factors.
Spontaneous resistance to hygromycin is potentially a problem because M. tuberculosis only has a single rRNA operon, and therefore is prone to resistance due to chromosomal mutations. Although the spontaneous mutation rate was reasonably constant, considerable variation in the efficiency of recombination for a given suicide vector was observed between transformations. As a result, the magnitude of the background problem caused by spontaneous mutants varied between experiments. However, this problem was largely overcome by including a second screenable marker, lacZ, in the vector.
Differences in DNA pre-treatment
UV pre-treatment was the more successful and technically easiest method, generating larger numbers of HR transformants than alkali pre- treatment. However, a potential drawback of UV treatment is that recombination with a DNA-damaged vector might result in the inadvertent introduction of secondary mutations. Although we have not directly tested for this, the fact that no lacZ mutants were identified amongst the large number of SCOs characterized in this study argues against mutagenesis of the integrated DNA occurring at a high frequency. Little, if anything, is known about the mutagenesis pathways that may operate in M. tuberculosis in response to DNA damage. A major mechanism for UV-induced mutagenesis in E. coli is via the umuCD-dependent SOS-inducible pathway. No umuD has been identified in the M. tuberculosis genome (Cole et al., 1998 ; Mizrahi & Andersen, 1998
), suggesting that the SOS-inducible mutagenesis pathway is unlikely to be operative in this organism, although we cannot exclude the possibility that other inducible pathways exist (Humayun, 1998
).
Alkali treatment was also successful in obtaining both SCO and DCO mutants, albeit at a lower rate. The difference in numbers of recombinants may reflect loss of DNA during precipitation or differences in the DNA substrates participating in the recombination events in vivo.
Use of lacZ
The inclusion of lacZ into the suicide vector was an effective way of revealing colonies carrying plasmids integrated by SCOs. Thus, with lacZ it is possible to judge firstly if the experiment looks successful in terms of achieving any HR, and secondly, allows blue colonies to be ignored when looking for DCOs. If no DCOs are found, a blue (SCO) colony can be picked and plated for a second recombination event, although the inclusion of a negative selection marker such as sacB may be necessary (Pelicic et al., 1996 ).
Conclusion
We have developed a simple method for generating defined mutants of M. tuberculosis by HR and have demonstrated its general utility in this paper using 11 genes. This has allowed the isolation of four DCO mutants in amino acid biosynthesis genes.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 3 June 1999;
accepted 20 August 1999.