Abstract
The DNA damage response is crucial for bacterial survival. The transcriptional repressor LexA is a key component of the SOS response, the main mechanism for the regulation of DNA repair genes in many bacteria. In contrast, in mycobacteria gene induction by DNA damage is carried out by two mechanisms; a relatively small number of genes are thought to be regulated by LexA, and a larger number by an alternate, independent mechanism. In this study we have used ChIP-seq analysis to identify 25 in vivo LexA-binding sites, including nine regulating genes not previously known to be part of this regulon. Some of these binding sites were found to be internal to the predicted open reading frame of the gene they are thought to regulate; experimental analysis has confirmed that these LexA-binding sites regulate the expression of the expected genes, and transcriptional start site analysis has found that their apparent relative location is due to misannotation of these genes. We have also identified novel binding sites for LexA in the promoters of genes that show no apparent DNA damage induction, show positive regulation by LexA, and those encoding small RNAs.
Highlights
Mycobacterium tuberculosis LexA is thought to repress the expression of a small number of genes
The LexA-binding motif for M. tuberculosis was originally characterized by comparison with that of other bacteria and found to be similar to that of Bacillus subtilis [10], following which the consensus sequence was defined as TCGAAC(N4)GTTCGA by use of a mutagenic approach [9]
Quantitative PCR was performed to confirm that known LexA binding regions were enriched in the immunoprecipitated DNA samples using primers that amplified the SOS boxes of Rv3074, Rv3776, and dnaE2, in contrast to the control primers located within the coding regions of sigA and dnaE2, which showed no enrichment
Summary
Mycobacterium tuberculosis LexA is thought to repress the expression of a small number of genes. We have identified novel binding sites for LexA in the promoters of genes that show no apparent DNA damage induction, show positive regulation by LexA, and those encoding small RNAs. Tuberculosis, the result of infection by the bacterium Mycobacterium tuberculosis, causes more deaths worldwide than any other infectious disease [1]. The LexA-binding motif for M. tuberculosis was originally characterized by comparison with that of other bacteria and found to be similar to that of Bacillus subtilis [10], following which the consensus sequence was defined as TCGAAC(N4)GTTCGA by use of a mutagenic approach [9] This information enabled the identification of genes and operons that appeared to be LexA-regulated [9]; the number of these sites was relatively small, being 15, and some of the binding sites were found to be internal to the annotated coding sequences. Among the newly identified LexAbinding sites, some were associated with genes not previously known to be DNA damage-inducible, and one was found to be positively regulated by LexA, and two were linked to potential small RNAs
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