The authors have developed a simple and highly efficient system for generating allelic exchanges in both fast-and slow-growing mycobacteria. In this procedure a gene of interest, disrupted by a selectable marker, is cloned into a conditionally replicating (temperature-sensitive) shuttle phasmid to generate a specialized transducing mycobacteriophage. The temperaturesensitive mutations in the mycobacteriophage genome permit replication at the permissive temperature of 30 SC but prevent replication at the nonpermissive temperature of 37 SC. Transduction at a non-permissive temperature results in highly efficient delivery of the recombination substrate to virtually all cells in the recipient population. The deletion mutations in the targeted genes are marked with antibiotic-resistance genes that are flanked by γδ-res (resolvase recognition target) sites. The transductants which have undergone a homologous recombination event can be conveniently selected on antibiotic-containing media. To demonstrate the utility of this genetic system seven different targeted gene disruptions were generated in three substrains of Mycobacterium bovis BCG, three strains of Mycobacterium tuberculosis, and Mycobacterium smegmatis. Mutants in the lysA, nadBC, panC, panCD, leuCD, Rv3291c and Rv0867c genes or operons were isolated as antibiotic-resistant (and in some cases auxotrophic) transductants. Using a plasmid encoding the γδ-resolvase (tnpR), the resistance genes could be removed, generating unmarked deletion mutations. It is concluded from the high frequency of allelic exchange events observed in this study that specialized transduction is a very efficient technique for genetic manipulation of mycobacteria and is a method of choice for constructing isogenic strains of M. tuberculosis, BCG or M. smegmatis which differ by defined mutations.
Mycobacterium tuberculosis is a specialized intracellular pathogen that must regulate gene expression to overcome stresses produced by host defenses during infection. SigH is an alternative sigma factor that we have previously shown plays a role in the response to stress of the saprophyte Mycobacterium smegmatis. In this work we investigated the role of sigH in the M. tuberculosis response to heat and oxidative stress. We determined that a M. tuberculosis sigH mutant is more susceptible to oxidative stresses and that the inducible expression of the thioredoxin reductase/thioredoxin genes trxB2/trxC and a gene of unknown function, Rv2466c, is regulated by sigH via expression from promoters directly recognized by SigH. We also determined that the sigH mutant is more susceptible to heat stress and that inducible expression of the heat shock genes dnaK and clpB is positively regulated by sigH. The induction of these heat shock gene promoters but not of other SigH-dependent promoters was markedly greater in response to heat versus oxidative stress, consistent with their additional regulation by a heat-labile repressor. To further understand the role of sigH in the M. tuberculosis stress response, we investigated the regulation of the stress-responsive sigma factor genes sigE and sigB. We determined that inducible expression of sigE is regulated by sigH and that basal and inducible expression of sigB is dependent on sigE and sigH. These data indicate that sigH plays a central role in a network that regulates heat and oxidative-stress responses that are likely to be important in M. tuberculosis pathogenesis.Tuberculosis remains a major cause of human suffering, exacting an enormous toll of morbidity and mortality in much of the world (11). The cause of tuberculosis, the obligate pathogen Mycobacterium tuberculosis, is highly adapted for survival in the host organism. Following infection M. tuberculosis is ingested by macrophages and must persist in this environment in order to survive, either in a quiescent state or through active replication that results in tissue destruction and the disease of tuberculosis. In adapting to this intracellular environment, this bacterium must regulate its physiology to survive a variety of stresses produced by the macrophage, including reactive oxygen and reactive nitrogen species produced by these cells (1, 4, 28). In addition M. tuberculosis has been shown to alter the physiology of the macrophage to modulate host defenses (51). Although the sequencing of the M. tuberculosis genome and recent insights are beginning to shed light on pathogenic mechanisms of this organism (7,8,27,30), the means by which M. tuberculosis adapts to survive and replicate in the host remain poorly understood.Data from a number of laboratories have implicated several alternative sigma factors of mycobacteria, including SigB, SigE, SigF, and SigH, in the adaptation of the pathogen M. tuberculosis and the saprophyte Mycobacterium smegmatis to several stresses (5,9,12,20,29,53). M. smegmatis SigH has been shown ...
Transposon mutagenesis provides a direct selection for mutants and is an extremely powerful technique to analyze genetic functions in a variety of prokaryotes. Transposon mutagenesis of Mycobacterium tuberculosis has been limited in part because of the inefficiency of the delivery systems. This report describes the development of conditionally replicating shuttle phasmids from the mycobacteriophages D29 and TM4 that enable efficient delivery of transposons into both fast-and slow-growing mycobacteria. These shuttle phasmids consist of an Escherichia coli cosmid vector containing either a miniTn10(kan) or Tn5367 inserted into a nonessential region of the phage genome. Thermosensitive mutations were created in the mycobacteriophage genome that allow replication at 30°C but not at 37°C (
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