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 Transcriptional Regulation of theecpOperon by EcpR, IHF, and H-NS in Attaching and Effacing Escherichia coli

2012 ◽  
Vol 194 (18) ◽  
pp. 5020-5033 ◽  
Author(s):  
Verónica I. Martínez-Santos ◽  
Abraham Medrano-López ◽  
Zeus Saldaña ◽  
Jorge A. Girón ◽  
José L. Puente
Keyword(s):  
Escherichia Coli ◽  
Transcriptional Regulation ◽  
Dna Binding ◽  
Regulatory Protein ◽  
Integration Host Factor ◽  
Start Codon ◽  
Binding Motif ◽  
Epithelial Surface ◽  
Content Type ◽  
E Coli

ABSTRACTEnteropathogenic (EPEC) and enterohemorrhagic (EHEC)Escherichia coliare clinically important diarrheagenic pathogens that adhere to the intestinal epithelial surface. TheE. colicommon pili (ECP), or meningitis-associated and temperature-regulated (MAT) fimbriae, are ubiquitous among both commensal and pathogenicE. colistrains and play a role as colonization factors by promoting the interaction between bacteria and host epithelial cells and favoring interbacterial interactions in biofilm communities. The first gene of theecpoperon encodes EcpR (also known as MatA), a proposed regulatory protein containing a LuxR-like C-terminal helix-turn-helix (HTH) DNA-binding motif. In this work, we analyzed the transcriptional regulation of theecpgenes and the role of EcpR as a transcriptional regulator. EHEC and EPECecpRmutants produce less ECP, while plasmids expressing EcpR increase considerably the expression of EcpA and production of ECP. Theecpgenes are transcribed as an operon from a promoter located 121 bp upstream of the start codon ofecpR. EcpR positively regulates this promoter by binding to two TTCCT boxes distantly located upstream of theecppromoter, thus enhancing expression of downstreamecpgenes, leading to ECP production. EcpR mutants in the putative HTH DNA-binding domain are no longer able to activateecpexpression or bind to the TTCCT boxes. EcpR-mediated activation is aided by integration host factor (IHF), which is essential for counteracting the repression exerted by histone-like nucleoid-structuring protein (H-NS) on theecppromoter. This work demonstrates evidence about the interplay between a novel member of a diverse family of regulatory proteins and global regulators in the regulation of a fimbrial operon.

scholarly journals Genomewide Stabilization of mRNA during a “Feast-to-Famine” Growth Transition in Escherichia coli

mSphere ◽  
2020 ◽  
Vol 5 (3) ◽  
Author(s):  
Manon Morin ◽  
Brice Enjalbert ◽  
Delphine Ropers ◽  
Laurence Girbal ◽  
Muriel Cocaign-Bousquet
Keyword(s):  
Gene Expression ◽  
Transcriptional Regulation ◽  
Mrna Stability ◽  
Posttranscriptional Regulation ◽  
Carbon Starvation ◽  
Fine Tuning ◽  
Content Type ◽  
E Coli ◽  
Transcript Stability

ABSTRACT Bacteria have to continuously adjust to nutrient fluctuations from favorable to less-favorable conditions and in response to carbon starvation. The glucose-acetate transition followed by carbon starvation is representative of such carbon fluctuations observed in Escherichia coli in many environments. Regulation of gene expression through fine-tuning of mRNA pools constitutes one of the regulation levels required for such a metabolic adaptation. It results from both mRNA transcription and degradation controls. However, the contribution of transcript stability regulation in gene expression is poorly characterized. Using combined transcriptome and mRNA decay analyses, we investigated (i) how transcript stability changes in E. coli during the glucose-acetate-starvation transition and (ii) if these changes contribute to gene expression changes. Our work highlights that transcript stability increases with carbon depletion. Most of the stabilization occurs at the glucose-acetate transition when glucose is exhausted, and then stabilized mRNAs remain stable during acetate consumption and carbon starvation. Meanwhile, expression of most genes is downregulated and we observed three times less gene expression upregulation. Using control analysis theory on 375 genes, we show that most of gene expression regulation is driven by changes in transcription. Although mRNA stabilization is not the controlling phenomenon, it contributes to the emphasis or attenuation of transcriptional regulation. Moreover, upregulation of 18 genes (33% of our studied upregulated set) is governed mainly by transcript stabilization. Because these genes are associated with responses to nutrient changes and stress, this underscores a potentially important role of posttranscriptional regulation in bacterial responses to nutrient starvation. IMPORTANCE The ability to rapidly respond to changing nutrients is crucial for E. coli to survive in many environments, including the gut. Reorganization of gene expression is the first step used by bacteria to adjust their metabolism accordingly. It involves fine-tuning of both transcription (transcriptional regulation) and mRNA stability (posttranscriptional regulation). While the forms of transcriptional regulation have been extensively studied, the role of mRNA stability during a metabolic switch is poorly understood. Investigating E. coli genomewide transcriptome and mRNA stability during metabolic transitions representative of the carbon source fluctuations in many environments, we have documented the role of mRNA stability in the response to nutrient changes. mRNAs are globally stabilized during carbon depletion. For a few genes, this leads directly to expression upregulation. As these genes are regulators of stress responses and metabolism, our work sheds new light on the likely importance of posttranscriptional regulations in response to environmental stress.

scholarly journals Novel Transcriptional Regulons for Autotrophic Cycle Genes in Crenarchaeota

2015 ◽  
Vol 197 (14) ◽  
pp. 2383-2391 ◽  
Author(s):  
Semen A. Leyn ◽  
Irina A. Rodionova ◽  
Xiaoqing Li ◽  
Dmitry A. Rodionov
Keyword(s):  
Carbon Dioxide ◽  
Transcriptional Regulation ◽  
Comparative Genomics ◽  
Dna Binding ◽  
Transcriptional Control ◽  
Binding Assays ◽  
Promoter Regions ◽  
Content Type ◽  
Genome Context

ABSTRACTAutotrophic microorganisms are able to utilize carbon dioxide as their only carbon source, or, alternatively, many of them can grow heterotrophically on organics. Different variants of autotrophic pathways have been identified in various lineages of the phylumCrenarchaeota. Aerobic members of the orderSulfolobalesutilize the hydroxypropionate-hydroxybutyrate cycle (HHC) to fix inorganic carbon, whereas anaerobicThermoprotealesuse the dicarboxylate-hydroxybutyrate cycle (DHC). Knowledge of transcriptional regulation of autotrophic pathways inArchaeais limited. We applied a comparative genomics approach to predict novel autotrophic regulons in theCrenarchaeota. We report identification of two novel DNA motifs associated with the autotrophic pathway genes in theSulfolobales(HHC box) andThermoproteales(DHC box). Based on genome context evidence, the HHC box regulon was attributed to a novel transcription factor from the TrmB family named HhcR. Orthologs of HhcR are present in allSulfolobalesgenomes but were not found in other lineages. A predicted HHC box regulatory motif was confirmed byin vitrobinding assays with the recombinant HhcR protein fromMetallosphaera yellowstonensis. For the DHC box regulon, we assigned a different potential regulator, named DhcR, which is restricted to the orderThermoproteales. DhcR inThermoproteus neutrophilus(Tneu_0751) was previously identified as a DNA-binding protein with high affinity for the promoter regions of two autotrophic operons. The global HhcR and DhcR regulons reconstructed by comparative genomics were reconciled with available omics data inMetallosphaeraandThermoproteusspp. The identified regulons constitute two novel mechanisms for transcriptional control of autotrophic pathways in theCrenarchaeota.IMPORTANCELittle is known about transcriptional regulation of carbon dioxide fixation pathways inArchaea. We previously applied the comparative genomics approach for reconstruction of DtxR family regulons in diverse lineages ofArchaea. Here, we utilize similar computational approaches to identify novel regulatory motifs for genes that are autotrophically induced in microorganisms from two lineages ofCrenarchaeotaand to reconstruct the respective regulons. The predicted novel regulons in archaeal genomes control the majority of autotrophic pathway genes and also other carbon and energy metabolism genes. The HhcR regulon was experimentally validated by DNA-binding assays inMetallosphaeraspp. Novel regulons described for the first time in this work provide a basis for understanding the mechanisms of transcriptional regulation of autotrophic pathways inArchaea.

scholarly journals Fast and Efficient 5′P Degradome Library Preparation for Analysis of Co-Translational Decay in Arabidopsis

Plants ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 466
Author(s):  
Marie-Christine Carpentier ◽  
Cécile Bousquet-Antonelli ◽  
Rémy Merret
Keyword(s):  
Gene Expression ◽  
Quality Control ◽  
Transcriptional Regulation ◽  
High Throughput ◽  
Library Preparation ◽  
Working Day ◽  
Set Up ◽  
Post Transcriptional Regulation ◽  
Commercial Kit

The recent development of high-throughput technologies based on RNA sequencing has allowed a better description of the role of post-transcriptional regulation in gene expression. In particular, the development of degradome approaches based on the capture of 5′monophosphate decay intermediates allows the discovery of a new decay pathway called co-translational mRNA decay. Thanks to these approaches, ribosome dynamics could now be revealed by analysis of 5′P reads accumulation. However, library preparation could be difficult to set-up for non-specialists. Here, we present a fast and efficient 5′P degradome library preparation for Arabidopsis samples. Our protocol was designed without commercial kit and gel purification and can be easily done in one working day. We demonstrated the robustness and the reproducibility of our protocol. Finally, we present the bioinformatic reads-outs necessary to assess library quality control.

scholarly journals Cyclic Diguanylate Regulates Virulence Factor Genes via Multiple Riboswitches inClostridium difficile

mSphere ◽  
2018 ◽  
Vol 3 (5) ◽  
Author(s):  
Robert W. McKee ◽  
Carissa K. Harvest ◽  
Rita Tamayo
Keyword(s):  
Gene Expression ◽  
Clostridium Difficile ◽  
Biofilm Formation ◽  
Toxin Production ◽  
Intestinal Colonization ◽  
Cyclic Diguanylate ◽  
Signaling Molecule ◽  
Content Type ◽  
Surface Motility

ABSTRACTThe intracellular signaling molecule cyclic diguanylate (c-di-GMP) regulates many processes in bacteria, with a central role in controlling the switch between motile and nonmotile lifestyles. Recent work has shown that inClostridium difficile(also calledClostridioides difficile), c-di-GMP regulates swimming and surface motility, biofilm formation, toxin production, and intestinal colonization. In this study, we determined the transcriptional regulon of c-di-GMP inC. difficile,employing overexpression of a diguanylate cyclase gene to artificially manipulate intracellular c-di-GMP. Consistent with prior work, c-di-GMP regulated the expression of genes involved in swimming and surface motility. c-di-GMP also affected the expression of multiple genes encoding cell envelope proteins, several of which affected biofilm formationin vitro. A substantial proportion of the c-di-GMP regulon appears to be controlled either directly or indirectly via riboswitches. We confirmed the functionality of 11 c-di-GMP riboswitches, demonstrating their effects on downstream gene expression independent of the upstream promoters. The class I riboswitches uniformly functioned as “off” switches in response to c-di-GMP, while class II riboswitches acted as “on” switches. Transcriptional analyses of genes 3′ of c-di-GMP riboswitches over a broad range of c-di-GMP levels showed that relatively modest changes in c-di-GMP levels are capable of altering gene transcription, with concomitant effects on microbial behavior. This work expands the known c-di-GMP signaling network inC. difficileand emphasizes the role of the riboswitches in controlling known and putative virulence factors inC. difficile.IMPORTANCEInClostridium difficile, the signaling molecule c-di-GMP regulates multiple processes affecting its ability to cause disease, including swimming and surface motility, biofilm formation, toxin production, and intestinal colonization. In this study, we used RNA-seq to define the transcriptional regulon of c-di-GMP inC. difficile. Many new targets of c-di-GMP regulation were identified, including multiple putative colonization factors. Transcriptional analyses revealed a prominent role for riboswitches in c-di-GMP signaling. Only a subset of the 16 previously predicted c-di-GMP riboswitches were functionalin vivoand displayed potential variability in their response kinetics to c-di-GMP. This work underscores the importance of studying c-di-GMP riboswitches in a relevant biological context and highlights the role of the riboswitches in controlling gene expression inC. difficile.

scholarly journals CsrA (BB0184) Is Not Involved in Activation of the RpoN-RpoS Regulatory Pathway in Borrelia burgdorferi

2014 ◽  
Vol 82 (4) ◽  
pp. 1511-1522 ◽  
Author(s):  
Zhiming Ouyang ◽  
Jianli Zhou ◽  
Michael V. Norgard
Keyword(s):  
Gene Expression ◽  
Borrelia Burgdorferi ◽  
Animal Studies ◽  
Expression System ◽  
Regulatory Pathway ◽  
Content Type ◽  
Mouse Tissues ◽  
Differential Gene ◽  
Carbon Storage Regulator

ABSTRACTBorrelia burgdorferiencodes a homologue of the bacterial carbon storage regulator A (CsrA). Recently, it was reported that CsrA contributes toB. burgdorferiinfectivity and is required for the activation of the central RpoN-RpoS regulatory pathway. However, many questions concerning the function of CsrA inB. burgdorferigene regulation remain unanswered. In particular, there are conflicting reports concerning the molecular details of how CsrA may modulaterpoSexpression and, thus, how CsrA may influence the RpoN-RpoS pathway inB. burgdorferi. To address these key discrepancies, we examined the role of CsrA in differential gene expression in the Lyme disease spirochete. Upon engineering an induciblecsrAexpression system inB. burgdorferi, controlled hyperexpression of CsrA in a merodiploid strain did not significantly alter the protein and transcript levels ofbosR,rpoS, and RpoS-dependent genes (such asospCanddbpA). In addition, we constructed isogeniccsrAmutants in two widely used infectiousB. burgdorferistrains. When expression ofbosR,rpoS,ospC, anddbpAwas compared between thecsrAmutants and their wild-type counterparts, no detectable differences were observed. Finally, animal studies indicated that thecsrAmutants remained infectious for and virulent in mice. Analyses ofB. burgdorferigene expression in mouse tissues showed comparable levels ofrpoStranscripts by thecsrAmutants and the parental strains. Taken together, these results constitute compelling evidence that CsrA is not involved in activation of the RpoN-RpoS pathway and is dispensable for mammalian infectious processes carried out byB. burgdorferi.

scholarly journals RstA Is a Major Regulator ofClostridioides difficileToxin Production and Motility

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
Author(s):  
Adrianne N. Edwards ◽  
Brandon R. Anjuwon-Foster ◽  
Shonna M. McBride
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Toxin Production ◽  
Toxin Gene ◽  
Dna Binding Domain ◽  
Content Type ◽  
Toxin Genes ◽  
Clostridioides Difficile ◽  
Species Specific ◽  
Toxin Gene Expression

ABSTRACTClostridioides difficileinfection (CDI) is a toxin-mediated diarrheal disease. Several factors have been identified that influence the production of the two majorC. difficiletoxins, TcdA and TcdB, but prior published evidence suggested that additional unknown factors were involved in toxin regulation. Previously, we identified aC. difficileregulator, RstA, that promotes sporulation and represses motility and toxin production. We observed that the predicted DNA-binding domain of RstA was required for RstA-dependent repression of toxin genes, motility genes, andrstAtranscription. In this study, we further investigated the regulation of toxin and motility gene expression by RstA. DNA pulldown assays confirmed that RstA directly binds therstApromoter via the predicted DNA-binding domain. Through mutational analysis of therstApromoter, we identified several nucleotides that are important for RstA-dependent transcriptional regulation. Further, we observed that RstA directly binds and regulates the promoters of the toxin genestcdAandtcdB, as well as the promoters for thesigDandtcdRgenes, which encode regulators of toxin gene expression. Complementation analyses with theClostridium perfringensRstA ortholog and a multispecies chimeric RstA protein revealed that theC. difficileC-terminal domain is required for RstA DNA-binding activity, suggesting that species-specific signaling controls RstA function. Our data demonstrate that RstA is a transcriptional repressor that autoregulates its own expression and directly inhibits transcription of the two toxin genes and two positive toxin regulators, thereby acting at multiple regulatory points to control toxin production.IMPORTANCEClostridioides difficileis an anaerobic, gastrointestinal pathogen of humans and other mammals.C. difficileproduces two major toxins, TcdA and TcdB, which cause the symptoms of the disease, and forms dormant endospores to survive the aerobic environment outside the host. A recently discovered regulatory factor, RstA, inhibits toxin production and positively influences spore formation. Herein, we determine that RstA directly binds its own promoter DNA to repress its own gene transcription. In addition, our data demonstrate that RstA directly represses toxin gene expression and gene expression of two toxin gene activators, TcdR and SigD, creating a complex regulatory network to tightly control toxin production. This study provides a novel regulatory link betweenC. difficilesporulation and toxin production. Further, our data suggest thatC. difficiletoxin production is regulated through a direct, species-specific sensing mechanism.

scholarly journals Overexpression of mitogen-activated protein kinase (ERK1) enhances T- cell cytokine gene expression: role of AP1, NF-AT, and NF-KB

Blood ◽  
1993 ◽  
Vol 82 (8) ◽  
pp. 2470-2477 ◽  
Author(s):  
JH Park ◽  
L Levitt
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Dna Binding ◽  
Map Kinase ◽  
Binding Activity ◽  
Cytokine Gene Expression ◽  
Cytokine Gene ◽  
Dna Binding Activity ◽  
Mitogen Activated Protein

Abstract Transfected Jurkat cells overexpressing extracellular signal-regulated kinase (ERK1), also referred to as mitogen-activated protein (MAP) kinase, were selected by Western blotting assay using anti-ERK1 and antiphosphotyrosine antibodies in combination with a functional MAP kinase assay. We then asked whether enhanced ERK1 expression had any effect on induction of T-cell cytokine genes. The results show that overexpression of ERK1 enhances expression of T-cell interleukin-2 (IL- 2), IL-3, and granulocyte-macrophage colony-stimulating factor mRNA; no change was seen in expression of the alpha-actin gene. DNA-binding activities of the transcription factors AP1, NF-AT, and NF-kB were specifically increased twofold to fourfold in ERK1-overexpressing clones relative to nontransformed or vector-transformed cells, whereas no enhancement of CK1-CK2 protein DNA binding activity was detected after ERK1 overexpression. Additionally, increased NF-AT DNA binding activity was associated with functional enhancement of NF-AT transactivating activity in ERK1-overexpressing cells. These results provide direct evidence for the role of MAP kinase in the regulation of cytokine gene expression and indicate that such regulation is likely mediated through the enhanced DNA binding activity of specific nuclear transcription factors.

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