SigG Does Not Control Gene Expression in Response to DNA Damage in Mycobacterium tuberculosis H37Rv

Abstract

Expression of the Mycobacterium tuberculosis sigG sigma factor was induced by a variety of DNA-damaging agents, but inactivation of sigG did not affect induction of gene expression or bacterial survival under these conditions. Therefore, SigG does not control the DNA repair response of M. tuberculosis H37Rv.Mycobacterium tuberculosis has at least two mechanisms regulating gene expression following DNA damage (6): the SOS response mediated by LexA and RecA (5,9,17) and an alternative mechanism independent of both RecA and LexA (6, 21). It is not known what controls the RecA-independent response, although a potential promoter motif common to a number of genes in this regulon that suggests the possible involvement of an alternative sigma factor has been identified (11). Of the 13 sigma factors in M. tuberculosis (2), SigG was the most highly induced following DNA damage and macrophage infection (1, 21). Moreover, a recent study suggested that SigG was responsible for transcription of lexA in a clinical isolate, CDC1551 (14).Here, we addressed the role of SigG in M. tuberculosis H37Rv in response to DNA damage by comparing the global transcriptional profile of a sigG mutant strain with that of the wild type. Our results indicate that the absence of SigG does not affect the induction of known DNA damage response genes. In addition, we could find no evidence for the involvement of SigG in the transcription of lexA in vivo. Furthermore, the sigG mutant was no more susceptible to DNA damage than the parental strain. Therefore, we conclude that SigG does not control expression of either the RecA-dependent or the RecAindependent genes in response to DNA damage in M. tuberculosis H37Rv.A potential role for SigG in regulating the response to DNA damage was initially proposed based on its induction following exposure to mitomycin C (21), which causes interstrand crosslinks and alkylation damage. Basing our analysis on the pelleting properties of the bacteria, we suspect mitomycin C also affects the mycobacterial cell surface. Therefore, we measured the levels of sigG mRNA following other treatments predicted to damage DNA, namely, bleomycin exposure, ofloxacin exposure, and UV irradiation, resulting in breaks in DNA, inhibition of DNA gyrase, and induction of the formation of thymine dimers, respectively. The effect of cell surface stress on sigG expression was also assessed by treatment with ethambutol and exposure to sodium dodecyl sulfate (SDS). M. tuberculosis H37Rv cultures (See Table 1 for strains and plasmids used) were grown under Advisory Committee on Dangerous Pathogens (ACDP) containment level 3 conditions as described previously (6). Cultures were divided into aliquots at an optical density at 600 nm (OD 600 ) of 0.3 to 0.4; samples were induced with mitomycin C (0.2 g ml Ϫ1 ), ofloxacin (7.5 g ml Ϫ1 ), or bleomycin (1.5 g ml Ϫ1 ) for 5 h at 37°C, exposed to 23.5 J m Ϫ2UV irradiation followed by 5 h of recovery at 37°C, or induced with SDS (0.05% for 5 h at 37°C) or ethambutol (15 g ml Ϫ1for 24 h at 37°C). Anothe...

Characterisation of the Mycobacterium tuberculosis alternative sigma factor SigG: Its operon and regulon

“…Previous investigations examining the gene expression profiles of a sigG mutant strain with and without DNA damage induction found no significant differences from the wild-type H37Rv strain 39 . Therefore, in this investigation we compared the expression profiles of M. tuberculosis H37Rv containing pKS09, a SigG over-expression construct, to that of H37Rv containing an empty vector (pKS12) by microarray (Table 1).…”

“…The mycobacterial strains used were Mycobacterium smegmatis mc 2 155, 42 M. tuberculosis wild-type strain H37Rv 31 sigG mutant strains in H37Rv Δ sigG 1 and Δ sigG 2, 39 and a sigG operon deletion in H37Rv Δ sigG WO (this study). Mycobacterial cultures grown in Dubos medium (Difco) supplemented with albumin and 0.2% glycerol or on Difco Middlebrook 7H11 agar plates (Beckton Dickenson) supplemented with 4% albumin and 0.5% glycerol.…”

“…In our previous work we attempted to compare gene expression in a sigG mutant strain to wild-type M. tuberculosis H37Rv but found no significant differences, 39 possibly due to SigG being the lowest expressed of all sigma factors under normal growth conditions 25 . Therefore, in this study we examined the regulon of sigG using an over-expression strain.…”

“…A DNA motif resembling a non-canonical promoter common to a number of genes regulated in this way has been identified leading to the suggestion that these genes were controlled by an alternative sigma factor 21 . However, SigG was found not to control transcription of either the SOS or RecA-independent response genes, and mutation of sigG did not increase sensitivity to DNA damage 39 . The RecA-independent DNA damage response was subsequently found to be regulated by the Clp protease regulator ClpR 45 …”

Virulence factors of theMycobacterium tuberculosiscomplex

“…It has been proposed that SigG is also involved in the SOS response of M. tuberculosis , 416 but recent evidence suggests the opposite 417 . Consistently with an essential role of sigG during intracellular growth of M. tuberculosis , an M. tuberculosis deleted in sigG gene displayed impaired survival in a macrophage infection model 416 .…”