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ABSTRACT: A simple and efficient delivery system was developed for making targeted gene knockouts in Mycobacterium smegmatis. This delivery system relies on the use of a pair of replicating plasmids, which are incompatible. Incompatible plasmids share elements of the same replication machinery and so compete with each other during both replication and partitioning into daughter cells. Such plasmids can be maintained together in the presence of antibiotics; however, removal of selection leads to the loss of one or both plasmids. For mutagenesis, two replicating plasmids based on pAL5000 are introduced; one of these plasmids carries a mutated allele of the targeted gene. Homologous recombination is allowed to take place, and either one or both of the vectors are lost through the pressure of incompatibility, allowing the phenotypic effects of the mutant to be studied. Several different plasmid combinations were tested to optimize loss in the absence of antibiotic selection. pAL5000 carries two replication genes (repA and repB), which act in trans, and the use of vectors that each lack one rep gene and complement each other resulted in the loss of both plasmids in M. smegmatis and Mycobacterium bovis BCG. The rate of loss was increased by the incorporation of an additional incompatibility region in one of the plasmids. To facilitate cloning when the system was used, we constructed plasmid vector pairs that allow simple addition of selection and screening genes on flexible gene cassettes. Using this system, we demonstrated that M. smegmatis pyrF mutants could be isolated at high frequency. This method should also be useful in other species in which pAL5000 replicates, including Mycobacterium tuberculosis.
Applied and Environmental Microbiology 02/2003; 69(1):517-23. · 3.83 Impact Factor
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Ruth A McAdam,
Selwyn Quan,
Debbie A Smith,
Stoyan Bardarov,
Joanna C Betts,
Fiona C Cook,
Elizabeth U Hooker,
Alan P Lewis,
Peter Woollard,
Martin J Everett,
Pauline T Lukey,
Gregory J Bancroft,
William R Jacobs Jr,
Ken Duncan
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ABSTRACT: A library of Mycobacterium tuberculosis insertional mutants was generated with the transposon Tn5370. The junction sequence between the transposon and the mycobacterial chromosome was determined, revealing the positions of 1329 unique insertions, 1189 of which were located in 351 different ORFs. Transposition was not completely random and examination of the most susceptible genome regions revealed a lower-than-average G+C content ranging from 54 to 62 mol%. Mutants were obtained in all of the recognized M. tuberculosis functional protein-coding gene classes. About 30% of the disrupted ORFs had matches elsewhere in the genome that suggested redundancy of function. The effect of gene disruption on the virulence of a selected set of defined mutants was investigated in a severe combined immune deficiency (SCID) mouse model. A range of phenotypes was observed in these mutants, the most notable being the severe attenuation in virulence of a strain disrupted in the Rv1290c gene, which encodes a protein of unknown function. The library described in this study provides a resource of defined mutant strains for use in functional analyses aimed at investigating the role of particular M. tuberculosis genes in virulence and defining their potential as targets for new anti-mycobacterial drugs or as candidates for deletion in a rationally attenuated live vaccine.
Microbiology 11/2002; 148(Pt 10):2975-86. · 3.06 Impact Factor
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ABSTRACT: A mutant of Mycobacterium tuberculosis defective in the metabolism of L-arginine was constructed by allelic exchange mutagenesis. The argF mutant strain required exogenous L-arginine for growth in vitro, and in the presence of 0.96 mM L-arginine, it achieved a growth rate and cell density in stationary phase comparable to those of the wild type. The mutant strain was also able to grow in the presence of high concentrations of argininosuccinate, but its auxotrophic phenotype could not be rescued by L-citrulline, suggesting that the DeltaargF::hyg mutation exerted a polar effect on the downstream argG gene but not on argH. The mutant strain displayed reduced virulence in immunodeficient SCID mice and was highly attenuated in immunocompetent DBA/2 mice, suggesting that L-arginine availability is restricted in vivo.
Infection and Immunity 07/2002; 70(6):3080-4. · 4.16 Impact Factor
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ABSTRACT: The search for new TB drugs that rapidly and effectively sterilize the tissues and are thus able to shorten the duration of chemotherapy from the current 6 months has been hampered by a lack of understanding of the metabolism of the bacterium when in a 'persistent' or latent form. Little is known about the condition in which the bacilli survive, although laboratory models have shown that Mycobacterium tuberculosis can exist in a non-growing, drug-resistant state that may mimic persistence in vivo. Using nutrient starvation, we have established a model in which M. tuberculosis arrests growth, decreases its respiration rate and is resistant to isoniazid, rifampicin and metronidazole. We have used microarray and proteome analysis to investigate the response of M. tuberculosis to nutrient starvation. Proteome analysis of 6-week-starved cultures revealed the induction of several proteins. Microarray analysis enabled us to monitor gene expression during adaptation to nutrient starvation and confirmed the changes seen at the protein level. This has provided evidence for slowdown of the transcription apparatus, energy metabolism, lipid biosynthesis and cell division in addition to induction of the stringent response and several other genes that may play a role in maintaining long-term survival within the host. Thus, we have generated a model with which we can search for agents active against persistent M. tuberculosis and revealed a number of potential targets expressed under these conditions.
Molecular Microbiology 03/2002; 43(3):717-31. · 5.01 Impact Factor
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ABSTRACT: A secretion reporter system based on Staphylococcus aureus nuclease (nuc) was developed for use in mycobacteria. Fusion of secretion signals to the reporter cloned in a shuttle vector, pBPnuc1, resulted in halo formation around colonies of Mycobacterium smegmatis and Mycobacterium tuberculosis grown on DNase agar plates containing Methyl Green indicator dye. This in-situ detection system was used to identify secreted proteins by screening a pBPnuc1::H37Rv nuc gene fusion library in M. smegmatis. The clones identified in this screen all formed colony halos when present in M. tuberculosis grown on indicator media. The proteins corresponded to DesA2, a stearoyl-acyl carrier protein desaturase, PepA, a putative serine protease and the Apa antigen, which is the ATP-binding subunit of an ABC transport system. Of these proteins, only PepA and Apa contained recognizable leader peptides.
Gene 12/1999; · 2.34 Impact Factor
