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Christian Ruckert - One of the best experts on this subject based on the ideXlab platform.
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monitoring global protein thiol oxidation and protein s mycothiolation in mycobacterium smegmatis under hypochlorite stress
Scientific Reports, 2017Co-Authors: Melanie Hillion, Jorg Bernhardt, Tobias Busche, Martina Rossius, Sandra Maas, Dorte Becher, Mamta Rawat, Markus Wirtz, Rudiger Hell, Christian RuckertAbstract:Mycothiol (MSH) is the major low molecular weight (LMW) thiol in Actinomycetes. Here, we used shotgun proteomics, OxICAT and RNA-seq transcriptomics to analyse protein S-mycothiolation, reversible thiol-oxidations and their impact on gene expression in Mycobacterium smegmatis under hypochlorite stress. In total, 58 S-mycothiolated proteins were identified under NaOCl stress that are involved in energy metabolism, fatty acid and mycolic acid biosynthesis, protein translation, redox regulation and detoxification. Protein S-mycothiolation was accompanied by MSH depletion in the thiol-metabolome. Quantification of the redox state of 1098 Cys residues using OxICAT revealed that 381 Cys residues (33.6%) showed >10% increased oxidations under NaOCl stress, which overlapped with 40 S-mycothiolated Cys-peptides. The absence of MSH resulted in a higher basal oxidation level of 338 Cys residues (41.1%). The RseA and RshA Anti-Sigma Factors and the Zur and NrdR repressors were identified as NaOCl-sensitive proteins and their oxidation resulted in an up-regulation of the SigH, SigE, Zur and NrdR regulons in the RNA-seq transcriptome. In conclusion, we show here that NaOCl stress causes widespread thiol-oxidation including protein S-mycothiolation resulting in induction of antioxidant defense mechanisms in M. smegmatis. Our results further reveal that MSH is important to maintain the reduced state of protein thiols.
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monitoring global protein thiol oxidation and protein s mycothiolation in mycobacterium smegmatis under hypochlorite stress
Scientific Reports, 2017Co-Authors: Melanie Hillion, Jorg Bernhardt, Tobias Busche, Martina Rossius, Sandra Maas, Dorte Becher, Mamta Rawat, Markus Wirtz, Rudiger Hell, Christian RuckertAbstract:Mycothiol (MSH) is the major low molecular weight (LMW) thiol in Actinomycetes. Here, we used shotgun proteomics, OxICAT and RNA-seq transcriptomics to analyse protein S-mycothiolation, reversible thiol-oxidations and their impact on gene expression in Mycobacterium smegmatis under hypochlorite stress. In total, 58 S-mycothiolated proteins were identified under NaOCl stress that are involved in energy metabolism, fatty acid and mycolic acid biosynthesis, protein translation, redox regulation and detoxification. Protein S-mycothiolation was accompanied by MSH depletion in the thiol-metabolome. Quantification of the redox state of 1098 Cys residues using OxICAT revealed that 381 Cys residues (33.6%) showed >10% increased oxidations under NaOCl stress, which overlapped with 40 S-mycothiolated Cys-peptides. The absence of MSH resulted in a higher basal oxidation level of 338 Cys residues (41.1%). The RseA and RshA Anti-Sigma Factors and the Zur and NrdR repressors were identified as NaOCl-sensitive proteins and their oxidation resulted in an up-regulation of the SigH, SigE, Zur and NrdR regulons in the RNA-seq transcriptome. In conclusion, we show here that NaOCl stress causes widespread thiol-oxidation including protein S-mycothiolation resulting in induction of antioxidant defense mechanisms in M. smegmatis. Our results further reveal that MSH is important to maintain the reduced state of protein thiols.
Claude Bruand - One of the best experts on this subject based on the ideXlab platform.
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a putative bifunctional histidine kinase phosphatase of the hwe family exerts positive and negative control on the sinorhizobium meliloti general stress response
Journal of Bacteriology, 2014Co-Authors: Laurent Sauviac, Claude BruandAbstract:The EcfG-type sigma factor RpoE2 is the regulator of the general stress response in Sinorhizobium meliloti. RpoE2 activity is negatively regulated by two NepR-type Anti-Sigma Factors (RsiA1/A2), themselves under the control of two anti-Anti-Sigma Factors (RsiB1/B2) belonging to the PhyR family of response regulators. The current model of RpoE2 activation suggests that in response to stress, RsiB1/B2 are activated by phosphorylation of an aspartate residue in their receiver domain. Once activated, RsiB1/B2 become able to interact with the Anti-Sigma Factors and release RpoE2, which can then associate with the RNA polymerase to transcribe its target genes. The purpose of this work was to identify and characterize proteins involved in controlling the phosphorylation status of RsiB1/B2. Using in vivo approaches, we show that the putative histidine kinase encoded by the rsiC gene (SMc01507), located downstream from rpoE2, is able to both positively and negatively regulate the general stress response. In addition, our data suggest that the negative action of RsiC results from inhibition of RsiB1/B2 phosphorylation. From these observations, we propose that RsiC is a bifunctional histidine kinase/phosphatase responsible for RsiB1/B2 phosphorylation or dephosphorylation in the presence or absence of stress, respectively. Two proteins were previously proposed to control PhyR phosphorylation in Caulobacter crescentus and Sphingomonas sp. strain FR1. However, these proteins contain a Pfam:HisKA_2 domain of dimerization and histidine phosphotransfer, whereas S. meliloti RsiC harbors a Pfam:HWE_HK domain instead. Therefore, this is the first report of an HWE_HK-containing protein controlling the general stress response in Alphaproteobacteria.
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dual control of sinorhizobium meliloti rpoe2 sigma factor activity by two phyr type two component response regulators
Journal of Bacteriology, 2010Co-Authors: Benedicte Bastiat, Laurent Sauviac, Claude BruandAbstract:RpoE2 is an extracytoplasmic function (ECF) sigma factor involved in the general stress response of Sinorhizobium meliloti, the nitrogen-fixing symbiont of the legume plant alfalfa. RpoE2 orthologues are widely found among alphaproteobacteria, where they play various roles in stress resistance and/or host colonization. In this paper, we report a genetic and biochemical investigation of the mechanisms of signal transduction leading to S. meliloti RpoE2 activation in response to stress. We showed that RpoE2 activity is negatively controlled by two paralogous Anti-Sigma Factors, RsiA1 (SMc01505) and RsiA2 (SMc04884), and that RpoE2 activation by stress requires two redundant paralogous PhyR-type response regulators, RsiB1 (SMc01504) and RsiB2 (SMc00794). RsiB1 and RsiB2 do not act at the level of rpoE2 transcription but instead interact with the Anti-Sigma Factors, and we therefore propose that they act as anti-Anti-Sigma Factors to relieve RpoE2 inhibition in response to stress. This model closely resembles a recently proposed model of activation of RpoE2-like sigma Factors in Methylobacterium extorquens and Bradyrhizobium japonicum, but the existence of two pairs of anti- and anti-Anti-Sigma Factors in S. meliloti adds an unexpected level of complexity, which may allow the regulatory system to integrate multiple stimuli. The capacity to sense and respond to environmental changes is essential for every living organism. In bacteria, a part of these responses occurs through modulation of initiation of gene transcription by changing the sigma factor associated with the core RNA polymerase. Sigma Factors are dissociable subunits which provide the specificity of promoter recognition to RNA polymerase. Association of different sigma Factors with the core enzyme makes it possible for the holoenzyme to recognize different promoters and express different sets of target genes. Sigma Factors thus provide efficient mechanisms for simultaneous regulation of large numbers of genes (18). The so-called sigma 70 family of sigma Factors includes primary sigma Factors, which direct the transcription of housekeeping genes, as well as related alternative sigma Factors which associate with the core RNA polymerase under various conditions, including stresses. The most abundant class of such alternative sigma Factors is composed of structurally related proteins called extra-cytoplasmic function (ECF) sigma Factors, as many of them control functions associated with various aspects of the cell surface or transport (20, 35). How stress stimuli are sensed and transduced to ECF sigma Factors has been the subject of numerous studies. It appears that most ECF sigma Factors share the important property of specifically interacting with a protein called Anti-Sigma factor, which plays a pivotal role in the control of sigma factor activity. In the absence of stimulus, the ECF sigma factor is kept inactive by interaction with its cognate Anti-Sigma factor. In the presence of stimulus, the Anti-Sigma factor gets inactivated, either via a mechanism involving successive steps of proteolysis or through conformational changes of the protein (for reviews, see references 2, 20, and 35). Sinorhizobium meliloti is a Gram-negative bacterium belonging to the alpha subclass of proteobacteria. This bacterium lives in the soil and can establish a symbiotic associa
Melanie Hillion - One of the best experts on this subject based on the ideXlab platform.
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monitoring global protein thiol oxidation and protein s mycothiolation in mycobacterium smegmatis under hypochlorite stress
Scientific Reports, 2017Co-Authors: Melanie Hillion, Jorg Bernhardt, Tobias Busche, Martina Rossius, Sandra Maas, Dorte Becher, Mamta Rawat, Markus Wirtz, Rudiger Hell, Christian RuckertAbstract:Mycothiol (MSH) is the major low molecular weight (LMW) thiol in Actinomycetes. Here, we used shotgun proteomics, OxICAT and RNA-seq transcriptomics to analyse protein S-mycothiolation, reversible thiol-oxidations and their impact on gene expression in Mycobacterium smegmatis under hypochlorite stress. In total, 58 S-mycothiolated proteins were identified under NaOCl stress that are involved in energy metabolism, fatty acid and mycolic acid biosynthesis, protein translation, redox regulation and detoxification. Protein S-mycothiolation was accompanied by MSH depletion in the thiol-metabolome. Quantification of the redox state of 1098 Cys residues using OxICAT revealed that 381 Cys residues (33.6%) showed >10% increased oxidations under NaOCl stress, which overlapped with 40 S-mycothiolated Cys-peptides. The absence of MSH resulted in a higher basal oxidation level of 338 Cys residues (41.1%). The RseA and RshA Anti-Sigma Factors and the Zur and NrdR repressors were identified as NaOCl-sensitive proteins and their oxidation resulted in an up-regulation of the SigH, SigE, Zur and NrdR regulons in the RNA-seq transcriptome. In conclusion, we show here that NaOCl stress causes widespread thiol-oxidation including protein S-mycothiolation resulting in induction of antioxidant defense mechanisms in M. smegmatis. Our results further reveal that MSH is important to maintain the reduced state of protein thiols.
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monitoring global protein thiol oxidation and protein s mycothiolation in mycobacterium smegmatis under hypochlorite stress
Scientific Reports, 2017Co-Authors: Melanie Hillion, Jorg Bernhardt, Tobias Busche, Martina Rossius, Sandra Maas, Dorte Becher, Mamta Rawat, Markus Wirtz, Rudiger Hell, Christian RuckertAbstract:Mycothiol (MSH) is the major low molecular weight (LMW) thiol in Actinomycetes. Here, we used shotgun proteomics, OxICAT and RNA-seq transcriptomics to analyse protein S-mycothiolation, reversible thiol-oxidations and their impact on gene expression in Mycobacterium smegmatis under hypochlorite stress. In total, 58 S-mycothiolated proteins were identified under NaOCl stress that are involved in energy metabolism, fatty acid and mycolic acid biosynthesis, protein translation, redox regulation and detoxification. Protein S-mycothiolation was accompanied by MSH depletion in the thiol-metabolome. Quantification of the redox state of 1098 Cys residues using OxICAT revealed that 381 Cys residues (33.6%) showed >10% increased oxidations under NaOCl stress, which overlapped with 40 S-mycothiolated Cys-peptides. The absence of MSH resulted in a higher basal oxidation level of 338 Cys residues (41.1%). The RseA and RshA Anti-Sigma Factors and the Zur and NrdR repressors were identified as NaOCl-sensitive proteins and their oxidation resulted in an up-regulation of the SigH, SigE, Zur and NrdR regulons in the RNA-seq transcriptome. In conclusion, we show here that NaOCl stress causes widespread thiol-oxidation including protein S-mycothiolation resulting in induction of antioxidant defense mechanisms in M. smegmatis. Our results further reveal that MSH is important to maintain the reduced state of protein thiols.
Kelly T Hughes - One of the best experts on this subject based on the ideXlab platform.
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the anti sigma Factors
Annual Review of Microbiology, 1998Co-Authors: Kelly T Hughes, Kalai MatheeAbstract:▪ Abstract A mechanism for regulating gene expression at the level of transcription utilizes an antagonist of the sigma transcription factor known as the Anti-Sigma (anti-σ) factor. The cytoplasmic class of anti-σ Factors has been well characterized. The class includes AsiA form bacteriophage T4, which inhibits Escherichia coli σ70; FlgM, present in both gram-positive and gram-negative bacteria, which inhibits the flagella sigma factor σ28; SpoIIAB, which inhibits the sporulation-specific sigma Factors, σF and σG, of Bacillus subtilis; RbsW of B. subtilis, which inhibits stress response sigma factor σB; and DnaK, a general regulator of the heat shock response, which in bacteria inhibits the heat shock sigma factor σ32. In addition to this class of well-characterized cytoplasmic Anti-Sigma Factors, a new class of homologous, inner-membrane–bound anti-σ Factors has recently been discovered in a variety of eubacteria. This new class of anti-σ Factors regulates the expression of so-called extracytoplasmic fun...
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the role of anti sigma Factors in gene regulation
Molecular Microbiology, 1995Co-Authors: Kit L Brown, Kelly T HughesAbstract:: Despite the isolation of an Anti-Sigma factor over 20 years ago, it is only recently that the concept of an Anti-Sigma factor emerged as a general mechanism of transcriptional regulation in prokaryotic systems. Anti-Sigma Factors bind to sigma Factors and inhibit their transcriptional activity. Studies on the mechanism of action of Anti-Sigma Factors has shed new light on the regulation of gene expression in bacteria, as the Anti-Sigma Factors add another layer to transcriptional control via negative regulation. Their cellular roles are as diverse as FIgM of Salmonella typhimurium, which can be exported to sense the structural state of the flagellar organelle, to SpoIIAB of Bacillus subtilis participating in the switch from one cell type to another during the process of sporulation. Additionally, the bacteriophage T4 uses an Anti-Sigma factor to sabotage the Escherichia coli E.sigma 70 RNA polymerase in order to direct exclusive transcription of its own genes. Cross-linking, co-immunoprecipitations, and co-purification indicate that the Anti-Sigma Factors directly interact with their corresponding sigma factor to negatively regulate transcription. In B. subtilis, anti Anti-Sigma Factors regulate Anti-Sigma Factors by preventing an Anti-Sigma factor from interacting with its cognate sigma Factors, thereby allowing transcription to occur.
Seyed E Hasnain - One of the best experts on this subject based on the ideXlab platform.
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the extracytoplasmic function sigma Factors role in bacterial pathogenesis
Infection Genetics and Evolution, 2004Co-Authors: Murali D. Bashyam, Seyed E HasnainAbstract:Bacteria utilize a distinct subfamily of sigma Factors to regulate extra cytoplasmic function (thus termed as ECF subfamily). Eubacteria appear to have evolved to incorporate extensive genetic diversity into their repertoire of ECF sigma Factors (some species have more than 60 ECF sigma Factors), while maintaining three major themes common to all members including: (1) they regulate and respond to extracytoplasmic functions; (2) they are themselves regulated by Anti-Sigma and/or anti-Anti-Sigma Factors; and (3) most of them control a relatively small regulon. The cell wall is the first bacterial structure that comes in contact with the host during infection by pathogenic bacteria. The cell wall components are often associated with functions related to host cell invasion. It is therefore, likely that the ECF sigma Factors regulate the bacterial response to host insult. Moreover, in some cases, virulence Factors have been shown to be regulated directly by the ECF sigma Factors. Unfortunately, this facet of the ECF sigma Factors has not been an important area of study by researchers. The present review attempts to highlight the important role played by ECF sigma Factors in bacterial pathogenesis and highlights several areas of future study involving the genetics of ECF sigma Factors vis-a-vis bacterial pathogenesis.