Interactions of anti-sigma factor antagonists of Mycobacterium tuberculosis in the yeast two-hybrid system

Parida, Bikram Kumar; Douglas, Trevor; Nino, Celina; Dhandayuthapani, Subramanian

doi:10.1016/j.tube.2005.08.001

Cited by 40 publications

(56 citation statements)

References 30 publications

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“…A combination of two-hybrid and far-Western analyses was therefore employed to demonstrate the self-interaction of BldG. Although RsbV and SpoIIAA do not form homodimers in solution (16,34,35,57), other homologous anti-anti-sigma factors, including TM1442 of Thermotoga maritima (22,38) and several examples in Mycobacterium tuberculosis (48), exist in solution in a dimeric form. Based on our results, we therefore propose that BldG also forms homodimers.…”

Section: Discussionmentioning

confidence: 99%

“…The similarity of BldG to proteins known to form homodimers (22,38,48), together with preliminary identification by chemical cross-linking of a BldGcontaining complex whose size was consistent with dimerization (not shown), led to examination of BldG self-interaction. First, bacterial two-hybrid analysis was performed using bldGcontaining recombinant plasmids (pAU377 to pAU379).…”

Section: Vol 191 2009 Interaction Of S Coelicolor Bldg With Sco354mentioning

confidence: 99%

“…Anti-sigma factors typically form homodimers (16,21,48,57); therefore, based on similarity to this family of proteins, SCO3548 would be predicted to self-interact. Bacterial two-hybrid analysis, using the sco3548-containing recombinant plasmids pAU380 to pAU382, was employed to examine this interaction.…”

Section: Vol 191 2009 Interaction Of S Coelicolor Bldg With Sco354mentioning

confidence: 99%

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BldG and SCO3548 Interact Antagonistically To Control Key Developmental Processes inStreptomyces coelicolor

et al. 2009

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The similarity of BldG and the downstream coexpressed protein SCO3548 to anti-anti-sigma and anti-sigma factors, respectively, together with the phenotype of a bldG mutant, suggests that BldG and SCO3548 interact as part of a regulatory system to control both antibiotic production and morphological differentiation in Streptomyces coelicolor. A combination of bacterial two-hybrid, affinity purification, and far-Western analyses demonstrated that there was self-interaction of both BldG and SCO3548, as well as a direct interaction between the two proteins. Furthermore, a genetic complementation experiment demonstrated that SCO3548 antagonizes the function of BldG, similar to other anti-anti-sigma/anti-sigma factor pairs. It is therefore proposed that BldG and SCO3548 form a partner-switching pair that regulates the function of one or more sigma factors in S. coelicolor. The conservation of bldG and sco3548 in other streptomycetes demonstrates that this system is likely a key regulatory switch controlling developmental processes throughout the genus Streptomyces.Streptomyces coelicolor A3(2), the well-studied model organism for processes of bacterial multicellular development and antibiotic production, possesses a large genome (8.67 Mbp) with a high degree of regulatory complexity (6). A large proportion of the coding sequence (12.3%) is predicted to encode the multitude of regulatory factors required to support a complex life cycle, involving the formation of sporulating aerial hyphae, that responds to the changing soil environment. Of particular note is the presence of 64 sigma factors, which are thought to play a critical role in the modulation of gene expression; this group is comprised of 4 housekeeping sigma factors, as well as 50 extracytoplasmic function sigma factors and 9 group 3 subfamily sigma factors (6, 23). The activity of alternative sigma factors is typically regulated by a number of mechanisms, including phosphorylation-dependent partner switching by antagonistic proteins. The best-studied examples of this regulatory mechanism, which is active against the group 3 sigma factors, are found in Bacillus subtilis, where partner switching controls the activity of both the sporulation-specific factor F and the general stress response factor B (1,17,18,44,50,57,59). In these systems, an anti-sigma factor protein (SpoIIAB and RsbW, respectively) sequesters the cognate sigma factor, preventing the expression of target genes. Upon sensing some activating signal, an anti-sigma factor antagonist (or anti-anti-sigma factor; SpoIIAA and RsbV, respectively) binds to the anti-sigma factor, mediating release of the active sigma factor to direct regulon transcription.Partner-switching systems are also thought to play a critical role in sigma factor regulation in S. coelicolor. The first characterized example in this organism is the RsbV-RsbA partnerswitching pair that controls the activity of the osmotic-stressresponsive factor B (36). Many genes encoding additional putative paralogues of these regulatory factors are...

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Section: Discussionmentioning

confidence: 99%

Section: Vol 191 2009 Interaction Of S Coelicolor Bldg With Sco354mentioning

confidence: 99%

Section: Vol 191 2009 Interaction Of S Coelicolor Bldg With Sco354mentioning

confidence: 99%

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BldG and SCO3548 Interact Antagonistically To Control Key Developmental Processes inStreptomyces coelicolor

et al. 2009

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“…In addition, several other proteins have been shown to interact with SigF or UsfX of M. tuberculosis and these are thought to exert some control on the activity of SigF (Parida et al, 2005). There appears to be no consensus regarding the expression of sigF from M. tuberculosis in response to different stress conditions such as infection of macrophages (Graham & Clark-Curtiss, 1999;Mariani et al, 2000), nutrient starvation (Betts et al, 2002;DeMaio et al, 1996;Michele et al, 1999), stationary-phase growth (DeMaio et al, 1996;Graham & Clark-Curtiss, 1999;Manganelli et al, 1999), or exposure to oxidative stress, alcohol, antibiotics and temperature shock (DeMaio et al, 1996;Manganelli et al, 1999;Michele et al, 1999).…”

Section: Introductionmentioning

confidence: 99%

The alternative sigma factor SigF of Mycobacterium smegmatis is required for survival of heat shock, acidic pH and oxidative stress

et al. 2008

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The alternative sigma factor SigF of Mycobacterium tuberculosis has been characterized in detail as a general-stress, stationary-phase sigma factor involved in the virulence of the bacterium. While a homologous gene has been annotated in the genome of the fast-growing Mycobacterium smegmatis, little experimental evidence is available on the function of this gene. Here, we demonstrate that SigF of M. smegmatis is required for resistance to hydrogen peroxide, heat shock and acidic pH, but not for survival in human neutrophils. No difference in sensitivity to isoniazid was observed between the wild-type strain and the DsigF mutant, suggesting that SigFmediated resistance to hydrogen peroxide was via a pathway independent of KatG or AhpC. RT-PCR and 59-RACE (rapid amplification of cDNA ends) analyses showed that sigF of M. smegmatis was co-transcribed with rsbW (thought to encode an anti-sigma factor for SigF) and MSMEG_1802 (unknown function) and was expressed from two promoters, one upstream of MSMEG_1802 and the second upstream of rsbW. Analysis of transcriptional lacZ fusion constructs in the sigF-deletion background revealed that the MSMEG_1802 promoter was dependent on SigF for expression. Moreover, MSMEG_1802-lacZ was induced twofold upon entry into stationary phase, while exposure of exponentially growing cultures to various stress conditions (e.g. heat, cold, ethanol, hydrogen peroxide or different pH values) did not lead to induction of MSMEG_1802-lacZ. Expression of rsbW-lacZ was independent of SigF and remained constant throughout the growth cycle and under various stress conditions unless the bacteria were challenged with D-cycloserine.

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“…five anti-sigma factor antagonists, RsfA, RsfB, Rv1364c, Rv1904, and Rv2638 (4,27). The latter are not part of the usfX-sigF operon but are dispersed across the genome.…”

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confidence: 99%

The SigF Regulon in Mycobacterium smegmatis Reveals Roles in Adaptation to Stationary Phase, Heat, and Oxidative Stress

et al. 2010

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SigF is an alternative sigma factor that is highly conserved among species of the genus Mycobacterium. In this study we identified the SigF regulon in Mycobacterium smegmatis using whole-genome microarray and promoter consensus analyses. In total, 64 genes in exponential phase and 124 genes in stationary phase are SigF dependent (P < 0.01, >2-fold expression change). Our experimental data reveal the SigF-dependent promoter consensus GTTT-N (15-17) -GGGTA for M. smegmatis, and we propose 130 potential genes under direct control of SigF, of which more than 50% exhibited reduced expression in a ⌬sigF strain. We previously reported an increased susceptibility of the ⌬sigF strain to heat and oxidative stress, and our expression data indicate a molecular basis for these phenotypes. We observed SigF-dependent expression of several genes purportedly involved in oxidative stress defense, namely, a heme-containing catalase, a manganese-containing catalase, a superoxide dismutase, the starvation-induced DNA-protecting protein MsDps1, and the biosynthesis genes for the carotenoid isorenieratene. Our data suggest that SigF regulates the biosynthesis of the thermoprotectant trehalose, as well as an uptake system for osmoregulatory compounds, and this may explain the increased heat susceptibility of the ⌬sigF strain. We identified the regulatory proteins SigH3, PhoP, WhiB1, and WhiB4 as possible genes under direct control of SigF and propose four novel anti-sigma factor antagonists that could be involved in the posttranslational regulation of SigF in M. smegmatis. This study emphasizes the importance of this sigma factor for stationary-phase adaptation and stress response in mycobacteria.The success of Mycobacterium tuberculosis as a pathogen can be attributed to its capacity to adapt to environmental changes throughout the course of infection. These changes include nutrient deprivation, hypoxia, various exogenous stress conditions, and the intraphagosomal environment. A large cohort of genes that facilitate this adaptation has been identified, and among them are many genes for transcriptional regulators such as sigma factors that modulate gene expression in response to different physiological cues. Sigma factors interact with the RNA polymerase to allow binding to specific promoter sequences and initiation of gene transcription. Mycobacteria harbor sigma factors of only the 70 family, which fall into four different categories. SigA (group 1) is the essential primary sigma factor in mycobacteria, and SigB (group 2) is its nonessential paralog. SigF (group 3) and extracytoplasmic function (ECF) sigma factors (group 4) are alternative sigma factors, which allow adaptation to a wide range of internal and external stimuli. Alternative sigma factors vary considerably in type and numbers between species, mirroring their different requirements for stress response (15).Thirteen sigma factors have been identified in M. tuberculosis (23,29). Eleven of these are classified as alternative sigma factors, and many of them are recognized as ...

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Interactions of anti-sigma factor antagonists of Mycobacterium tuberculosis in the yeast two-hybrid system

Cited by 40 publications

References 30 publications

BldG and SCO3548 Interact Antagonistically To Control Key Developmental Processes inStreptomyces coelicolor

BldG and SCO3548 Interact Antagonistically To Control Key Developmental Processes inStreptomyces coelicolor

The alternative sigma factor SigF of Mycobacterium smegmatis is required for survival of heat shock, acidic pH and oxidative stress

The SigF Regulon in Mycobacterium smegmatis Reveals Roles in Adaptation to Stationary Phase, Heat, and Oxidative Stress

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