Evidence for complex interactions of stress-associated regulons in an mprAB deletion mutant of Mycobacterium tuberculosis

Abstract

Two-component systems are important constituents of bacterial regulatory networks. Results of this investigation into the role of the MprAB two-component system of Mycobacterium tuberculosis indicate that it is associated with the regulation of several stress-responsive regulons. Using a deletion mutant lacking portions of the response regulator, MprA, and the histidine kinase, MprB, it was demonstrated by real-time PCR, primer extension analyses and DNA microarrays that MprAB activates sigma factor genes sigE and sigB, under SDS stress and during exponential growth. SDS-inducible, MprA-dependent transcriptional start points were identified for mprA, sigE and sigB, and variations in distance between these points and MprA-binding sites suggest that MprA is involved in different mechanisms of promoter activation. Although most of the SigE regulon was downregulated in the deletion mutant, the cluster of genes Rv1129c, Rv1130 and Rv1131, which is associated with growth in monoctyes, was upregulated in the deletion mutant under SDS stress, and this upregulation was dependent upon atmospheric growth conditions. Multiple stress-associated genes of the DosR, SigD and IdeR regulons were also upregulated in the deletion mutant, during exponential growth and/or in the presence of SDS. Surprisingly, the deletion mutant had increased resistance to SDS compared to the parental strain, and enhanced growth in human peripheral blood monocytes, characteristics which may result from a loss of repression of stress-associated genes. INTRODUCTIONAs evidenced by its historical and current impact on human populations (Corbett & Raviglione, 2005;Zink et al., 2005), Mycobacterium tuberculosis is a highly successful pathogen. To withstand the challenges of aerosol transmission, growth within host macrophages, and prolonged encapsulation within lung granuloma, these organisms require the ability to respond to different types of stress. M. tuberculosis has 13 sigma factors (Cole et al., 1998), and DNA microarray analyses have shown the importance of several of these in regulating the changes in gene expression patterns associated with various stresses (Geiman et al., 2004;Manganelli et al., 2001Manganelli et al., , 2002. In addition to sigma factors, the response regulators of some two-component systems (TCSs) (Rison et al., 2005), such as DosR (Kendall et al., 2004;Park et al., 2003) and PhoP Perez et al., 2001;Walters et al., 2006), and other transcriptional regulators, such as IdeR (Dussurget et al., 1999;Manabe et al., 2005) and EmbR (Sharma et al., 2006), have been shown to modulate mycobacterial gene expression in response to particular stresses. However, in general, the mechanisms by which M. tuberculosis senses a particular stress, and activates the appropriate regulatory factor(s) while inhibiting others, are largely unknown. The GEO accession numbers for the array data associated with this paper are GSM155424-155447 (series record no. GSE6750).Four supplementary tables are available with the online version of this paper.Abbreviati...

Regulation of the biosynthesis of thiopeptide antibiotic cyclothiazomycin by the transcriptional regulator SHJG8833 in Streptomyces hygroscopicus 5008

“…Primer extension was performed essentially as described by Pang et al (2007). Briefly, 10 mg of S. hygroscopicus 5008 RNA was used to generate cDNA by reverse transcription, using a high-temperature-tolerant Thermoscript reverse transcriptase (Invitrogen) to overcome possible secondary structure due to the high G+C content of Streptomyces strains.…”

“…Reverse transcription was carried out as described previously (Pang et al, 2007). Real-time PCR assays were performed with SYBR Premix Ex Taq (TaKaRa).…”

Transcription factors Rv0081 and Rv3334 connect the early and the enduring hypoxic response of Mycobacterium tuberculosis

“…Using this system, it was shown that an early bacterial response (2 hr) was the coordinated upregulation of 48  M. tb genes under the control of two sensor kinases (DosS and DosT) and the response regulator (DosR), known as the DosR regulon. [22–24] It was subsequently shown that the DosR regulon is induced by other stress conditions including exposure to nitric oxide, [25] carbon monoxide, [26] SDS, [27] and low pH [28]. The DosR regulon is also induced upon M. tb infection of macrophages [29] and mice.…”

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

“…The M. tuberculosis DNA microarray consisted of 4295 70-mer oligonucleotides representing the 3924 predicted ORFs of the H37Rv strain (http://www.sanger.ac.uk) and 371 probes designed to detect sequences in the CDC1551 strain (http://www.tigr.org). The arrays were prepared by spotting oligonucleotides (Tuberculosis Genome Set version 1.0, Operon Biotechnologies) onto poly-L-lysine-coated glass microscope slides, as described previously (Pang et al, 2007). Total RNA from two independent experiments was prepared from M. tuberculosis harvested from infected MonoMac6 cells, as described above.…”

“…Total RNA from two independent experiments was prepared from M. tuberculosis harvested from infected MonoMac6 cells, as described above. cDNA synthesis, labelling, hybridization, scanning, image acquisition, normalization, filtering and fold-change calculations were performed as described previously (Pang et al, 2007), and for each experiment, a dye flip was performed. A one-class t test was run using the MEV software (Saeed et al, 2003), with P values based on t distribution and an overall alpha set to 0.05.…”