scholarly journals The Influence of Repressor DNA Binding Site Architecture on Transcriptional Control

mBio ◽  
2014 ◽  
Vol 5 (5) ◽  
Author(s):  
Dan M. Park ◽  
Patricia J. Kiley
Keyword(s):  
Escherichia Coli ◽  
Dna Binding ◽  
Binding Site ◽  
Information Content ◽  
Fine Tuning ◽  
Carbon Oxidation ◽  
Content Type ◽  
Architectural Features ◽  
Recognition Elements ◽  
Insight Into

ABSTRACTHow the architecture of DNA binding sites dictates the extent of repression of promoters is not well understood. Here, we addressed the importance of the number and information content of the three direct repeats (DRs) in the binding and repression of theicdApromoter by the phosphorylated form of the globalEscherichia colirepressor ArcA (ArcA-P). We show that decreasing the information content of the two sites with the highest information (DR1 and DR2) eliminated ArcA binding to all three DRs and ArcA repression oficdA. Unexpectedly, we also found that DR3 occupancy functions principally in repression, since mutation of this low-information-content site both eliminated DNA binding to DR3 and significantly weakenedicdArepression, despite the fact that binding to DR1 and DR2 was intact. In addition, increasing the information content of any one of the three DRs or addition of a fourth DR increased ArcA-dependent repression but perturbed signal-dependent regulation of repression. Thus, our data show that the information content and number of DR elements are critical architectural features for maintaining a balance between high-affinity binding and signal-dependent regulation oficdApromoter function in response to changes in ArcA-P levels. Optimization of such architectural features may be a common strategy to either dampen or enhance the sensitivity of DNA binding among the members of the large OmpR/PhoB family of regulators as well as other transcription factors.IMPORTANCEInEscherichia coli, the response regulator ArcA maintains homeostasis of redox carriers under O2-limiting conditions through a comprehensive repression of carbon oxidation pathways that require aerobic respiration to recycle redox carriers. Although a binding site architecture comprised of a variable number of sequence recognition elements has been identified within the promoter regions of ArcA-repressed operons, it is unclear how this variable architecture dictates transcriptional regulation. By dissecting the role of multiple sequence elements within theicdApromoter, we provide insight into the design principles that allow ArcA to repress transcription within diverse promoter contexts. Our data suggest that the arrangement of recognition elements is tailored to achieve sufficient repression of a given promoter while maintaining appropriate signal-dependent regulation of repression, providing insight into how diverse binding site architectures link changes in O2with the fine-tuning of carbon oxidation pathway levels.

scholarly journals The Nucleoid Occlusion Protein SlmA Binds to Lipid Membranes

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Miguel Ángel Robles-Ramos ◽  
William Margolin ◽  
Marta Sobrinos-Sanguino ◽  
Carlos Alfonso ◽  
Germán Rivas ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Dna Binding ◽  
Division Ring ◽  
Binding Sites ◽  
Lipid Membranes ◽  
Fine Tuning ◽  
Chromosomal Dna ◽  
Content Type ◽  
Binding Partners

ABSTRACT Protection of the chromosome from scission by the division machinery during cytokinesis is critical for bacterial survival and fitness. This is achieved by nucleoid occlusion, which, in conjunction with other mechanisms, ensures formation of the division ring at midcell. In Escherichia coli, this mechanism is mediated by SlmA, a specific DNA binding protein that antagonizes assembly of the central division protein FtsZ into a productive ring in the vicinity of the chromosome. Here, we provide evidence supporting direct interaction of SlmA with lipid membranes, tuned by its binding partners FtsZ and SlmA binding sites (SBS) on chromosomal DNA. Reconstructions in minimal membrane systems that mimic cellular environments show that SlmA binds to lipid-coated microbeads or locates at the edge of microfluidic-generated microdroplets, inside which the protein is encapsulated. DNA fragments containing SBS sequences do not seem to be recruited to the membrane by SlmA but instead compete with SlmA’s ability to bind lipids. The interaction of SlmA with FtsZ modulates this behavior, ultimately triggering membrane localization of the SBS sequences alongside the two proteins. The ability of SlmA to bind lipids uncovered in this work extends the interaction network of this multivalent regulator beyond its well-known protein and nucleic acid recognition, which may have implications in the overall spatiotemporal control of division ring assembly. IMPORTANCE Successful bacterial proliferation relies on the spatial and temporal precision of cytokinesis and its regulation by systems that protect the integrity of the nucleoid. In Escherichia coli, one of these protectors is SlmA protein, which binds to specific DNA sites around the nucleoid and helps to shield the nucleoid from inappropriate bisection by the cell division septum. Here, we discovered that SlmA not only interacts with the nucleoid and septum-associated cell division proteins but also binds directly to cytomimetic lipid membranes, adding a novel putative mechanism for regulating the local activity of these cell division proteins. We find that interaction between SlmA and lipid membranes is regulated by SlmA’s DNA binding sites and protein binding partners as well as chemical conditions, suggesting that the SlmA-membrane interactions are important for fine-tuning the regulation of nucleoid integrity during cytokinesis.

scholarly journals YjjQ Represses Transcription offlhDCand Additional Loci in Escherichia coli

2015 ◽  
Vol 197 (16) ◽  
pp. 2713-2720 ◽  
Author(s):  
Helene Wiebe ◽  
Doreen Gürlebeck ◽  
Jana Groß ◽  
Katrin Dreck ◽  
Derk Pannen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Binding ◽  
Binding Site ◽  
Transcriptional Repressor ◽  
Farm Animals ◽  
Sequence Motif ◽  
Content Type ◽  
E Coli ◽  
Dna Binding Site ◽  
K 12

ABSTRACTThe presumptive transcriptional regulator YjjQ has been identified as being virulence associated in avian pathogenicEscherichia coli(APEC). In this work, we characterize YjjQ as transcriptional repressor of theflhDCoperon, encoding the master regulator of flagellar synthesis, and of additional loci. The latter includegfc(capsule 4 synthesis),ompC(outer membrane porin C),yfiRNB(regulated c-di-GMP synthesis), and loci of poorly defined function (ybhLandymiA-yciX). We identify the YjjQ DNA-binding sites at theflhDCandgfcpromoters and characterize a DNA-binding sequence motif present at all promoters found to be repressed by YjjQ. At theflhDCpromoter, the YjjQ DNA-binding site overlaps the RcsA-RcsB DNA-binding site. RcsA-RcsB likewise represses theflhDCpromoter, but the repression by YjjQ and that by RcsA-RcsB are independent of each other. These data suggest that YjjQ is an additional regulator involved in the complex control offlhDCat the level of transcription initiation. Furthermore, we show that YjjQ represses motility of theE. coliK-12 laboratory strain and of uropathogenicE. coli(UPEC) strains CFT073 and 536. Regulation offlhDC,yfiRNB, and additional loci by YjjQ may be features relevant for pathogenicity.IMPORTANCEEscherichia coliis a commensal and pathogenic bacterium causing intra- and extraintestinal infections in humans and farm animals. The pathogenicity ofE. colistrains is determined by their particular genome content, which includes essential and associated virulence factors that control the cellular physiology in the host environment. However, the gene pools of commensal and pathogenicE. coliare not clearly differentiated, and the function of virulence-associated loci needs to be characterized. In this study, we characterize the function ofyjjQ, encoding a transcription regulator that was identified as being virulence associated in avian pathogenicE. coli(APEC). We characterize YjjQ as transcriptional repressor of flagellar motility and of additional loci related to pathogenicity.

scholarly journals Histone-like Nucleoid-Structuring Protein (H-NS) Paralogue StpA Activates the Type I-E CRISPR-Cas System against Natural Transformation in Escherichia coli

2020 ◽  
Vol 86 (14) ◽  
Author(s):  
Dongchang Sun ◽  
Xudan Mao ◽  
Mingyue Fei ◽  
Ziyan Chen ◽  
Tingheng Zhu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Natural Transformation ◽  
Inducible Promoter ◽  
Regulation Mechanism ◽  
Type I ◽  
Content Type ◽  
E Coli ◽  
Double Stranded Dna ◽  
Dna Binding Site

ABSTRACT Working mechanisms of CRISPR-Cas systems have been intensively studied. However, far less is known about how they are regulated. The histone-like nucleoid-structuring protein H-NS binds the promoter of cas genes (Pcas) and suppresses the type I-E CRISPR-Cas system in Escherichia coli. Although the H-NS paralogue StpA also binds Pcas, its role in regulating the CRISPR-Cas system remains unidentified. Our previous work established that E. coli is able to take up double-stranded DNA during natural transformation. Here, we investigated the function of StpA in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli. We first documented that although the activated type I-E CRISPR-Cas system, due to hns deletion, interfered with CRISPR-Cas-targeted plasmid transfer, stpA inactivation restored the level of natural transformation. Second, we showed that inactivating stpA reduced the transcriptional activity of Pcas. Third, by comparing transcriptional activities of the intact Pcas and the Pcas with a disrupted H-NS binding site in the hns and hns stpA null deletion mutants, we demonstrated that StpA activated transcription of cas genes by binding to the same site as H-NS in Pcas. Fourth, by expressing StpA with an arabinose-inducible promoter, we confirmed that StpA expressed at a low level stimulated the activity of Pcas. Finally, by quantifying the level of mature CRISPR RNA (crRNA), we demonstrated that StpA was able to promote the amount of crRNA. Taken together, our work establishes that StpA serves as a transcriptional activator in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli. IMPORTANCE StpA is normally considered a molecular backup of the nucleoid-structuring protein H-NS, which was reported as a transcriptional repressor of the type I-E CRISPR-Cas system in Escherichia coli. However, the role of StpA in regulating the type I-E CRISPR-Cas system remains elusive. Our previous work uncovered a new route for double-stranded DNA (dsDNA) entry during natural transformation of E. coli. In this study, we show that StpA plays a role opposite to that of its paralogue H-NS in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli. Our work not only expands our knowledge on CRISPR-Cas-mediated adaptive immunity against extracellular nucleic acids but also sheds new light on understanding the complex regulation mechanism of the CRISPR-Cas system. Moreover, the finding that paralogues StpA and H-NS share a DNA binding site but play opposite roles in transcriptional regulation indicates that higher-order compaction of bacterial chromatin by histone-like proteins could switch prokaryotic transcriptional modes.

scholarly journals Exploring the Amino Acid Residue Requirements of the RNA Polymerase (RNAP) α Subunit C-Terminal Domain for Productive Interaction between Spx and RNAP of Bacillus subtilis

2017 ◽  
Vol 199 (14) ◽  
Author(s):  
Cierra A. Birch ◽  
Madison J. Davis ◽  
Lea Mbengi ◽  
Peter Zuber
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
Dna Binding ◽  
Rna Polymerase ◽  
Α Subunit ◽  
Stress Induction ◽  
Content Type ◽  
Terminal Domain ◽  
Codon Substitution

ABSTRACT Bacillus subtilis Spx is a global transcriptional regulator that is conserved among Gram-positive bacteria, in which Spx is required for preventing oxidatively induced proteotoxicity. Upon stress induction, Spx engages RNA polymerase (RNAP) through interaction with the C-terminal domain of the rpoA-encoded RNAP α subunit (αCTD). Previous mutational analysis of rpoA revealed that substitutions of Y263 in αCTD severely impaired Spx-activated transcription. Attempts to substitute alanine for αCTD R261, R268, R289, E255, E298, and K294 were unsuccessful, suggesting that these residues are essential. To determine whether these RpoA residues were required for productive Spx-RNAP interaction, we ectopically expressed the putatively lethal rpoA mutant alleles in the rpoAY263C mutant, where “Y263C” indicates the amino acid change that results from mutation of the allele. By complementation analysis, we show that Spx-bound αCTD amino acid residues are not essential for Spx-activated transcription in vivo but that R261A, E298A, and E255A mutants confer a partial defect in NaCl-stress induction of Spx-controlled genes. In addition, strains expressing rpoAE255A are defective in disulfide stress resistance and produce RNAP having a reduced affinity for Spx. The E255 residue corresponds to Escherichia coli αD259, which has been implicated in αCTD-σ70 interaction (σ70 R603, corresponding to R362 of B. subtilis σA). However, the combined rpoAE255A and sigAR362A mutations have an additive negative effect on Spx-dependent expression, suggesting the residues' differing roles in Spx-activated transcription. Our findings suggest that, while αCTD is essential for Spx-activated transcription, Spx is the primary DNA-binding determinant of the Spx-αCTD complex. IMPORTANCE Though extensively studied in Escherichia coli, the role of αCTD in activator-stimulated transcription is largely uncharacterized in Bacillus subtilis. Here, we conduct phenotypic analyses of putatively lethal αCTD alanine codon substitution mutants to determine whether these residues function in specific DNA binding at the Spx-αCTD-DNA interface. Our findings suggest that multisubunit RNAP contact to Spx is optimal for activation while Spx fulfills the most stringent requirement of upstream promoter binding. Furthermore, several αCTD residues targeted for mutagenesis in this study are conserved among many bacterial species and thus insights on their function in other regulatory systems may be suggested herein.

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 Ligand-Controlled Proteolysis of the Escherichia coli Transcriptional Regulator ZntR

2007 ◽  
Vol 189 (8) ◽  
pp. 3017-3025 ◽  
Author(s):  
Mihaela Pruteanu ◽  
Saskia B. Neher ◽  
Tania A. Baker
Keyword(s):  
Escherichia Coli ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Transcriptional Regulator ◽  
Zinc Homeostasis ◽  
Fine Tuning ◽  
Physical Analysis ◽  
Cellular Environment ◽  
High Zinc

ABSTRACT Proteases play a crucial role in remodeling the bacterial proteome in response to changes in cellular environment. Escherichia coli ZntR, a zinc-responsive transcriptional regulator, was identified by proteomic experiments as a likely ClpXP substrate, suggesting that protein turnover may play a role in regulation of zinc homeostasis. When intracellular zinc levels are high, ZntR activates expression of ZntA, an ATPase essential for zinc export. We find that ZntR is degraded in vivo in a manner dependent on both the ClpXP and Lon proteases. However, ZntR degradation decreases in the presence of high zinc concentrations, the level of ZntR rises, and transcription of the zntA exporter is increased. Mutagenesis experiments reveal that zinc binding does not appear to be solely responsible for the zinc-induced protection from proteolysis. Therefore, we tested whether DNA binding was important in the zinc-induced stabilization of ZntR by mutagenesis of the DNA binding helices. Replacement of a conserved arginine (R19A) in the DNA binding domain both enhances ZntR degradation and abolishes zinc-induced transcriptional activation of zntA. Biochemical and physical analysis of ZntRR19A demonstrates that it is structurally similar to, and binds zinc as well as does, the wild-type protein but is severely defective in binding DNA. Thus, we conclude that two different ligands—zinc and DNA—function together to increase ZntR stability and that ligand-controlled proteolysis of ZntR plays an important role in fine-tuning zinc homeostasis in bacteria.

scholarly journals Searching for Genes Responsible for Patulin Degradation in a Biocontrol Yeast Provides Insight into the Basis for Resistance to This Mycotoxin

2013 ◽  
Vol 79 (9) ◽  
pp. 3101-3115 ◽  
Author(s):  
G. Ianiri ◽  
A. Idnurm ◽  
S. A. I. Wright ◽  
R. Durán-Patrón ◽  
L. Mannina ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Reactive Oxygen Species ◽  
Genetic Basis ◽  
Degradation Process ◽  
Oxygen Species ◽  
Content Type ◽  
Pome Fruits ◽  
Multistep Process ◽  
External Stresses ◽  
Insight Into

ABSTRACTPatulin is a mycotoxin that contaminates pome fruits and derived products worldwide. Basidiomycete yeasts belonging to the subphylumPucciniomycotinahave been identified to have the ability to degrade this molecule efficiently and have been explored through different approaches to understand this degradation process. In this study,Sporobolomycessp. strain IAM 13481 was found to be able to degrade patulin to form two different breakdown products, desoxypatulinic acid and (Z)-ascladiol. To gain insight into the genetic basis of tolerance and degradation of patulin, more than 3,000 transfer DNA (T-DNA) insertional mutants were generated in strain IAM 13481 and screened for the inability to degrade patulin using a bioassay based on the sensitivity ofEscherichia colito patulin. Thirteen mutants showing reduced growth in the presence of patulin were isolated and further characterized. Genes disrupted in patulin-sensitive mutants included homologs ofSaccharomyces cerevisiae YCK2,PAC2,DAL5, andVPS8. The patulin-sensitive mutants also exhibited hypersensitivity to reactive oxygen species as well as genotoxic and cell wall-destabilizing agents, suggesting that the inactivated genes are essential for tolerating and overcoming the initial toxicity of patulin. These results support a model whereby patulin degradation occurs through a multistep process that includes an initial tolerance to patulin that utilizes processes common to other external stresses, followed by two separate pathways for degradation.

scholarly journals VirB, a key transcriptional regulator of virulence plasmid genes in Shigella flexneri, forms DNA-binding site dependent foci in the bacterial cytoplasm

2021 ◽  
Author(s):  
Jillian N. Soceaa ◽  
Grant R. Bowmanb ◽  
Helen J. Wing
Keyword(s):  
Dna Binding ◽  
Subcellular Localization ◽  
Binding Site ◽  
Shigella Flexneri ◽  
Transcriptional Regulator ◽  
Virulence Plasmid ◽  
Focus Formation ◽  
Dna Binding Site ◽  
Bacterial Cytoplasm ◽  
Insight Into

VirB is a key regulator of genes located on the large virulence plasmid (pINV) in the bacterial pathogen Shigella flexneri. VirB is unusual; it is not related to other transcriptional regulators, instead, it belongs to a family of proteins that primarily function in plasmid and chromosome partitioning; exemplified by ParB. Despite this, VirB does not function to segregate DNA, but rather counters transcriptional silencing mediated by the nucleoid structuring protein, H-NS. Since ParB localizes subcellularly as discrete foci in the bacterial cytoplasm, we chose to investigate the subcellular localization of VirB to gain novel insight into how VirB functions as a transcriptional anti-silencer. To do this, a GFP-VirB fusion that retains the regulatory activity of VirB and yet, does not undergo significant protein degradation in S. flexneri, was used. Surprisingly, discrete fluorescent foci were observed in live wild-type S. flexneri cells and an isogenic virB mutant using fluorescence microscopy. In contrast, foci were rarely observed (<10%) in pINV-cured cells or in cells expressing a GFP-VirB fusion carrying amino acid substitutions in the VirB DNA binding domain. Finally, the 25 bp VirB-binding site was demonstrated to be sufficient and necessary for GFP-VirB focus formation using a set of small surrogate plasmids. Combined, these data demonstrate that the VirB:DNA interactions required for the transcriptional anti-silencing activity of VirB on pINV are a prerequisite for the subcellular localization of VirB in the bacterial cytoplasm. The significance of these findings, in light of the anti-silencing activity of VirB, is discussed. Importance This study reveals the subcellular localization of VirB, a key transcriptional regulator of virulence genes found on the large virulence plasmid (pINV) in Shigella. Fluorescent signals generated by an active GFP-VirB fusion form 2, 3, or 4 discrete foci in the bacterial cytoplasm, predominantly at the quarter cell position. These signals are completely dependent upon VirB interacting with its DNA binding site found either on the virulence plasmid or an engineered surrogate. Our findings: 1) provide novel insight into VirB:pINV interactions, 2) suggest that VirB may have utility as a DNA marker, and 3) raise questions about how and why this anti-silencing protein that controls virulence gene expression on pINV of Shigella spp. forms discrete foci/hubs within the bacterial cytoplasm.

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