Identification of a promoter motif regulating the major DNA damage response mechanism of<i>Mycobacterium tuberculosis</i>

Gamulin, Vera; Ćetković, Helena; Ahel, Ivan

doi:10.1111/j.1574-6968.2004.tb09737.x

Cited by 30 publications

(48 citation statements)

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“…Analysis of LexA binding in vivo revealed that the mycobacterial SOS box is specified by the consensus sequence TCGAACnnnnGTTCGA . However, many of the DNA repair genes of M. tuberculosis have been shown to be DNA-damage-inducible in a LexA/RecAindependent manner, revealing the existence of an overlapping stress response system in mycobacteria (Gamulin et al, 2004;Rand et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

Genetic makeup of the Corynebacterium glutamicum LexA regulon deduced from comparative transcriptomics and in vitro DNA band shift assays

et al. 2009

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The lexA gene of Corynebacterium glutamicum ATCC 13032 was deleted to create the mutant strain C. glutamicum NJ2114, which has an elongated cell morphology and an increased doubling time. To characterize the SOS regulon in C. glutamicum, the transcriptomes of NJ2114 and a DNA-damage-induced wild-type strain were compared with that of a wild-type control using DNA microarray hybridization. The expression data were combined with bioinformatic pattern searches for LexA binding sites, leading to the detection of 46 potential SOS boxes located upstream of differentially expressed transcription units. Binding of a hexahistidyl-tagged LexA protein to 40 double-stranded oligonucleotides containing the potential SOS boxes was demonstrated in vitro by DNA band shift assays. It turned out that LexA binds not only to SOS boxes in the promoteroperator region of upregulated genes, but also to SOS boxes detected upstream of downregulated genes. These results demonstrated that LexA controls directly the expression of at least 48 SOS genes organized in 36 transcription units. The deduced genes encode a variety of physiological functions, many of them involved in DNA repair and survival after DNA damage, but nearly half of them have hitherto unknown functions. Alignment of the LexA binding sites allowed the corynebacterial SOS box consensus sequence TcGAA(a/c)AnnTGTtCGA to be deduced. Furthermore, the common intergenic region of lexA and the differentially expressed divS-nrdR operon, encoding a cell division suppressor and a regulator of deoxyribonucleotide biosynthesis, was characterized in detail. Promoter mapping revealed differences in divS-nrdR expression during SOS response and normal growth conditions. One of the four LexA binding sites detected in the intergenic region is involved in regulating divS-nrdR transcription, whereas the other sites are apparently used for negative autoregulation of lexA expression.

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Section: Introductionmentioning

confidence: 99%

Genetic makeup of the Corynebacterium glutamicum LexA regulon deduced from comparative transcriptomics and in vitro DNA band shift assays

et al. 2009

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“…The basis for the downregulation of the RecA-NDp and ESX-1 genes is unknown, but we speculate that these genes are regulated by an alternative sigma factor that is at a competitive disadvantage in the presence of increased levels of SigA, or has reduced expression in TB294-pSigA. Promoters for both sigG and sigH contain the RecA-NDp motif (Gamulin et al, 2004), and additional evidence links sigG to the DNA-damage response (Lee et al, 2008). However, sigH actually showed a slight increase in expression (1.5-fold) in TB294-pSigA (data not shown), and sigG (Rv0182c) expression was below detectable levels in our array analysis, although its neighbouring genes (Rv0181c, Rv0184 and Rv0185) did have lower expression levels in TB294-pSigA (Supplementary Table S2).…”

Section: Discussionmentioning

confidence: 99%

“…These genes include the DNA repair genes recA, recX, radA, ogt and alkA. The RecA nondependent promoter (RecA-NDp) motif was identified upstream of genes that are activated in response to DNA damage, independently of RecA and LexA (Gamulin et al, 2004). Most DNA repair genes in M. tuberculosis that are induced by DNA damage are regulated independently of RecA (Rand et al, 2003), and promoter sequences containing the RecA-NDp motif have been shown to be required for the RecA-independent induction of recA itself and Rv2719c (Brooks et al, 2006;Davis et al, 2002;Gopaul et al, 2003), both of which are expressed at lower levels in TB294-pSigA.…”

Section: Discussionmentioning

confidence: 99%

Section: Expression Analysis Of Recombinant Strains Growing In Monomamentioning

confidence: 99%

“…However, the eis/ Rv2415c cluster was one of only five clusters that were expressed at higher levels in TB294-pSigA (Supplementary Table S2). Most of the differentially expressed genes had lower expression in TB294-pSigA (Supplementary Tables S1 and S2), and interestingly, as described in the Discussion, the majority of these genes are regulated by RecA-NDp promoters (Supplementary Table S2), a type of promoter associated with RecA-independent activation in response to DNA damage (Gamulin et al, 2004).To confirm that eis was expressed at higher levels in TB294-pSigA, we performed three additional experiments, in which TB294-pSigA and TB294-pCV were grown in MonoMac6 cells for 6 days or in 7H9 medium, and then eis mRNA was quantified by real-time PCR. In 7H9 medium, eis expression was over 200-fold greater in TB294-pSigA than in TB294-pCV (Fig.…”

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Activation of the eis gene in a W-Beijing strain of Mycobacterium tuberculosis correlates with increased SigA levels and enhanced intracellular growth

Barnes

Samten

et al. 2009

Microbiology

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There is growing evidence that strains of Mycobacterium tuberculosis differ in pathogenicity and transmissibility, but little is understood about the contributory factors. We have previously shown that increased expression of the principal sigma factor, SigA, mediates the capacity of M. tuberculosis strain 210 to grow more rapidly in human monocytes, compared with other strains. Strain 210 is part of the widespread W-Beijing family of M. tuberculosis strains and includes clinical isolate TB294. To identify genes that respond to changes in SigA levels and that might enhance intracellular growth, we examined RNA and protein expression patterns in TB294-pSigA, a recombinant strain of TB294 that overexpresses sigA from a multicopy plasmid. Lysates from broth-grown cultures of TB294-pSigA contained high levels of Eis, a protein known to modulate host-pathogen interactions. DNA microarray analysis indicated that the eis gene, Rv2416c, was expressed at levels in TB294-pSigA 40-fold higher than in the vector control strain TB294-pCV, during growth in the human monocyte cell line MonoMac6. Other genes with elevated expression in TB294-pSigA showed much smaller changes from TB294-pCV, and the majority of genes with expression differences between the two strains had reduced expression in TB294-pSigA, including an unexpected number of genes associated with the DNA-damage response. Real-time PCR analyses confirmed that eis was expressed at very high levels in TB294-pSigA in monocytes as well as in broth culture, and further revealed that, like sigA, eis was also more highly expressed in wild-type TB294 than in the laboratory strain H37Rv, during growth in monocytes. These findings suggested an association between increased SigA levels and eis activation, and results of chromatin immunoprecipitation confirmed that SigA binds the eis promoter in live TB294 cells. Deletion of eis reduced growth of TB294 in monocytes, and complementation of eis reversed this effect. We conclude that SigA regulates eis, that there is a direct correlation between upregulation of SigA and high expression levels of eis, and that eis contributes to the enhanced capacity of a clinical isolate of M. tuberculosis strain 210 to grow in monocytes.Abbreviation: ChIP, chromatin immunoprecipitation. Two supplementary tables, listing the results of DNA microarray analyses of recombinant strains grown for 6 days in human monocyte cells and features of selected genes showing differential expression in TB294-pSigA compared with TB294-pCV, with associated references, are available with the online version of this paper.The microarray data used in this study have been deposited in the NCBI Gene Expression Omnibus and are accessible through GEO Series accession number GSE13780 http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=pbsltuqwkukqexu&acc=GSE13780. Bishai et al., 1998;Small et al., 1994), implying that some strains spread more effectively than others. M. tuberculosis strain 210 is widely distributed in the south-west and south-central USA (Barnes et al., 1...

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Genome Instability in Bacteria and Archaea

Messling

Williams

2016

Genome Stability

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Identification of a promoter motif regulating the major DNA damage response mechanism ofMycobacterium tuberculosis

Cited by 30 publications

References 31 publications

Genetic makeup of the Corynebacterium glutamicum LexA regulon deduced from comparative transcriptomics and in vitro DNA band shift assays

Genetic makeup of the Corynebacterium glutamicum LexA regulon deduced from comparative transcriptomics and in vitro DNA band shift assays

Activation of the eis gene in a W-Beijing strain of Mycobacterium tuberculosis correlates with increased SigA levels and enhanced intracellular growth

Genome Instability in Bacteria and Archaea

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