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Bacterial chromosomally encoded type II toxin-antitoxin (TA) loci may be involved in survival upon exposure to stress and have been linked to persistence and dormancy. Therefore, understanding the role of the numerous predicted TA loci within the human pathogen Mycobacterium tuberculosis has become a topic of great interest. Antitoxin proteins are known to autoregulate TA expression under normal growth conditions, but it is unknown whether they have a more global role in transcriptional regulation. This study focuses on analyzing the regulatory role of the M. tuberculosis HigA antitoxin. We first show that the M. tuberculosis higBA locus is functional within its native organism, as higB, higA, and Rv1957 were successfully deleted from the genome together while the deletion of higA alone was not possible. The effects of higB-Rv1957 deletion on M. tuberculosis global gene expression were investigated, and a number of potential HigA-regulated genes were identified. Transcriptional fusion and protein-DNA-binding assays were utilized to confirm the direct role of HigA in Rv1954A-Rv1957 repression, and the M. tuberculosis HigA DNA-binding motif was defined as ATATAGG([N.sub.6])CCTATAT. As HigA failed to bind to the next-most-closely related motif within the M. tuberculosis genome, HigA may not directly regulate any other genes in addition to its own operon. doi: 10.1128/JB.00454-10

Mycobaeterium tuberculosis ruvC was induced by DNA damage in a ArecA strain despite having an appropriately positioned SOS box to which LexA binds in vitro. An inducible transcript start mapped within the SOS box, and transcriptional fusions identified the promoter. Disruption of the SOS box did not prevent induction, indicating that an alternative mechanism plays a significant role in the control of ruvC expression. doi: 10.1128/JB.01066-09

In this study, we investigated the role of the nucleotide excision repair (NER) pathway in mycobacterial DNA repair. Mycobacterium smegmatis lacking the NER excinuclease component uvrB or the helicase uvrD1 gene and a double knockout lacking both genes were constructed, and their sensitivities to a series of DNA-damaging agents were analyzed. As anticipated, the mycobacterial NER system was shown to be involved in the processing of bulky DNA adducts and interstrand cross-links. In addition, it could be shown to exert a protective effect against oxidizing and nitrosating agents. Interestingly, inactivation of uvrB and uvrD1 significantly increased marker integration frequencies in gene conversion assays. This implies that in mycobacteria (which lack the postreplicative mismatch repair system) NER, and particularly the UvrD1 helicase, is involved in the processing of a subset of recombination-associated mismatches.

UvrD is a helicase that is widely conserved in gram-negative bacteria. A uvrD homologne was identified in Mycobacterium tuberculosis on the basis of the homology of its encoded protein with Escherichia coli UvrD, with which it shares 39% amino acid identity, distributed throughout the protein. The gene was cloned, and a histidine-tagged form of the protein was expressed and purified to homogeneity. The purified protein had in vitro ATPase activity that was dependent upon the presence of DNA. Oligonucleotides as short as four nucleotides were sufficient to promote the ATPase activity. The DNA helicase activity of the enzyme was only fueled by ATP and dATP. UvrD preferentially unwound 3'-single-stranded tailed duplex substrates over 5'-single-stranded ones, indicating that the protein had a duplex-unwinding activity with 3'-to-5' polarity. A 3' single-stranded DNA tail of 18 nucleotides was required for effective unwinding. By using a series of synthetic oligonucleotide substrates, we demonstrated that M. tuberculosis UvrD has an unwinding preference towards nicked DNA duplexes and stalled replication forks, representing the likely sites of action in vivo. The potential role of M. tuberculosis UvrD in maintenance of bacterial genomic integrity makes it a promising target for drug design against M. tuberculosis.

The mycobacterium-specific gene Rv2719c was found to be expressed primarily from a promoter that was clearly DNA damage inducible independently of RecA. Upstream of the transcriptional start site for this promoter, sequence motifs resembling those observed previously at the RecA-independent, DNA damage-inducible recA promoter were identified, and the -10 motif was demonstrated by mutational analysis in transcriptional fusion constructs to be important for expression of Rv2719c.

The role of the serine/threonine kinase PknH in the physiology and virulence of Mycobacterium tuberculosis was assessed by the construction of a pknH deletion mutant. Deletion of the pknH gene did not affect sensitivity to the antimycobacterial drug ethambutol, although it was previously thought to be involved in regulating expression of emb genes encoding arabinosyl transferases, the targets of ethambutol. Nevertheless, transcription analyses revealed that genes associated with mycobacterial cell wall component synthesis, such as emb and ini operons, are downstream substrates of the PknH signaling cascade. In vitro survival studies revealed that a mutant with a deletion of the pknH gene displayed increased resistance to acidified nitrite stress, suggesting that nitric oxide is one of the potential environmental triggers for PknH activation. The effect of pknH deletion on mycobacterial virulence was investigated in BALB/c mice. In this model, the [delta]pknH mutant was found to survive and replicate to a higher bacillary load in mouse organs than its parental strain and the pknH-complemented strain. In contrast, another closely related kinase mutant, the [delta]pknE mutant, obtained from the same parental strain, was not affected in its virulence phenotype. Infection of THP-1 cells or in vitro growth studies in 7H9 medium did not reveal a significant in vitro growth advantage phenotype for the [delta]pknH mutant. In conclusion, we propose that the serine/threonine kinase PknH plays a role in regulating bacillary load in mouse organs to facilitate adaptation to the host environment, possibly by enabling a regulated chronic infection by M. tuberculosis.

The recA gene of Mycobacterium tuberculosis is unusual in that it is expressed from two promoters, one of which, P1, is DNA damage inducible independently of LexA and RecA, while the other, P2, is regulated by LexA in the classical way (E. O. Davis, B. Springer, K. K. Gopaul, K. G. Papavinasasundaram, P. Sander, and E. C. Bottger, Mol. Microbiol. 46:791-800, 2002). In this study we characterized these two promoters in more detail. Firstly, we localized the promoter elements for each of the promoters, and in so doing we identified a mutation in each promoter which eliminates promoter activity. Interestingly, a motif with similarity to Escherichia coli [[sigma].sup.70] -35 elements but located much closer to the -10 element is important for optimal expression of P1, whereas the sequence at the -35 location is not. Secondly, we found that the sequences flanking the promoters can have a profound effect on the expression level directed by each of the promoters. Finally, we examined the contribution of each of the promoters to recA expression and compared their kinetics of induction following DNA damage.

Results indicate that the mycobacterial SOS box exceeds a length of 2 basepairs in each half of the palindromic site. Single base changes in the SOS box on LexA binding in vivo reveals 12 genes regulated by LexA along with those from DNA damage.

Research examines the role of DNA damage-inducible genes in Mycobacterium tuberculosis with reference to recA gene and LexA repressor binding to specific sequences upstream of the recA and lexA genes. Results do not show of any such binding, suggesting an alternative mechanism for DNA damage repair in M. tuberculosis.

The expression of the recA gene of Mycobacterium tuberculosis was found to be inducible in response to a number of DNA-damaging agents by applying a transcriptional fusion to the reporter gene encoding chloramphenicol acetyltransferase. A segment of DNA about 300 bp upstream of the coding region was found to be needed for expression. However, primer extension analysis showed that the transcriptional start sites are 47 and 93 bp upstream of the translational initiation codon.

Research has been conducted on the Mycobacterium leprae and Mycobacterium tuberculosis RecA proteins. Results indicate that M. leprae RecA is functionally spliced in M. smegmatis and is resistant to DNA-damaging agents.