Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK1
Department of Medical Microbiology, Barts and the London, Queen Marys School of Medicine and Dentistry, 32 Newark Street, London E1 2AA, UK2
Author for correspondence: Tanya Parish. Tel: +44 20 7377 0444. Fax: +44 20 7247 3428. e-mail: t.parish{at}qmul.ac.uk
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ABSTRACT |
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Keywords: chorismate biosynthesis, auxotrophs, gene replacement, bacteriophage L5, excisionase
Abbreviations: BCG, bacille CalmetteGuérin; DAHP, 3-deoxy-D-arabino-heptulosonate-7-phosphate
a Present address: Department of Pathology and Infectious Diseases, Royal Veterinary College, Royal College Street, London NW1 0TU, UK.
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INTRODUCTION |
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The shikimate biosynthetic pathway, in which 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) is converted to chorismate, is required for synthesis of all aromatic amino acids, as well as other important metabolites in bacteria (Fig. 1; Pittard, 1987
). Disruption of genes in this pathway has been successfully employed in a wide range of bacterial species to generate strains that are attenuated in models of infection (Gunel-Ozcan et al., 1997
; Hoiseth & Stocker, 1981
; Ingham et al., 2002
; Simmons et al., 1997
; Vaughan et al., 1993
). Such strains have been used as live attenuated vaccines (Dilts et al., 2000
). With this in mind, the aroA, aroB and aroQ genes were among the first M. tuberculosis genes to be cloned and sequenced, and proposed as attractive targets for gene disruption in order to generate a rationally attenuated vaccine to replace BCG (Garbe et al., 1990
, 1991
).
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METHODS |
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Construction of vectors.
The aroK::hyg disrupted allele from plasmid pAROB2 (Parish et al., 1999
) was cloned into p2NIL (Parish & Stoker, 2000a
), which does not replicate in mycobacteria, to make pAROB11. This allele has a 324 bp NruI internal fragment of the gene replaced with the hyg gene. The vector carries 0·8 kb of the 3' end of the 1·2 kb aroC gene. The marker gene Phsp60-sacB from pGOAL13 (Parish & Stoker, 2000a
) was then cloned into the unique PacI site of pAROB11 to make the final non-replicating delivery vector pAROB14 (Fig. 2b
). The wild-type aroK+ allele (Fig. 2a
) was cloned as an EcoRV fragment from cosmid Y159 into pUC-Gm-INT (Mahenthiralingam et al., 1998
) which carries the L5 integrase (int) and attachment site (attP) and a gentamicin-resistance gene (aacC1), to make the integrating vector pAINT1 (Fig. 2c
).
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Isolation of double cross-over strains from Aroma1.
The single cross-over strain (Aroma1) was streaked out onto media containing aro supplement but without antibiotics and incubated for 2 weeks at 37 °C. A loopful of cells was resuspended in liquid media by vortexing with 1 mm glass beads. Serial dilutions were plated onto 2% (w/v) sucrose plates plus aro supplement (and hygromycin where required). Plates were incubated at 37 °C for 46 weeks. Sucrose-resistant colonies were then patch-tested for kanamycin and hygromycin resistance on plates containing aro supplement.
Construction of merodiploid strain (Aroma3) and isolation of double cross-over strains.
Aroma1 (single cross-over strain) was electroporated with the integrating vector pAINT1. Transformants carrying the vector were selected on gentamicin, kanamycin and hygromycin without aro supplement. An individual colony was picked for further manipulation (Aroma3). Isolation of double cross-over strains was carried out essentially as for the single cross-over strain. Briefly, Aroma3 was streaked out onto media containing aro supplement but without antibiotics and incubated for 2 weeks at 37 °C. A loopful of cells was resuspended in liquid media by vortexing with 1 mm glass beads. Serial dilutions were plated onto 2% (w/v) sucrose plates plus aro supplement (and hygromycin where required). Plates were incubated at 37 °C for 46 weeks. Sucrose-resistant colonies were then patch-tested for kanamycin and hygromycin resistance on plates containing aro supplement.
Excision of L5-integrated plasmids.
Aroma5 and Aroma6 strains were electroporated with 0·5 µg plasmid pJL28 and plated onto kanamycin plus aro supplement to select for cells carrying the xis plasmid (Parish et al., 2001 ). Kanamycin-resistant colonies were then patch-tested for gentamicin resistance to determine whether the integrated vector was still present.
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RESULTS |
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There is some confusion over the nomenclature of the aro genes. We have designated the chorismate synthase (Rv2540c) gene as aroC to avoid confusion (it is described as aroF in the original Sanger Centre annotation), since it is annotated as aroC in other bacteria and in the well-characterized Escherichia coli pathway, aroF encodes one of the three DAHP synthases. The dehydroquinate synthase gene (Rv2537c) is designated as aroD in the annotation, but we prefer aroQ as named in the original characterization (Garbe et al., 1991 ) to reflect the fact that it has homology to fungal catabolic 3-dehydroquinases (aroQ) rather than the prokaryotic biosynthetic 3-dehydroquinases (aroD).
Construction of a single cross-over strain (Aroma1)
We were interested in constructing mutants of M. tuberculosis for the aro pathway, since in other bacteria mutants are attenuated (Gunel-Ozcan et al., 1997 ; Hoiseth & Stocker, 1981
; Ingham et al., 2002
; Simmons et al., 1997
; Vaughan et al., 1993
). We have previously attempted to construct aroK double cross-over strains where the chromosomal copy is replaced by a hygromycin-disrupted allele using a one-step strategy (Parish et al., 1999
). This was unsuccessful and demonstrated that there was a low frequency of recombination at this locus (Parish et al., 1999
). We therefore changed to a two-step strategy (Parish & Stoker, 2000a
) and tried to isolate double cross-over strains from a single cross-over strain. A two-step strategy is more efficient when there is a low frequency of homologous recombination at a locus, since only one single cross-over is required at each step.
pAROB14 (Fig. 2) was electroporated into M. tuberculosis and single cross-over strains selected using hygromycin and kanamycin. Using 2 µg DNA, only one single cross-over colony was obtained. PCR analysis (Fig. 4
) and Southern blotting confirmed that it had the single cross-over genotype and demonstrated that recombination had occurred within the shorter 5' region of homology (1·0 kb as compared to 1·5 kb). In our previous work we had obtained a single cross-over strain where recombination had taken place within the longer 3' region (Parish et al., 1999
). This showed that recombination could occur on either side to generate viable strains. This strain (Aroma1) is not an aro mutant since it could be grown in the absence of aro supplement.
Second step selection for double cross-over strains from Aroma1
The single cross-over strain (Aroma1) was then used to try to isolate double cross-over strains. Aroma1 was streaked onto a fresh agar plate (containing aro supplement, but in the absence of antibiotics) to allow the second cross-over to occur. Since the delivery vector contained Phsp60-sacB, the strain is sensitive to sucrose. Double cross-over strains can lead to wild-type or mutant cells, depending on the location of the second cross-over. We therefore plated the cells onto plates containing sucrose, hygromycin and aro supplement to select for the double cross-overs leading to the mutant allele. This confirmed that the strain was sucrose-sensitive since there was a reduction of approximately 104 c.f.u. on plates containing sucrose as compared to those without sucrose. Colonies were then tested for kanamycin resistance to distinguish double cross-overs (kanS) from spontaneous sucrose-resistant (kanR) mutants. Sixty-seven colonies were tested and all were spontaneous sucrose-resistant, single cross-over strains. Thus, no mutant double cross-over strains were isolated. The absence of any mutants using this method suggested that the gene is essential.
We repeated the sucrose selection step in the absence of hygromycin to confirm that homologous recombination was occurring at this locus at a frequency that could be observed. Of 40 sucrose-resistant colonies tested, 32 were spontaneous sucrose-resistant strains (kanR hygR) and eight were wild-type double cross-overs (kanS, hygS). None were mutant double cross-overs (kanS, hygR). This confirmed that we were seeing the second cross-over events, so the absence of mutants was not due to a complete lack of homologous recombination.
Construction of a merodiploid strain (Aroma3) and second step selection for double cross-over strains
We had already demonstrated the essentiality of another gene, glnE, by generating a merodiploid strain, which demonstrates that the lack of mutants isolated is not due to the lack of cross-overs leading to mutants, but rather to the viability of the mutants (Parish & Stoker, 2000b ). We used a similar strategy to determine whether aroK is essential. First, we constructed a merodiploid strain (Aroma3) from the single cross-over strain Aroma1, which carried a second wild-type copy of the gene in a different region of the chromosome. An L5 phage-based integrating vector carrying an intact copy of aroK (pAINT1; Fig. 2
) was electroporated into Aroma1. The construct was designed to carry the upstream region of aroC as well and therefore contained the natural promoter of the operon. Gentamicin-resistant colonies were isolated and the presence of the integrating plasmid confirmed by Southern analysis. We then plated Aroma3 onto sucrose plates to isolate double cross-over strains as we had done previously with Aroma1. No hygromycin was included, so that both wild-type and mutant double cross-over strains could be isolated. Out of 40 sucrose-resistant colonies tested, nine were wild-type double cross-over strains, one was a mutant double cross-over strain and the remaining 30 were spontaneous sucrose-resistant mutants (single cross-overs). The mutant (Aroma5) was analysed by Southern blotting showing that in the mutant double cross-over strain, the wild-type aroK allele had indeed been replaced, whilst the L5-integrated copy was retained. These results demonstrated that homologous recombination was occurring at this locus to obtain both double cross-over strains although the frequency to give rise to the disrupted allele was low.
Excision of L5-integrated plasmid from double cross-over strain
Since the number of double cross-over strains obtained was very low in both experiments as compared to the background of spontaneous sucrose resistance, the results were not statistically significant. The bias of the second recombination event towards the wild-type strains means that obtaining enough double cross-over strains to obtain statistical significance would be extremely difficult and laborious. We therefore used a second approach to confirm that the gene was indeed essential (Fig. 5), in which the L5-integrated plasmid is efficiently excised using the L5 excisionase gene (Parish et al., 2001
).
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DISCUSSION |
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We have used both one- (Parish et al., 1999 ) and two-step recombination strategies to construct a mutant strain carrying a disrupted allele of aroK. In each approach, the disrupted allele was the same, but the delivery vector backbone was different. Using either vector we have seen a very low frequency of recombination as compared to other loci. Previous work has demonstrated that the frequency of recombination does vary greatly between loci (Parish et al., 1999
) and these data support the hypothesis that there is an inherently low frequency of recombination at the aroK locus. It is unlikely that the low frequency reflects an inability to get plasmids into the cells as the transformation efficiency using a replicating plasmid is 107 per µg DNA.
The possibility that the aroK::hyg mutation results in a polar effect on downstream genes can be discounted. The aroK gene is found in a cluster of four closely linked shikimate pathway genes with the structure aroCaroKaroBaroQ, which probably form a single transcription unit. Although the insertion of the hyg gene may interrupt transcription from the natural promoter, the aroB and aroQ genes should still be expressed from the hyg promoter. This is confirmed by the fact that the region used in the complementing integrated vector contained aroC and aroK, but not the two downstream genes. Thus the inability to obtain gene replacement cannot be attributed to disruption of the function of aroB and/or aroQ.
The end product of the common aromatic amino acid pathway is chorismate, the precursor for biosynthesis of the aromatic amino acids tyrosine, tryptophan and phenylalanine, as well as folate and ubiquinone (Fig. 1). We expected that the aromatic supplements used would be sufficient to permit the growth of aroK mutants, as it is in other bacteria that have been studied. Fig. 1
shows the supplements added to the media in relation to the pathway. Several possibilities exist to explain the essentiality of this pathway. The first is that the supplements are not transported into the cell. We have already isolated a tryptophan auxotroph, which can be grown in the presence of L-tryptophan, showing that this amino acid is transported into the cell (Parish et al., 1999
). M. tuberculosis has a homologue of AroP2, an aromatic amino acid transporter (Cole et al., 1998
), so we would expect that the other aromatic amino acids are transported into the cell as well.
The second possibility is that M. tuberculosis relies on a constant supply of the final product of one or more downstream pathway(s) for its survival. It is feasible that there is another, as yet uncharacterized, branch from the aromatic pathway in mycobacteria. The end product of this pathway would have to be added to the supplements in order for aroK mutants to be viable. We also tried to isolate mutants in the presence of chorismate supplementation, but again were unsuccessful, suggesting that a branch point after chorismate itself is unlikely.
There are several differences between the well-characterized E. coli genes and the M. tuberculosis homologues. The first step of the pathway is catalysed by three enzymes in E. coli, each of which is independently regulated, whereas M. tuberculosis possesses only one DAHP synthase (aroG). E. coli also has two shikimate kinase genes (aroK and aroL), whilst M. tuberculosis only has one. Therefore there appears to be less redundancy in the M. tuberculosis pathway. Vinella et al. (1996) identified a second function for the E. coli aroK gene product that is distinct and unrelated to its shikimate kinase activity. Should the M. tuberculosis enzyme possess a similar activity it is possible that disruption of this activity would explain the inability of the bacteria to grow in the absence of a functional copy of the gene.
Analysis of the genetic organization of the aro genes between slow- and fast-growing mycobacteria reveals differences in the main aro operon. This may reflect differences in the regulation or the function of the pathway. We do not know if the common aromatic amino acid pathway is essential in all mycobacteria or whether this phenomenon is confined to M. tuberculosis alone.
The genes and pathways that are essential for the growth of M. tuberculosis make attractive drug targets since inhibiting their function may kill the organism. The chorismate pathway is one of great interest in that it makes a good target for drug development. The pathway is absent from the human host, thus the products cannot be gleaned from the host environment and inhibition of any of the mycobacterial enzymes is unlikely to have a toxic side effect on the host. The evidence that this pathway is essential for M. tuberculosis even in the presence of exogenous supplements reinforces its attractiveness as a drug target. It is interesting to note that the M. tuberculosis shikimate kinase (AroK) has been crystallized (Gu et al., 2001 ) and the structure of the dehydroquinase (AroQ) has been determined (Gourley et al., 1999
), opening the possibility of rational design of inhibitors.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 27 March 2002;
revised 26 June 2002;
accepted 27 June 2002.