DNA binding and gene regulatory functions of MSMEG_2295, a repressor encoded by the dinB2 operon of Mycobacterium smegmatis

Microbiology ◽  
2021 ◽  
Vol 167 (10) ◽  
Author(s):  
Madhu Manti Patra ◽  
Poulami Ghosh ◽  
Shreya Sengupta ◽  
Sujoy K. Das Gupta
Keyword(s):  
Oxidative Stress ◽  
Dna Binding ◽  
Mycobacterium Smegmatis ◽  
Gene Knockout ◽  
Growth Inhibitor ◽  
Vital Role ◽  
Repeat Sequence ◽  
Binding Studies ◽  
Pyruvate Metabolism ◽  
Content Type

MSMEG_2295 is a TetR family protein encoded by the first gene of a Mycobacterium smegmatis (Msm) operon that expresses the gene for DinB2 (MSMEG_2294), a translesion DNA repair enzyme. We have carried out investigations to understand its function by performing DNA binding studies and gene knockout experiments. We found that the protein binds to a conserved inverted repeat sequence located upstream of the dinB2 operon and several other genes. Using a knockout of MSMEG_2295, we show that MSMEG_2295 controls the expression of at least five genes, the products of which could potentially influence carbohydrate and fatty acid metabolism as well as antibiotic and oxidative stress resistance. We have demonstrated that MSMEG_2295 is a repressor by performing complementation analysis. Knocking out of MSMEG_2295 had a significant impact on pyruvate metabolism. Pyruvate dehydrogenase activity was virtually undetectable in ΔMSMEG_2295, although in the complemented strain, it was high. We also show that knocking out of MSMEG_2295 causes resistance to H2O2, reversed in the complemented strain. We have further found that the mycobacterial growth inhibitor plumbagin, a compound of plant origin, acts as an inducer of MSMEG_2295 regulated genes. We, therefore, establish that MSMEG_2295 functions by exerting its role as a repressor of multiple Msm genes and that by doing so, it plays a vital role in controlling pyruvate metabolism and response to oxidative stress.

scholarly journals Control of Thioredoxin Reductase Gene (trxB) Transcription by SarA in Staphylococcus aureus

2009 ◽  
Vol 192 (1) ◽  
pp. 336-345 ◽  
Author(s):  
Anand Ballal ◽  
Adhar C. Manna
Keyword(s):  
Oxidative Stress ◽  
Staphylococcus Aureus ◽  
Dna Binding ◽  
Target Genes ◽  
Thioredoxin Reductase ◽  
Negative Regulator ◽  
Binding Studies ◽  
Oxidizing Agents

ABSTRACT Thioredoxin reductase (encoded by trxB) protects Staphylococcus aureus against oxygen or disulfide stress and is indispensable for growth. Among the different sarA family mutants analyzed, transcription of trxB was markedly elevated in the sarA mutant under conditions of aerobic as well as microaerophilic growth, indicating that SarA acts as a negative regulator of trxB expression. Gel shift analysis showed that purified SarA protein binds directly to the trxB promoter region DNA in vitro. DNA binding of SarA was essential for repression of trxB transcription in vivo in S. aureus. Northern blot analysis and DNA binding studies of the purified wild-type SarA and the mutant SarAC9G with oxidizing agents indicated that oxidation of Cys-9 reduced the binding of SarA to the trxB promoter DNA. Oxidizing agents, in particular diamide, could further enhance transcription of the trxB gene in the sarA mutant, suggesting the presence of a SarA-independent mode of trxB induction. Analysis of two oxidative stress-responsive sarA regulatory target genes, trxB and sodM, with various mutant sarA constructs showed a differential ability of the SarA to regulate expression of the two above-mentioned genes in vivo. The overall data demonstrate the important role played by SarA in modulating expression of genes involved in oxidative stress resistance in S. aureus.

scholarly journals Ergothioneine Is a Secreted Antioxidant in Mycobacterium smegmatis

2013 ◽  
Vol 57 (7) ◽  
pp. 3202-3207 ◽  
Author(s):  
Carine Sao Emani ◽  
Monique J. Williams ◽  
Ian J. Wiid ◽  
Nicholas F. Hiten ◽  
Albertus J. Viljoen ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Mycobacterium Smegmatis ◽  
Double Mutant ◽  
Culture Media ◽  
Growth Cycle ◽  
Low Molecular Weight ◽  
Deficient Mutant ◽  
Wild Type ◽  
Content Type

ABSTRACTErgothioneine (ERG) and mycothiol (MSH) are two low-molecular-weight thiols synthesized by mycobacteria. The role of MSH has been extensively investigated in mycobacteria; however, little is known about the role of ERG in mycobacterial physiology. In this study, quantification of ERG at various points in the growth cycle ofMycobacterium smegmatisrevealed that a significant portion of ERG is found in the culture media, suggesting that it is actively secreted. A mutant ofM. smegmatislackingegtD(MSMEG_6247) was unable to synthesize ERG, confirming its role in ERG biosynthesis. Deletion ofegtDfrom wild-typeM. smegmatisand an MSH-deficient mutant did not affect their susceptibility to antibiotics tested in this study. The ERG- and MSH-deficient double mutant was significantly more sensitive to peroxide than either of the single mutants lacking either ERG or MSH, suggesting that both thiols play a role in protectingM. smegmatisagainst oxidative stress and that ERG is able to partly compensate for the loss of MSH.

scholarly journals Rules of Expansion: an Updated Consensus Operator Site for the CopR-CopY Family of Bacterial Copper Exporter System Repressors

mSphere ◽  
2020 ◽  
Vol 5 (3) ◽  
Author(s):  
Henrik O’Brien ◽  
Joseph W. Alvin ◽  
Sanjay V. Menghani ◽  
Yamil Sanchez-Rosario ◽  
Koenraad Van Doorslaer ◽  
...  
Keyword(s):  
Dna Binding ◽  
Consensus Sequence ◽  
Species Interaction ◽  
Copper Stress ◽  
Copper Chaperone ◽  
Binding Studies ◽  
Content Type ◽  
Operator Sequence ◽  
Export System

ABSTRACT Copper is broadly toxic to bacteria. As such, bacteria have evolved specialized copper export systems (cop operons) often consisting of a DNA-binding/copper-responsive regulator (which can be a repressor or activator), a copper chaperone, and a copper exporter. For those bacteria using DNA-binding copper repressors, few studies have examined the regulation of this operon regarding the operator DNA sequence needed for repressor binding. In Streptococcus pneumoniae (the pneumococcus), CopY is the copper repressor for the cop operon. Previously, homologs of pneumococcal CopY have been characterized to bind a 10-base consensus sequence T/GACANNTGTA known as the cop box. Using this motif, we sought to determine whether genes outside the cop operon are also regulated by the CopY repressor, which was previously shown in Lactococcus lactis. We found that S. pneumoniae CopY did not bind to cop operators upstream of these candidate genes in vitro. During this process, we found that the cop box sequence is necessary but not sufficient for CopY binding. Here, we propose an updated operator sequence for the S. pneumoniae cop operon to be ATTGACAAATGTAGAT binding CopY with a dissociation constant (Kd) of ∼28 nM. We demonstrate strong cross-species interaction between some CopY proteins and CopY operators, suggesting strong evolutionary conservation. Taken together with our binding studies and bioinformatics data, we propose the consensus operator RNYKACANNYGTMRNY for the bacterial CopR-CopY copper repressor homologs. IMPORTANCE Many Gram-positive bacteria respond to copper stress by upregulating a copper export system controlled by a copper-sensitive repressor, CopR-CopY. The previous operator sequence for this family of proteins had been identified as TACANNTGTA. Here, using several recombinant proteins and mutations in various DNA fragments, we define those 10 bases as necessary but not sufficient for binding and in doing so, refine the cop operon operator to the 16-base sequence RNYKACANNTGTMRNY. Due to the sheer number of repressors that have been said to bind to the original 10 bases, including many antibiotic resistance repressors such as BlaI and MecI, we feel that this study highlights the need to reexamine many of these sites of the past and use added stringency for verifying operators in the future.

scholarly journals Phosphatidylinositol-Specific Phospholipase C Contributes to Survival of Staphylococcus aureus USA300 in Human Blood and Neutrophils

2014 ◽  
Vol 82 (4) ◽  
pp. 1559-1571 ◽  
Author(s):  
Mark J. White ◽  
Jeffrey M. Boyd ◽  
Alexander R. Horswill ◽  
William M. Nauseef
Keyword(s):  
Oxidative Stress ◽  
Staphylococcus Aureus ◽  
Phospholipase C ◽  
Whole Blood ◽  
Dependent Manner ◽  
Binding Studies ◽  
Content Type ◽  
Positive Regulators

ABSTRACTStaphylococcus aureusis an important human pathogen that employs a large repertoire of secreted virulence factors to promote disease pathogenesis. Many strains ofS. aureuspossess aplcgene that encodes a phosphatidylinositol (PI)-specific phospholipase C (PI-PLC) capable of hydrolyzing PI and cleaving glycosyl-PI (GPI)-linked proteins from cell surfaces. Despite being secreted by virulent staphylococci, the contribution of PI-PLC to the capacity ofS. aureusto cause disease remains undefined. Our goal in these studies was to understand PI-PLC in the context ofS. aureusbiology. Among a collection of genetically diverse clinical isolates ofS. aureus, community-associated methicillin-resistantS. aureus(CA-MRSA) USA300 secreted the most PI-PLC. Screening a collection of two-component system (TCS) mutants ofS. aureus, we identified both theagrquorum-sensing system and the SrrAB TCS to be positive regulators ofplcgene expression. Real-time PCR and PI-PLC enzyme assays of the TCS mutants, coupled with SrrA promoter binding studies, demonstrated that SrrAB was the predominant transcriptional activator ofplc. Furthermore,plcregulation was linked to oxidative stress bothin vitroandin vivoin a SrrAB-dependent manner. A Δplcmutant in a CA-MRSA USA300 background exhibited a survival defect in human whole blood and in isolated neutrophils. However, the same mutant strain displayed no survival defect in murine models of infection or murine whole blood. Overall, these data identify potential links between bacterial responses to the host innate immune system and to oxidative stress and suggest how PI-PLC could contribute to the pathogenesis ofS. aureusinfections.

scholarly journals Mechanisms of Action of Escapin, a Bactericidal Agent in the Ink Secretion of the Sea Hare Aplysia californica: Rapid and Long-Lasting DNA Condensation and Involvement of the OxyR-Regulated Oxidative Stress Pathway

2012 ◽  
Vol 56 (4) ◽  
pp. 1725-1734 ◽  
Author(s):  
Ko-Chun Ko ◽  
Phang C. Tai ◽  
Charles D. Derby
Keyword(s):  
Oxidative Stress ◽  
Carboxylic Acid ◽  
Dna Binding ◽  
Binding Protein ◽  
Aplysia Californica ◽  
Dna Binding Protein ◽  
Bactericidal Effect ◽  
Dna Condensation ◽  
Maximal Effect ◽  
Content Type

ABSTRACTThe marine snailAplysia californicaproduces escapin, anl-amino acid oxidase, in its defensive ink. Escapin usesl-lysine to produce diverse products called escapin intermediate products ofl-lysine (EIP-K), including α-amino-ε-caproic acid, Δ1-piperidine-2-carboxylic acid, and Δ2-piperidine-2-carboxylic acid. EIP-K and H2O2together, but neither alone, is a powerful bactericide. Here, we report bactericidal mechanisms of escapin products onEscherichia coli. We show that EIP-K and H2O2together cause rapid and long-lasting DNA condensation: 2-min treatment causes significant DNA condensation and killing, and 10-min treatment causes maximal effect, lasting at least 70 h. We isolated two mutants resistant to EIP-K plus H2O2, both having a single missense mutation in the oxidation regulatory gene,oxyR. A complementation assay showed that the mutated gene,oxyR(A233V), renders resistance to EIP-K plus H2O2, and a gene dosage effect leads to reduction of resistance for strains carrying wild-typeoxyR. Temperature stress with EIP-K does not produce the bactericidal effect, suggesting the effect is due to a specific response to oxidative stress. The null mutant for any single DNA-binding protein—Dps, H-NS, Hup, Him, or MukB—was not resistant to EIP-K plus H2O2, suggesting that no single DNA-binding protein is necessary to mediate this bactericidal effect, but allowing for the possibility that EIP-K plus H2O2could function through a combination of DNA-binding proteins. The bactericidal effect of EIP-K plus H2O2was eliminated by the ferrous ion chelator 1,10-phenanthroline, and it was reduced by the hydroxyl radical scavenger thiourea, suggesting hydroxyl radicals mediate the effects of EIP-K plus H2O2.

scholarly journals Lsr2 and Its Novel Paralogue Mediate the Adjustment of Mycobacterium smegmatis to Unfavorable Environmental Conditions

mSphere ◽  
2021 ◽  
Vol 6 (3) ◽  
Author(s):  
Marta Kołodziej ◽  
Tomasz Łebkowski ◽  
Przemysław Płociński ◽  
Joanna Hołówka ◽  
Mariola Paściak ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcriptional Regulation ◽  
Dna Binding ◽  
Mycobacterium Smegmatis ◽  
Integration Host Factor ◽  
Loop Formation ◽  
Content Type ◽  
Dna Loop ◽  
Dna Coating

ABSTRACT Lsr2 is a nucleoid-associated protein (NAP) that has been found strictly in actinobacteria, including mycobacteria. It is a functional homolog of histone-like nucleoid-structuring protein (H-NS); it acts as a DNA-bridging protein that plays a role in chromosomal organization and transcriptional regulation. To date, the studies on Lsr2 have focused mainly on Mycobacterium tuberculosis. In this study, we analyze the role of Lsr2 as a transcription factor in Mycobacterium smegmatis, a saprophytic bacterium whose natural habitat (soil and water) substantially differs from those of the obligatory mycobacterial pathogens. Our chromatin immunoprecipitation-sequencing (ChIP-seq) data revealed that Lsr2 binds preferentially to AT-rich regions of the M. smegmatis chromosome. We found that Lsr2 acts mainly as a repressor, controlling gene expression either directly by binding promoter regions or indirectly through DNA loop formation and DNA coating. One of the Lsr2-repressed genes encodes polyketide synthase (MSMEG_4727), which is involved in the synthesis of lipooligosaccharides (LOSs). An M. smegmatis strain deprived of Lsr2 produces more LOSs, which is mirrored by changes in the smoothness of cells and their susceptibilities to antibiotics. Unlike M. tuberculosis, M. smegmatis additionally encodes a paralogue of Lsr2, MSMEG_1060, which is a novel member of the mycobacterial NAP family. The Lsr2 and MSMEG_1060 proteins exhibit different DNA binding specificities and chromosomal localizations. Our results suggest that these proteins help M. smegmatis cells cope with stress conditions, including hypoxia and exposure to antibiotics. Thus, the present work provides novel insight into the role of Lsr2 paralogues in the ability of a saprophytic mycobacterial species to adjust to environmental changes. IMPORTANCE Nucleoid-associated proteins (NAPs) are the most abundant proteins involved in bacterial chromosome organization and global transcription regulation. The mycobacterial NAP family includes many diverse proteins; some are unique to actinobacteria, and many are crucial for survival under stress (e.g., HupB and Lsr2) and/or optimal growth conditions (e.g., mycobacterial integration host factor [mIHF]). Here, we present a comprehensive study concerning two functional homologues of mycobacterial H-NS: Lsr2 and its paralogue from M. smegmatis, MSMEG_1060. We found that Lsr2 plays a role in transcriptional regulation, mainly by repressing gene expression via DNA loop formation and/or DNA-coating mechanisms. Intriguingly, the number of Lsr2-mediated genes was found to increase under hypoxia. Compared to Lsr2, MSMEG_1060 exhibits a different DNA binding specificity and chromosomal localization. Since tuberculosis remains a serious worldwide health problem, studies on stress response-mediating agents, such as Lsr2, may contribute to the development of novel antituberculosis drugs.

scholarly journals Inactivation of the Organic Hydroperoxide Stress Resistance Regulator OhrR Enhances Resistance to Oxidative Stress and Isoniazid in Mycobacterium smegmatis

2014 ◽  
Vol 197 (1) ◽  
pp. 51-62 ◽  
Author(s):  
Sankaralingam Saikolappan ◽  
Kishore Das ◽  
Subramanian Dhandayuthapani
Keyword(s):  
Oxidative Stress ◽  
Mutant Strain ◽  
Stress Resistance ◽  
Mycobacterium Smegmatis ◽  
Cumene Hydroperoxide ◽  
Wild Type ◽  
Mobility Shift ◽  
Organic Hydroperoxide ◽  
Content Type ◽  
Protein Levels

The organic hydroperoxide stress resistance regulator (OhrR) is a MarR type of transcriptional regulator that primarily regulates the expression of organic hydroperoxide reductase (Ohr) in bacteria. In mycobacteria, the genes encoding these proteins exist in only a few species, which include the fast-growing organismMycobacterium smegmatis. To delineate the roles of Ohr and OhrR in defense against oxidative stress inM. smegmatis, strains lacking the expression of these proteins were constructed by deleting theohrRandohrgenes, independently and together, through homologous recombination. The OhrR mutant strain (MSΔohrR) showed severalfold upregulation of Ohr expression, which could be observed at both the transcript and protein levels. Similar upregulation of Ohr expression was also noticed in anM. smegmatiswild-type strain (MSWt) induced with cumene hydroperoxide (CHP) andt-butyl hydroperoxide (t-BHP). The elevated Ohr expression in MSΔohrR correlated with heightened resistance to oxidative stress due to CHP andt-BHP and to inhibitory effects due to the antituberculosis drug isoniazid (INH). Further, this mutant strain exhibited significantly enhanced survival in the intracellular compartments of macrophages. In contrast, the strains lacking either Ohr alone (MSΔohr) or both Ohr and OhrR (MSΔohr-ohrR) displayed limited or no resistance to hydroperoxides and INH. Additionally, these strains showed no significant differences in intracellular survival from the wild type. Electrophoretic mobility shift assays (EMSAs) revealed that the overexpressed and purified OhrR interacts with theohr-ohrRintergenic region with a greater affinity and this interaction is contingent upon the redox state of the OhrR. These findings suggest that Ohr-OhrR is an important peroxide stress response system inM. smegmatis.

scholarly journals A Membrane-Bound Transcription Factor is Proteolytically Regulated by the AAA+ Protease FtsH in Staphylococcus aureus

2020 ◽  
Vol 202 (9) ◽  
Author(s):  
Won-Sik Yeo ◽  
Chiamara Anokwute ◽  
Philip Marcadis ◽  
Marcus Levitan ◽  
Mahmoud Ahmed ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Dna Binding ◽  
Cytoplasmic Membrane ◽  
Agar Plate ◽  
Repeat Sequence ◽  
Chromosomal Dna ◽  
Content Type ◽  
Membrane Bound

ABSTRACT In bacteria, chromosomal DNA resides in the cytoplasm, and most transcription factors are also found in the cytoplasm. However, some transcription factors, called membrane-bound transcription factors (MTFs), reside in the cytoplasmic membrane. Here, we report the identification of a new MTF in the Gram-positive pathogen Staphylococcus aureus and its regulation by the protease FtsH. The MTF, named MbtS (membrane-bound transcription factor of Staphylococcus aureus), is encoded by SAUSA300_2640 and predicted to have an N-terminal DNA binding domain and three transmembrane helices. The MbtS protein was degraded by membrane vesicles containing FtsH or by the purified FtsH. MbtS bound to an inverted repeat sequence in its promoter region, and the DNA binding was essential for its transcription. Transcriptional comparison between the ftsH deletion mutant and the ftsH mbtS double mutant showed that MbtS could alter the transcription of over 200 genes. Although the MbtS protein was not detected in wild-type (WT) cells grown in a liquid medium, the protein was detected in some isolated colonies on an agar plate. In a murine model of a skin infection, the disruption of mbtS increased the lesion size. Based on these results, we concluded that MbtS is a new S. aureus MTF whose activity is proteolytically regulated by FtsH. IMPORTANCE Staphylococcus aureus is an important pathogenic bacterium causing various diseases in humans. In the bacterium, transcription is typically regulated by the transcription factors located in the cytoplasm. In this study, we report an atypical transcription factor identified in S. aureus. Unlike most other transcription factors, the newly identified transcription factor is located in the cytoplasmic membrane, and its activity is proteolytically controlled by the membrane-bound AAA+ protease FtsH. The newly identified MTF, named MbtS, has the potential to regulate the transcription of over 200 genes. This study provides a molecular mechanism by which a protease affects bacterial transcription and illustrates the diversity of the bacterial transcriptional regulation.

scholarly journals A Novel DNA-Binding Protein Plays an Important Role in Helicobacter pylori Stress Tolerance and Survival in the Host

2014 ◽  
Vol 197 (5) ◽  
pp. 973-982 ◽  
Author(s):  
Ge Wang ◽  
Robert J. Maier
Keyword(s):  
Oxidative Stress ◽  
Helicobacter Pylori ◽  
Dna Binding ◽  
Stress Tolerance ◽  
Mutant Strain ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Wild Type ◽  
Content Type ◽  
H Pylori

The gastric pathogenHelicobacter pylorimust combat chronic acid and oxidative stress. It does so via many mechanisms, including macromolecule repair and gene regulation. Mitomycin C-sensitive clones from a transposon mutagenesis library were screened. One sensitive strain contained the insertion element at the locus ofhp119, a hypothetical gene. No homologous gene exists in any (non-H. pylori) organism. Nevertheless, the predicted protein has some features characteristic of histone-like proteins, and we showed that purified HP119 protein is a DNA-binding protein. A Δhp119strain was markedly more sensitive (viability loss) to acid or to air exposure, and these phenotypes were restored to wild-type (WT) attributes upon complementation of the mutant with the wild-type version ofhp119at a separate chromosomal locus. The mutant strain was approximately10-fold more sensitive to macrophage-mediated killing than the parent or the complemented strain. Of 12 mice inoculated with the wild type, all containedH. pylori, whereas 5 of 12 mice contained the mutant strain; the mean colonization numbers were 158-fold less for the mutant strain. A proteomic (two-dimensional PAGE with mass spectrometric analysis) comparison between the Δhp119mutant and the WT strain under oxidative stress conditions revealed a number of important antioxidant protein differences; SodB, Tpx, TrxR, and NapA, as well as the peptidoglycan deacetylase PgdA, were significantly less expressed in the Δhp119mutant than in the WT strain. This study identified HP119 as a putative histone-like DNA-binding protein and showed that it plays an important role inHelicobacter pyloristress tolerance and survival in the host.

scholarly journals Compounds with Potential Activity againstMycobacterium tuberculosis

2018 ◽  
Vol 62 (4) ◽  
pp. e02236-17 ◽  
Author(s):  
C. Sao Emani ◽  
M. J. Williams ◽  
I. J. Wiid ◽  
B. Baker ◽  
C. Carolis
Keyword(s):  
Oxidative Stress ◽  
Mycobacterium Smegmatis ◽  
High Throughput Screening ◽  
Fusaric Acid ◽  
New Drugs ◽  
Acquisition Rate ◽  
Content Type ◽  
Potential Activity ◽  
And Oxidative Stress ◽  
Increased Susceptibility

ABSTRACTThe high acquisition rate of drug resistance byMycobacterium tuberculosisnecessitates the ongoing search for new drugs to be incorporated in the tuberculosis (TB) regimen. Compounds used for the treatment of other diseases have the potential to be repurposed for the treatment of TB. In this study, a high-throughput screening of compounds against thiol-deficientMycobacterium smegmatisstrains and subsequent validation with thiol-deficientM. tuberculosisstrains revealed thatΔegtAandΔmshAmutants had increased susceptibility to azaguanine (Aza) and sulfaguanidine (Su);ΔegtBandΔegtEmutants had increased susceptibility to bacitracin (Ba); andΔegtA,ΔmshA, andΔegtBmutants had increased susceptibility to fusaric acid (Fu). Further analyses revealed that some of these compounds were able to modulate the levels of thiols and oxidative stress inM. tuberculosis. This study reports the activities of Aza, Su, Fu, and Ba againstM. tuberculosisand provides a rationale for further investigations.

Sign in / Sign up

Export Citation Format

Share Document