scholarly journals HrrSA orchestrates a systemic response to heme and determines prioritization of terminal cytochrome oxidase expression

2020 ◽  
Vol 48 (12) ◽  
pp. 6547-6562
Author(s):  
Marc Keppel ◽  
Max Hünnefeld ◽  
Andrei Filipchyk ◽  
Ulrike Viets ◽  
Cedric-Farhad Davoudi ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Response Regulator ◽  
Cell Envelope ◽  
Affinity Purification ◽  
Network Evolution ◽  
Dynamic Binding ◽  
Prosthetic Group ◽  
Time Resolved ◽  
Genes Encoding

Abstract Heme is a multifaceted molecule. While serving as a prosthetic group for many important proteins, elevated levels are toxic to cells. The complexity of this stimulus has shaped bacterial network evolution. However, only a small number of targets controlled by heme-responsive regulators have been described to date. Here, we performed chromatin affinity purification and sequencing to provide genome-wide insights into in vivo promoter occupancy of HrrA, the response regulator of the heme-regulated two-component system HrrSA of Corynebacterium glutamicum. Time-resolved profiling revealed dynamic binding of HrrA to more than 200 different genomic targets encoding proteins associated with heme biosynthesis, the respiratory chain, oxidative stress response and cell envelope remodeling. By repression of the extracytoplasmic function sigma factor sigC, which activates the cydABCD operon, HrrA prioritizes the expression of genes encoding the cytochrome bc1-aa3 supercomplex. This is also reflected by a significantly decreased activity of the cytochrome aa3 oxidase in the ΔhrrA mutant. Furthermore, our data reveal that HrrA also integrates the response to heme-induced oxidative stress by activating katA encoding the catalase. These data provide detailed insights in the systemic strategy that bacteria have evolved to respond to the versatile signaling molecule heme.

scholarly journals Influence of Oxidative Stress on Time-Resolved Oxygen Detection by [Ru(Phen)3]2+ In Vivo and In Vitro

Molecules ◽  
2021 ◽  
Vol 26 (2) ◽  
pp. 485
Author(s):  
Veronika Huntosova ◽  
Denis Horvath ◽  
Robert Seliga ◽  
Georges Wagnieres
Keyword(s):  
Oxidative Stress ◽  
Tissue Oxygenation ◽  
Intact Cell ◽  
Molecular Probes ◽  
Stress Factors ◽  
Time Resolved ◽  
Invasive Approach ◽  
Oxygen Detection

Detection of tissue and cell oxygenation is of high importance in fundamental biological and in many medical applications, particularly for monitoring dysfunction in the early stages of cancer. Measurements of the luminescence lifetimes of molecular probes offer a very promising and non-invasive approach to estimate tissue and cell oxygenation in vivo and in vitro. We optimized the evaluation of oxygen detection in vivo by [Ru(Phen)3]2+ in the chicken embryo chorioallantoic membrane model. Its luminescence lifetimes measured in the CAM were analyzed through hierarchical clustering. The detection of the tissue oxygenation at the oxidative stress conditions is still challenging. We applied simultaneous time-resolved recording of the mitochondrial probe MitoTrackerTM OrangeCMTMRos fluorescence and [Ru(Phen)3]2+ phosphorescence imaging in the intact cell without affecting the sensitivities of these molecular probes. [Ru(Phen)3]2+ was demonstrated to be suitable for in vitro detection of oxygen under various stress factors that mimic oxidative stress: other molecular sensors, H2O2, and curcumin-mediated photodynamic therapy in glioma cancer cells. Low phototoxicities of the molecular probes were finally observed. Our study offers a high potential for the application and generalization of tissue oxygenation as an innovative approach based on the similarities between interdependent biological influences. It is particularly suitable for therapeutic approaches targeting metabolic alterations as well as oxygen, glucose, or lipid deprivation.

scholarly journals The Skn7 Response Regulator ofSaccharomyces cerevisiaeInteracts with Hsf1 In Vivo and Is Required for the Induction of Heat Shock Genes by Oxidative Stress

2000 ◽  
Vol 11 (7) ◽  
pp. 2335-2347 ◽  
Author(s):  
Desmond C. Raitt ◽  
Anthony L. Johnson ◽  
Alexander M. Erkine ◽  
Kozo Makino ◽  
Brian Morgan ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Heat Shock ◽  
Response Regulator ◽  
Coiled Coil ◽  
Normal Growth ◽  
Temperature Sensitive ◽  
Regulatory Sequences ◽  
Heat Shock Genes

The Skn7 response regulator has previously been shown to play a role in the induction of stress-responsive genes in yeast, e.g., in the induction of the thioredoxin gene in response to hydrogen peroxide. The yeast Heat Shock Factor, Hsf1, is central to the induction of another set of stress-inducible genes, namely the heat shock genes. These two regulatory trans-activators, Hsf1 and Skn7, share certain structural homologies, particularly in their DNA-binding domains and the presence of adjacent regions of coiled-coil structure, which are known to mediate protein–protein interactions. Here, we provide evidence that Hsf1 and Skn7 interact in vitro and in vivo and we show that Skn7 can bind to the same regulatory sequences as Hsf1, namely heat shock elements. Furthermore, we demonstrate that a strain deleted for the SKN7 gene and containing a temperature-sensitive mutation in Hsf1 is hypersensitive to oxidative stress. Our data suggest that Skn7 and Hsf1 cooperate to achieve maximal induction of heat shock genes in response specifically to oxidative stress. We further show that, like Hsf1, Skn7 can interact with itself and is localized to the nucleus under normal growth conditions as well as during oxidative stress.

scholarly journals CovR Activation of the Dipeptide Permease Promoter (PdppA) in Group A Streptococcus

2006 ◽  
Vol 189 (4) ◽  
pp. 1407-1416 ◽  
Author(s):  
Asiya A. Gusa ◽  
Barbara J. Froehlich ◽  
Devak Desai ◽  
Virginia Stringer ◽  
June R. Scott
Keyword(s):  
Group A Streptococcus ◽  
Response Regulator ◽  
Activation Of Transcription ◽  
Genes Encoding ◽  
Group A ◽  
Transcription In Vitro ◽  
Dna Template ◽  
Regulated Promoters

ABSTRACT CovR, the two-component response regulator of Streptococcus pyogenes (group A streptococcus [GAS]) directly or indirectly represses about 15% of the genome, including genes encoding many virulence factors and itself. Transcriptome analyses also showed that some genes are activated by CovR. We asked whether the regulation by CovR of one of these genes, dppA, the first gene in an operon encoding a dipeptide permease, is direct or indirect. Direct regulation by CovR was suggested by the presence of five CovR consensus binding sequences (CBs) near the putative promoter. In this study, we identified the 5′ end of the dppA transcript synthesized in vivo and showed that the start of dppA transcription in vitro is the same. We found that CovR binds specifically to the dppA promoter region (PdppA) in vitro with an affinity similar to that at which it binds to other CovR-regulated promoters. Disruption of any of the five CBs by a substitution of GG for TT inhibited CovR binding to that site in vitro, and binding at two of the CBs appeared cooperative. In vivo, CovR activation of transcription was not affected by individual mutations of any of the four CBs that we could study. This suggests that the binding sites are redundant in vivo. In vitro, CovR did not activate transcription from PdppA in experiments using purified GAS RNA polymerase and either linear or supercoiled DNA template. Therefore, we propose that in vivo, CovR may interfere with the binding of a repressor of PdppA.

scholarly journals Oxidative Stress Sensor Keap1 Functions as an Adaptor for Cul3-Based E3 Ligase To Regulate Proteasomal Degradation of Nrf2

2004 ◽  
Vol 24 (16) ◽  
pp. 7130-7139 ◽  
Author(s):  
Akira Kobayashi ◽  
Moon-Il Kang ◽  
Hiromi Okawa ◽  
Makiko Ohtsuji ◽  
Yukari Zenke ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Stress Proteins ◽  
E3 Ligase ◽  
Genes Encoding ◽  
Cullin 3 ◽  
Ubiquitin Proteasome ◽  
A Subunit ◽  
Antioxidant Stress ◽  
And Oxidative Stress

ABSTRACT Transcription factor Nrf2 is a major regulator of genes encoding phase 2 detoxifying enzymes and antioxidant stress proteins in response to electrophilic agents and oxidative stress. In the absence of such stimuli, Nrf2 is inactive owing to its cytoplasmic retention by Keap1 and rapid degradation through the proteasome system. We examined the contribution of Keap1 to the rapid turnover of Nrf2 (half-life of less than 20 min) and found that a direct association between Keap1 and Nrf2 is required for Nrf2 degradation. In a series of domain function analyses of Keap1, we found that both the BTB and intervening-region (IVR) domains are crucial for Nrf2 degradation, implying that these two domains act to recruit ubiquitin-proteasome factors. Indeed, Cullin 3 (Cul3), a subunit of the E3 ligase complex, was found to interact specifically with Keap1 in vivo. Keap1 associates with the N-terminal region of Cul3 through the IVR domain and promotes the ubiquitination of Nrf2 in cooperation with the Cul3-Roc1 complex. These results thus provide solid evidence that Keap1 functions as an adaptor of Cul3-based E3 ligase. To our knowledge, Nrf2 and Keap1 are the first reported mammalian substrate and adaptor, respectively, of the Cul3-based E3 ligase system.

scholarly journals The Involvement of Mycobacterium Type III-A CRISPR-Cas System in Oxidative Stress

2021 ◽  
Vol 12 ◽  
Author(s):  
Fan Yang ◽  
Lingqing Xu ◽  
Lujie Liang ◽  
Wanfei Liang ◽  
Jiachen Li ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Envelope ◽  
Reduction Process ◽  
Intracellular Survival ◽  
Host Immunity ◽  
Type I ◽  
Type Iii

Type I and type II CRISPR-Cas systems are employed to evade host immunity by targeting interference of bacteria’s own genes. Although Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis, possesses integrated type III-A CRISPR-Cas system, its role in mycobacteria remains obscure. Here, we observed that seven cas genes (csm2∼5, cas10, cas6) were upregulated in Mycobacterium bovis BCG under oxidative stress treatment, indicating the role of type III-A CRISPR-Cas system in oxidative stress. To explore the functional role of type III-A CRISPR-Cas system, TCC (Type III-A CRISPR-Cas system, including cas6, cas10, and csm2-6) mutant was generated. Deletion of TCC results in increased sensitivity in response to hydrogen peroxide and reduced cell envelope integrity. Analysis of RNA-seq dataset revealed that TCC impacted on the oxidation-reduction process and the composition of cell wall which is essential for mycobacterial envelop integrity. Moreover, disrupting TCC led to poor intracellular survival in vivo and in vitro. Finally, we showed for the first time that TCC contributed to the regulation of regulatory T cell population, supporting a role of TCC in modulating host immunity. Our finding reveals the important role of TCC in cell envelop homeostasis. Our work also highlights type III-A CRISPR-Cas system as an important factor for intracellular survival and host immunoregulation in mycobacteria, thus may be a potential target for therapy.

scholarly journals Genetic and Biochemical Analyses of BvgA Interaction with the Secondary Binding Region of the fhaPromoter of Bordetella pertussis

2001 ◽  
Vol 183 (2) ◽  
pp. 536-544 ◽  
Author(s):  
Philip E. Boucher ◽  
Mei-Shin Yang ◽  
Deanna M. Schmidt ◽  
Scott Stibitz
Keyword(s):  
Bordetella Pertussis ◽  
Response Regulator ◽  
Sequence Specificity ◽  
Binding Region ◽  
Dna Sequence Specificity ◽  
Genes Encoding ◽  
Two Component Signal Transduction ◽  
Biochemical Analyses

ABSTRACT The BvgA-BvgS two-component signal transduction system regulates expression of virulence factors in Bordetella pertussis. The BvgA response regulator activates transcription by binding to target promoters, which include those for the genes encoding filamentous hemagglutinin (fha) and pertussis toxin (ptx). We have previously shown that at both promoters the phosphorylated form of BvgA binds multiple high- and low-affinity sites. Specifically, at the fha promoter, we proposed that there may be high- and a low-affinity binding sites for the BvgA dimer. In our present investigation, we used DNA binding analyses and in vitro and in vivo assays of promoters with substitutions and deletions to support and extend this hypothesis. Our observations indicate that (i) binding of BvgA∼P to a primary (high-affinity) site and a secondary binding region (lower affinity) is cooperative, (ii) although both the primary binding site and the secondary binding region are required for full activity of the wild-type (undeleted) promoter, deletion of two helical turns within the secondary binding region can produce a fully active or hyperactive promoter, and (iii) BvgA binding to the secondary binding region shows limited DNA sequence specificity.

scholarly journals Alternative Sigma Factor RpoE Is Important for Vibrio parahaemolyticus Cell Envelope Stress Response and Intestinal Colonization

2014 ◽  
Vol 82 (9) ◽  
pp. 3667-3677 ◽  
Author(s):  
Brandy Haines-Menges ◽  
W. Brian Whitaker ◽  
E. Fidelma Boyd
Keyword(s):  
Stress Response ◽  
Vibrio Parahaemolyticus ◽  
Response Regulator ◽  
Cell Envelope ◽  
Polymyxin B ◽  
Content Type ◽  
Alternative Sigma Factors ◽  
Envelope Stress

ABSTRACTVibrio parahaemolyticusis a halophile that inhabits brackish waters and a wide range of hosts, including crustaceans, fish, mollusks, and humans. In humans, it is the leading cause of bacterial seafood-borne gastroenteritis. The focus of this work was to determine the role of alternative sigma factors in the stress response ofV. parahaemolyticusRIMD2210633, an O3:K6 pandemic isolate. Bioinformatics identified five putative extracytoplasmic function (ECF) family of alternative sigma factors: VP0055, VP2210, VP2358, VP2578, and VPA1690. ECF factors typically respond to cell wall/cell envelope stress, iron levels, and the oxidation state of the cell. We have demonstrated here that one such sigma factor, VP2578, a homologue of RpoE fromEscherichia coli, is important for survival under a number of cell envelope stress conditions and in gastrointestinal colonization of a streptomycin-treated adult mouse. In this study, we determined that anrpoEdeletion mutant strain BHM2578 compared to the wild type (WT) was significantly more sensitive to polymyxin B, ethanol, and high-temperature stresses. We demonstrated that inin vivocompetition assays between therpoEmutant and the WT marked with the β-galactosidase genelacZ(WBWlacZ), the mutant strain was defective in colonization compared to the WT. In contrast, deletion of therpoSstress response regulator did not affectin vivosurvival. In addition, we examined the role of the outer membrane protein, OmpU, which inV. choleraeis proposed to be the sole activator of RpoE. We found that anompUdeletion mutant was sensitive to bile salt stress but resistant to polymyxin B stress, indicating OmpU is not essential for the cell envelope stress responses or RpoE function. Overall, these data demonstrate that RpoE is a key cell envelope stress response regulator and, similar toE. coli, RpoE may have several factors that stimulate its function.

scholarly journals Characterization of key enzymes involved in triacylglycerol biosynthesis in mycobacteria

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Agostina Crotta Asis ◽  
Franco Savoretti ◽  
Matías Cabruja ◽  
Hugo Gramajo ◽  
Gabriela Gago
Keyword(s):  
Phosphatidic Acid ◽  
Growth Phase ◽  
De Novo ◽  
Cell Envelope ◽  
Exponential Growth Phase ◽  
Genes Encoding ◽  
Concomitant Reduction ◽  
Tag Biosynthesis ◽  
Odd Chain Fatty Acids

AbstractPhosphatidic acid phosphatase (PAP) catalyzes the dephosphorylation of phosphatidic acid (PA) yielding diacylglycerol (DAG), the lipid precursor for triacylglycerol (TAG) biosynthesis. PAP activity has a key role in the regulation of PA flux towards TAG or glycerophospholipid synthesis. In this work we have characterized two Mycobacterium smegmatis genes encoding for functional PAP proteins. Disruption of both genes provoked a sharp reduction in de novo TAG biosynthesis in early growth phase cultures under stress conditions. In vivo labeling experiments demonstrated that TAG biosynthesis was restored in the ∆PAP mutant when bacteria reached exponential growth phase, with a concomitant reduction of phospholipid synthesis. In addition, comparative lipidomic analysis showed that the ∆PAP strain had increased levels of odd chain fatty acids esterified into TAGs, suggesting that the absence of PAP activity triggered other rearrangements of lipid metabolism, like phospholipid recycling, in order to maintain the wild type levels of TAG. Finally, the lipid changes observed in the ∆PAP mutant led to defective biofilm formation. Understanding the interaction between TAG synthesis and the lipid composition of mycobacterial cell envelope is a key step to better understand how lipid homeostasis is regulated during Mycobacterium tuberculosis infection.

scholarly journals The ChvG–ChvI and NtrY–NtrX two-component systems coordinately regulate growth of Caulobacter crescentus

2021 ◽  
Author(s):  
Benjamin J. Stein ◽  
Aretha Fiebig ◽  
Sean Crosson
Keyword(s):  
Gene Expression ◽  
Phosphatase Activity ◽  
Environmental Changes ◽  
Caulobacter Crescentus ◽  
Genetic Interaction ◽  
Response Regulator ◽  
Cell Envelope ◽  
Signaling Systems ◽  
Two Component

Two-component signaling systems (TCSs) are comprised of a sensory histidine kinase and a response regulator protein. In response to environmental changes, sensor kinases directly phosphorylate their cognate response regulator to affect gene expression. Bacteria typically express multiple TCSs that are insulated from one another and regulate distinct physiological processes. There are certainly examples of cross-regulation between TCSs, but this phenomenon remains relatively unexplored. We have identified regulatory links between the ChvG–ChvI (ChvGI) and NtrY–NtrX (NtrYX) TCSs, which control important and often overlapping processes in α-proteobacteria, including maintenance of the cell envelope. Deletion of chvG and chvI in Caulobacter crescentus limited growth in defined medium and a selection for genetic suppressors of this growth phenotype uncovered interactions among chvGI , ntrYX , and ntrZ , which encodes a previously uncharacterized periplasmic protein. Significant overlap in the experimentally-defined ChvI and NtrX transcriptional regulons provided support for the observed genetic connections between ntrYX and chvGI . Moreover, we present evidence that the growth defect of strains lacking chvGI is influenced by the phosphorylation state of NtrX and, to some extent, by levels of the TonB-dependent receptor ChvT. Measurements of NtrX phosphorylation in vivo indicated that NtrZ is an upstream regulator of NtrY, and that NtrY primarily functions as an NtrX phosphatase. We propose a model in which NtrZ functions in the periplasm to inhibit NtrY phosphatase activity; regulation of phosphorylated NtrX levels by NtrZ and NtrY provides a mechanism to modulate and balance expression of the NtrX and ChvI regulons under different growth conditions. Importance Two-component signaling systems (TCSs) enable bacteria to regulate gene expression in response to physiochemical changes in their environment. The ChvGI and NtrYX TCSs regulate diverse pathways associated with pathogenesis, growth, and cell envelope function in many α-proteobacteria. We used Caulobacter crescentus as a model to investigate regulatory connections between ChvGI and NtrYX. Our work defined the ChvI transcriptional regulon in C. crescentus and revealed a genetic interaction between ChvGI and NtrYX, whereby modulation of NtrYX signaling affects the survival of cells lacking ChvGI. In addition, we identified NtrZ as a periplasmic inhibitor of NtrY phosphatase activity in vivo . Our work establishes C. crescentus as an excellent model to investigate multi-level regulatory connections between ChvGI and NtrYX in α-proteobacteria.

scholarly journals Mapping and Identification of the Major Cell Wall-Associated Components of Mycobacterium leprae

1998 ◽  
Vol 66 (6) ◽  
pp. 2625-2631 ◽  
Author(s):  
Maria Angela M. Marques ◽  
Sadhana Chitale ◽  
Patrick J. Brennan ◽  
Maria Cristina V. Pessolani
Keyword(s):  
Cell Wall ◽  
Response Regulator ◽  
Cell Envelope ◽  
Elongation Factor ◽  
Mycobacterium Leprae ◽  
Monosaccharide Composition ◽  
Mycolic Acid ◽  
Diaminopimelic Acid ◽  
Two Dimensional Gel Electrophoresis

ABSTRACT Mycobacterium leprae, an obligate intracellular pathogen, can be derived only from host tissue and thus affords the opportunity to study in vivo-expressed products responsible for the particular pathogenesis of leprosy. Despite considerable progress in the characterization of the proteins and secondary gene products ofM. leprae, there is little information on the nature of the proteins associated with the cell envelope. M. leprae has been fractionated into its major subcellular components, cell wall, cytoplasmic membrane, and soluble cytosol. A number of biochemical markers, including diaminopimelic acid content, monosaccharide composition, mycolic acid, and glycolipid distribution, were applied to their characterization, and two-dimensional gel electrophoresis was used to map the component proteins. A total of 391 major proteins spots were resolved, and 8 proteins were identified based on their reactivity to a panel of monoclonal antibodies and/or relative pI size. Microsequencing of six protein spots present in the cell wall fraction allowed identification of new proteins, including the protein elongation factor EF-Tu and a homolog for the Mycobacterium tuberculosis MtrA response regulator. These results, together with previous studies, contribute to the progressive knowledge of the composition of the in vivo-expressed proteins of M. leprae.

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