scholarly journals Identification of the Regulon of AphB and Its Essential Roles in LuxR and Exotoxin Asp Expression in the Pathogen Vibrio alginolyticus

2017 ◽  
Vol 199 (20) ◽  
Author(s):  
Xiating Gao ◽  
Yang Liu ◽  
Huan Liu ◽  
Zhen Yang ◽  
Qin Liu ◽  
...  
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Vibrio Alginolyticus ◽  
Physiological Adaptation ◽  
Pcr Analysis ◽  
Growth Stages ◽  
Mobility Shift ◽  
Marine Animals ◽  
Content Type ◽  
Virulence Gene Expression

ABSTRACT In Vibrio species, AphB is essential to activate virulence cascades by sensing low-pH and anaerobiosis signals; however, its regulon remains largely unknown. Here, AphB is found to be a key virulence regulator in Vibrio alginolyticus, a pathogen for marine animals and humans. Chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) enabled the detection of 20 loci in the V. alginolyticus genome that contained AphB-binding peaks. An AphB-specific binding consensus was confirmed by electrophoretic mobility shift assays (EMSAs), and the regulation of genes flanking such binding sites was demonstrated using quantitative real-time PCR analysis. AphB binds directly to its own promoter and positively controls its own expression in later growth stages. AphB also activates the expression of the exotoxin Asp by binding directly to the promoter regions of asp and the master quorum-sensing (QS) regulator luxR. DNase I footprinting analysis uncovered distinct AphB-binding sites (BBS) in these promoters. Furthermore, a BBS in the luxR promoter region overlaps that of LuxR-binding site I, which mediates the positive control of luxR promoter activity by AphB. This study provides new insights into the AphB regulon and reveals the mechanisms underlying AphB regulation of physiological adaptation and QS-controlled virulence in V. alginolyticus. IMPORTANCE In this work, AphB is determined to play essential roles in the expression of genes associated with QS, physiology, and virulence in V. alginolyticus, a pathogen for marine animals and humans. AphB was found to bind directly to 20 genes and control their expression by a 17-bp consensus binding sequence. Among the 20 genes, the aphB gene itself was identified to be positively autoregulated, and AphB also positively controlled asp and luxR expression. Taken together, these findings improve our understanding of the roles of AphB in controlling physiological adaptation and QS-controlled virulence gene expression.

scholarly journals VqsA, a Novel LysR-Type Transcriptional Regulator, Coordinates Quorum Sensing (QS) and Is Controlled by QS To Regulate Virulence in the Pathogen Vibrio alginolyticus

2018 ◽  
Vol 84 (12) ◽  
Author(s):  
Xiating Gao ◽  
Xuetong Wang ◽  
Qiaoqiao Mao ◽  
Rongjing Xu ◽  
Xiaohui Zhou ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Cell Density ◽  
Binding Sites ◽  
Feedback Inhibition ◽  
Transcriptional Regulator ◽  
Transcriptional Factors ◽  
Vibrio Alginolyticus ◽  
Type Vi Secretion System ◽  
Mobility Shift ◽  
Content Type

ABSTRACT The quorum sensing (QS) system controls bacterial group behaviors in response to cell density. In vibrios, LuxR and AphA are two master QS regulators (MQSRs) controlling gene expression in response to high or low cell density. Other regulators involved in the regulation of these two MQSRs and QS pathways remain to be determined. Here, we performed bacterial one-hybrid (B1H)-assay-based screens of transcriptional factors (TFs) to identify TFs that can directly regulate the expression of luxR and aphA from a library of 285 TFs encoded by the fish pathogen Vibrio alginolyticus. A total of 7 TFs were identified to bind to the promoters of both luxR and aphA. Among these TFs, the novel LysR-type transcriptional regulator (LTTR) VqsA could activate LuxR and repress AphA transcription. Meanwhile, LuxR and AphA exerted feedback inhibition and activation of vqsA expression, respectively, indicating that VqsA coordinates QS and is also regulated by QS. In addition, VqsA inhibited its own expression by directly binding to its own promoter region. The VqsA-binding sites in the promoter regions of luxR and aphA as well as the binding sites of LuxR, AphA, and VqsA in the vqsA gene were uncovered by electrophoretic mobility shift assays (EMSAs) and DNase I footprinting analysis. Finally, VqsA was verified to play essential roles in QS-regulated phenotypes, i.e., type VI secretion system 2 (T6SS2)-dependent interbacterial competition, biofilm formation, exotoxin production, and in vivo virulence of V. alginolyticus. Collectively, our data showed that VqsA is an important QS regulator in V. alginolyticus. IMPORTANCE Investigation of the mechanism of regulation of quorum sensing (QS) systems will facilitate an understanding of bacterial pathogenesis and the identification of effective QS interference (QSI) targets. Here, we systematically screened transcriptional factors (TFs) that modulate the expression of the master QS regulators (MQSRs) LuxR and AphA, and a novel LysR-type transcriptional regulator, VqsA, was identified. Our data illuminated the mechanisms mediating the interaction among LuxR, AphA, and VqsA as well as the effects of these regulators on the expression and output of QS. The impaired expression of virulence genes as a result of vqsA disruption demonstrated that VqsA is an important player in QS regulation and pathogenesis and may be the third MQSR involved in sensing environmental signals by vibrios to coordinate QS responses. This study will facilitate the development of strategies to interfere with QS and effectively control this pathogen that plagues the aquaculture industry.

scholarly journals Chromatin Immunoprecipitation Sequencing Technology Reveals Global Regulatory Roles of Low-Cell-Density Quorum-Sensing Regulator AphA in the Pathogen Vibrio alginolyticus

2016 ◽  
Vol 198 (21) ◽  
pp. 2985-2999 ◽  
Author(s):  
Dan Gu ◽  
Huan Liu ◽  
Zhen Yang ◽  
Yuanxing Zhang ◽  
Qiyao Wang
Keyword(s):  
Quorum Sensing ◽  
Cell Density ◽  
Small Rna ◽  
Chromatin Immunoprecipitation ◽  
Specific Binding ◽  
Vibrio Alginolyticus ◽  
Binding Motif ◽  
Marine Animals ◽  
Content Type ◽  
Low Cell Density

ABSTRACTQuorum sensing (QS) is an important regulatory system in virulence expression and environmental adaptation in bacteria. The master QS regulators (MQSR) LuxR and AphA reciprocally control QS gene expression in vibrios. However, the molecular basis for the regulatory functions of AphA remains undefined. In this study, we characterized its regulatory roles inVibrio alginolyticus, an important zoonotic pathogen causing diseases in marine animals as well as in humans. AphA is involved in the motility ability, biofilm formation, andin vivosurvival ofV. alginolyticus. Specifically, AphA is expressed at low-cell-density growth phases. In addition, AphA negatively regulates the expression of the main virulence factor, alkaline serine protease (Asp), through LuxR. Chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) detected 49 enriched loci harboring AphA-binding peaks across theV. alginolyticusgenome. An AphA-specific binding motif was identified and further confirmed by electrophoretic mobility shift assay (EMSA) and mutagenesis analysis. A quantitative real-time PCR (qRT-PCR) assay further validated the regulation of AphA on these genes. AphA binds directly to theaphApromoter and negatively regulates its own expression. Moreover, AphA directly regulates genes encoding adenylate cyclase, anti-σD, FabR, and the small RNA CsrB, revealing versatile regulatory roles of AphA in its physiology and virulence. Furthermore, our data indicated that AphA modulates motility through the coordinated function of LuxR and CsrB. Collectively, the findings of this work contribute to better understanding of the regulatory roles of AphA in QS and non-QS genes.IMPORTANCEIn this work, we determined that AphA, the master regulator of QS at low cell density, plays essential roles in expression of genes associated with physiology and virulence inV. alginolyticus, a Gram-negative pathogen for humans and marine animals. We further uncovered that 49 genes could be directly regulated by AphA and a 19-bp consensus binding sequence was identified. Among the 49 genes, the QS and other non-QS-associated genes were identified to be regulated by AphA. Besides, the small RNA CsrB was negatively regulated by AphA, and AphA regulate motility abilities through both CsrB and LuxR. Taken together, the findings of this study improve our understanding of the complex regulation network of AphA and QS.

scholarly journals CidR and CcpA Synergistically Regulate Staphylococcus aureus cidABC Expression

2019 ◽  
Vol 201 (23) ◽  
Author(s):  
Marat R. Sadykov ◽  
Ian H. Windham ◽  
Todd J. Widhelm ◽  
Vijaya Kumar Yajjala ◽  
Sean M. Watson ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Carbon Metabolism ◽  
Protein A ◽  
Transcriptional Regulators ◽  
Extracellular Dna ◽  
Regulatory Control ◽  
Pcr Analysis ◽  
Mobility Shift ◽  
Content Type ◽  
Biofilm Development

ABSTRACT The death and lysis of a subpopulation of Staphylococcus aureus cells during biofilm development benefit the whole bacterial population through the release of an important component of the biofilm matrix, extracellular DNA. Previously, we have demonstrated that these processes are affected by the gene products of the cidABC operon, the expression of which is controlled by the LysR-type transcriptional regulator, CidR. In this study, we characterized cis- and trans-acting elements essential for the induction of the cidABC operon. In addition to a CidR-binding site located within the cidABC promoter region, sequence analysis revealed the presence of a putative catabolite responsive element (cre box), suggestive of the involvement of the catabolite control protein A (CcpA) in the regulation of cidABC expression. This was confirmed using electrophoretic mobility shift assays and real-time reverse transcriptase PCR analysis demonstrating the direct positive control of cidABC transcription by the master regulator of carbon metabolism. Furthermore, the importance of CcpA and the identified cre site for the induction of the cidABC operon was demonstrated by examining the expression of PcidABC-lacZ reporter fusions in various mutant strains in which the genes involved in carbon metabolism and carbon catabolite repression were disrupted. Together the results of this study demonstrate the necessity of both transcriptional regulators, CidR and CcpA, for the induction of the cidABC operon and reveal the complexity of molecular interactions controlling its expression. IMPORTANCE This work focuses on the characterization of cis- and trans-acting elements essential for the induction of the cidABC operon in S. aureus. The results of this study are the first to demonstrate the synergistic control of cidABC expression by transcriptional regulators CidR and CcpA during carbohydrate metabolism. We established that the full induction of cidABC expression depends on the metabolic state of bacteria and requires both CidR and CcpA. Together, these findings delineate regulatory control of cidABC expression under different metabolic conditions and provide important new insights into our understanding of cell death mechanisms during biofilm development in S. aureus.

scholarly journals Binding of Pseudomonas aeruginosa AlgZ to Sites Upstream of the algZ Promoter Leads to Repression of Transcription

2005 ◽  
Vol 187 (13) ◽  
pp. 4430-4443 ◽  
Author(s):  
Deborah M. Ramsey ◽  
Patricia J. Baynham ◽  
Daniel J. Wozniak
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Binding Sites ◽  
Transcriptional Control ◽  
Specific Binding ◽  
Transcriptional Start Site ◽  
Opportunistic Pathogen ◽  
Single Copy ◽  
Amino Acid Identity ◽  
Mobility Shift ◽  
Base Pairs

ABSTRACT Mucoid variants of the opportunistic pathogen Pseudomonas aeruginosa produce the exopolysaccharide alginate and colonize the respiratory tracts of cystic fibrosis patients. The genes encoding the alginate biosynthetic enzymes are clustered in a single operon, which is under tight transcriptional control. One essential activator of the alginate operon is AlgZ, a proposed ribbon-helix-helix DNA binding protein that shares 30% amino acid identity with the Mnt repressor of Salmonella enterica serovar Typhimurium bacteriophage P22. In the current study, we examined the role of AlgZ as an autoregulator. Using single-copy algZ-lacZ transcription fusions, an increase in algZ transcription was observed in an algZ mutant compared to the isogenic wild-type strain, suggesting that AlgZ may have an additional role as a repressor. To identify the AlgZ binding site, overlapping regions upstream of algZ were incubated with AlgZ and analyzed by electrophoretic mobility shift assays. Specific binding activity was localized to a region spanning from 66 to 185 base pairs upstream of the algZ transcriptional start site. Two AlgZ binding sites were defined using copper-phenanthroline footprinting and deletion analyses, with one site centered at 93 base pairs and the other centered at 161 base pairs upstream of the algZ promoter. Deletion of both binding sites resulted in the loss of AlgZ binding. These results indicate that AlgZ represses algZ transcription, and this activity is mediated by multiple AlgZ-DNA interactions.

scholarly journals Toxicity and Binding Studies of Bacillus thuringiensis Cry1Ac, Cry1F, Cry1C, and Cry2A Proteins in the Soybean Pests Anticarsia gemmatalis and Chrysodeixis (Pseudoplusia) includens

2017 ◽  
Vol 83 (11) ◽  
Author(s):  
Yolanda Bel ◽  
Joel J. Sheets ◽  
Sek Yee Tan ◽  
Kenneth E. Narva ◽  
Baltasar Escriche
Keyword(s):  
Bacillus Thuringiensis ◽  
Binding Sites ◽  
Specific Binding ◽  
Management Strategies ◽  
Anticarsia Gemmatalis ◽  
Cross Resistance ◽  
Binding Studies ◽  
Content Type ◽  
Bt Toxins ◽  
Competition Binding

ABSTRACT Anticarsia gemmatalis (velvetbean caterpillar) and Chrysodeixis includens (soybean looper, formerly named Pseudoplusia includens) are two important defoliating insects of soybeans. Both lepidopteran pests are controlled mainly with synthetic insecticides. Alternative control strategies, such as biopesticides based on the Bacillus thuringiensis (Bt) toxins or transgenic plants expressing Bt toxins, can be used and are increasingly being adopted. Studies on the insect susceptibilities and modes of action of the different Bt toxins are crucial to determine management strategies to control the pests and to delay outbreaks of insect resistance. In the present study, the susceptibilities of both soybean pests to the Bt toxins Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa have been investigated. Bioassays performed in first-instar larvae showed that both insects are susceptible to all these toxins. Competition-binding studies carried out with Cry1Ac and Cry1Fa 125-iodine labeled proteins demonstrated the presence of specific binding sites for both of them on the midgut brush border membrane vesicles (BBMVs) of both A. gemmatalis and C. includens. Competition-binding experiments and specific-binding inhibition studies performed with selected sugars and lectins indicated that Cry1Ac and Cry1Fa share some, but not all, binding sites in the midguts of both insects. Also, the Cry1Ac- or Cry1Fa-binding sites were not shared with Cry1Ca or Cry2Aa in either soybean pest. This study contributes to the knowledge of Bt toxicity and midgut toxin binding sites in A. gemmatalis and C. includens and sheds light on the cross-resistance potential of Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa Bt proteins as candidate proteins for Bt-pyramided crops. IMPORTANCE In the present study, the toxicity and the mode of action of the Bacillus thuringiensis (Bt) toxins Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa in Anticarsia gemmatalis and Chrysodeixis includens (important defoliating pests of soybeans) have been investigated. These studies are crucial for determining management strategies for pest control. Bioassays showed that both insects were susceptible to the toxins. Competition-binding studies demonstrated the presence of Cry1Fa- and Cry1Ac-specific binding sites in the midguts of both pests. These results, together with the results from binding inhibition studies performed with sugars and lectins, indicated that Cry1Ac and Cry1Fa share some, but not all, binding sites, and that they were not shared with Cry1Ca or Cry2Aa in either soybean pest. This study contributes to the knowledge of Bt toxicity in A. gemmatalis and C. includens and sheds light on the cross-resistance potential of Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa Bt proteins as candidate proteins for Bt-pyramided crops.

scholarly journals Mutations within themepAOperator Affect Binding of the MepR Regulatory Protein and Its Induction by MepA Substrates in Staphylococcus aureus

2015 ◽  
Vol 197 (6) ◽  
pp. 1104-1114 ◽  
Author(s):  
Bryan D. Schindler ◽  
Susan M. Seo ◽  
Ivan Birukou ◽  
Richard G. Brennan ◽  
Glenn W. Kaatz
Keyword(s):  
Staphylococcus Aureus ◽  
Binding Sites ◽  
Regulatory Protein ◽  
Titration Calorimetry ◽  
Multidrug Efflux ◽  
Mobility Shift ◽  
Content Type ◽  
Operator Mutant ◽  
Current Sequence

The expression ofmepA, encoding theStaphylococcus aureusMepA multidrug efflux protein, is repressed by the MarR homologue MepR. Repression occurs through binding of two MepR dimers to an operator with two homologous and closely approximated pseudopalindromic binding sites (site 1 [S1] and site 2 [S2]). MepR binding is impeded in the presence of pentamidine, a MepA substrate. The effects of variousmepAoperator mutations on MepR binding were determined using electrophoretic mobility shift assays and isothermal titration calorimetry, and anin vivoconfirmation of the effects observed was established for a fully palindromic operator mutant. Altering the S1-S2 spacing by 1 to 4 bp severely impaired S2 binding, likely due to a physical collision between adjacent MepR dimers. Extension of the spacing to 9 bp eliminated the S1 binding-mediated DNA allostery required for efficient S2 binding, consistent with positive cooperative binding of MepR dimers. Binding of a single dimer to S1 was maintained when S2 was disrupted, whereas disruption of S1 eliminated any significant binding to S2, also consistent with positive cooperativity. Palindromization of binding sites, especially S2, enhanced MepR affinity for themepAoperator and reduced MepA substrate-mediated MepR induction. As a result, the on-off equilibrium between MepR and its binding sites was shifted toward the on state, resulting in less free MepR being available for interaction with inducing ligand. The selective pressure(s) under whichmepAexpression is advantageous likely contributed to the accumulation of mutations in themepAoperator, resulting in the current sequence from which MepR is readily induced by MepA substrates.

scholarly journals Phenylacetic Acid Catabolism and Its Transcriptional Regulation in Corynebacterium glutamicum

2012 ◽  
Vol 78 (16) ◽  
pp. 5796-5804 ◽  
Author(s):  
Xi Chen ◽  
Thomas A. Kohl ◽  
Christian Rückert ◽  
Dmitry A. Rodionov ◽  
Ling-Hao Li ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Phenylacetic Acid ◽  
Specific Binding ◽  
Genomic Analysis ◽  
Negative Regulator ◽  
Comparative Genomic ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Content Type ◽  
Transcription Regulator

ABSTRACTThe industrially important organismCorynebacterium glutamicumhas been characterized in recent years for its robust ability to assimilate aromatic compounds. In this study,C. glutamicumstrain AS 1.542 was investigated for its ability to catabolize phenylacetic acid (PAA). Thepaagenes were identified; they are organized as a continuouspaagene cluster. The type strain ofC. glutamicum, ATCC 13032, is not able to catabolize PAA, but the recombinant strain ATCC 13032/pEC-K18mob2::paagained the ability to grow on PAA. ThepaaRgene, encoding a TetR family transcription regulator, was studied in detail. Disruption ofpaaRin strain AS 1.542 resulted in transcriptional increases of allpaagenes. Transcription start sites and putative promoter regions were determined. An imperfect palindromic motif (5′-ACTNACCGNNCGNNCGGTNAGT-3′; 22 bp) was identified in the upstream regions ofpaagenes. Electrophoretic mobility shift assays (EMSA) demonstrated specific binding of PaaR to this motif, and phenylacetyl coenzyme A (PA-CoA) blocked binding. It was concluded that PaaR is the negative regulator of PAA degradation and that PA-CoA is the PaaR effector. In addition, GlxR binding sites were found, and binding to GlxR was confirmed. Therefore, PAA catabolism inC. glutamicumis regulated by the pathway-specific repressor PaaR, and also likely by the global transcription regulator GlxR. By comparative genomic analysis, we reconstructed orthologous PaaR regulons in 57 species, including species ofActinobacteria,Proteobacteria, andFlavobacteria, that carry PAA utilization genes and operate by conserved binding motifs, suggesting that PaaR-like regulation might commonly exist in these bacteria.

scholarly journals Induction of Virulence Gene Expression in Staphylococcus aureus by Pulmonary Surfactant

2014 ◽  
Vol 82 (4) ◽  
pp. 1500-1510 ◽  
Author(s):  
Kenichi Ishii ◽  
Tatsuo Adachi ◽  
Jyunichiro Yasukawa ◽  
Yutaka Suzuki ◽  
Hiroshi Hamamoto ◽  
...  
Keyword(s):  
Gene Expression ◽  
Staphylococcus Aureus ◽  
Stress Responses ◽  
Pulmonary Surfactant ◽  
Virulence Gene ◽  
Pcr Analysis ◽  
Host Animal ◽  
Content Type ◽  
Disruption Mutant ◽  
Virulence Gene Expression

ABSTRACTWe performed a genomewide analysis using a next-generation sequencer to investigate the effect of pulmonary surfactant on gene expression inStaphylococcus aureus, a clinically important opportunistic pathogen. RNA sequence (RNA-seq) analysis of bacterial transcripts at late log phase revealed 142 genes that were upregulated >2-fold following the addition of pulmonary surfactant to the culture medium. Among these genes, we confirmed by quantitative reverse transcription-PCR analysis that mRNA amounts for genes encoding ESAT-6 secretion system C (EssC), an unknown hypothetical protein (NWMN_0246; also called pulmonary surfactant-inducible factor A [PsiA] in this study), and hemolysin gamma subunit B (HlgB) were increased 3- to 10-fold by the surfactant treatment. Among the major constituents of pulmonary surfactant, i.e., phospholipids and palmitate, only palmitate, which is the most abundant fatty acid in the pulmonary surfactant and a known antibacterial substance, stimulated the expression of these three genes. Moreover, these genes were also induced by supplementing the culture with detergents. The induction of gene expression by surfactant or palmitate was not observed in a disruption mutant of thesigBgene, which encodes an alternative sigma factor involved in bacterial stress responses. Furthermore, each disruption mutant of theessC,psiA, andhlgBgenes showed attenuation of both survival in the lung and host-killing ability in a murine pneumonia model. These findings suggest thatS. aureusresists membrane stress caused by free fatty acids present in the pulmonary surfactant through the regulation of virulence gene expression, which contributes to its pathogenesis within the lungs of the host animal.

scholarly journals Binding Sites for Bacillus thuringiensis Cry2Ae Toxin on Heliothine Brush Border Membrane Vesicles Are Not Shared with Cry1A, Cry1F, or Vip3A Toxin

2011 ◽  
Vol 77 (10) ◽  
pp. 3182-3188 ◽  
Author(s):  
C. Gouffon ◽  
A. Van Vliet ◽  
J. Van Rie ◽  
S. Jansens ◽  
J. L. Jurat-Fuentes
Keyword(s):  
Bacillus Thuringiensis ◽  
Brush Border ◽  
Binding Sites ◽  
Brush Border Membrane ◽  
Specific Binding ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Target Range ◽  
Content Type ◽  
Bt Toxins

ABSTRACTThe use of combinations ofBacillus thuringiensis(Bt) toxins with diverse modes of action for insect pest control has been proposed as the most efficient strategy to increase target range and delay the onset of insect resistance. Considering that most cases of cross-resistance to Bt toxins in laboratory-selected insect colonies are due to alteration of common toxin binding sites, independent modes of action can be defined as toxins sharing limited or no binding sites in brush border membrane vesicles (BBMV) prepared from the target insect larvae. In this paper, we report on the specific binding of Cry2Ae toxin to binding sites on BBMV from larvae of the three most commercially relevant heliothine species,Heliothis virescens,Helicoverpa zea, andHelicoverpa armigera. Using chromatographic purification under reducing conditions before labeling, we detected specific binding of radiolabeled Cry2Ae, which allowed us to perform competition assays using Cry1Ab, Cry1Ac, Cry1Fa, Vip3A, Cry2Ae, and Cry2Ab toxins as competitors. In these assays, Cry2Ae binding sites were shared with Cry2Ab but not with the tested Cry1 or Vip3A toxins. Our data support the use of Cry2Ae toxin in combination with Cry1 or Vip3A toxins in strategies to increase target range and delay the onset of heliothine resistance.

scholarly journals CodY Regulates SigD Levels and Activity by Binding to Three Sites in thefla/cheOperon

2015 ◽  
Vol 197 (18) ◽  
pp. 2999-3006 ◽  
Author(s):  
Qutaiba O. Ababneh ◽  
Jennifer K. Herman
Keyword(s):  
Binding Sites ◽  
Maximal Level ◽  
Phenotypic Switch ◽  
Mobility Shift ◽  
Content Type ◽  
Motile Cells ◽  
Important Regulator ◽  
Electrophoretic Mobility Shift Assays ◽  
Tip Cells

ABSTRACTExponentially growing cultures ofBacillus subtilis(PY79) are composed primarily of nonmotile, chained cells. The alternative sigma factor, SigD, promotes the phenotypic switch from nonmotile, chained cells to unchained, motile cells. In the present work, we investigated the role of the GTP-sensing protein CodY in the regulation of SigD. Deletion ofcodYresulted in a significant increase in SigD accumulation and activity and shifted the proportion of unchained cells up from ∼15% to ∼75%, suggesting that CodY is an important regulator of SigD. CodY was previously shown to bind to the PD3and Pfla/chepromoters located upstream of the first gene in thesigD-containingfla/cheoperon. Using electrophoretic mobility shift assays, we found that CodY also binds to two other previously uncharacterized sites within thefla/cheoperon. Mutations in any one of the three binding sites resulted in SigD levels similar to those seen with the ΔcodYmutant, suggesting that each site is sufficient to tip cells toward a maximal level of CodY-dependent SigD accumulation. However, mutations in all three sites were required to phenocopy the ΔcodYmutant's reduced level of cell chaining, consistent with the idea that CodY binding in thefla/cheoperon is also important for posttranslational control of SigD activity.IMPORTANCEOne way that bacteria adapt quickly and efficiently to changes in environmental quality is to employ global transcriptional regulators capable of responding allosterically to key cellular metabolites. In this study, we found that the conserved GTP-sensing protein CodY directly regulates cell motility and chaining inB. subtilisby controlling expression and activity of SigD. Our results suggest thatB. subtilisbecomes poised for cell dispersal as intracellular GTP levels are depleted.

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