scholarly journals SpdR, a Response Regulator Required for Stationary-Phase Induction of Caulobacter crescentus cspD

2010 ◽  
Vol 192 (22) ◽  
pp. 5991-6000 ◽  
Author(s):  
Carolina A. P. T. da Silva ◽  
Heloise Balhesteros ◽  
Ricardo R. Mazzon ◽  
Marilis V. Marques
Keyword(s):  
Stationary Phase ◽  
Cold Shock ◽  
Caulobacter Crescentus ◽  
Response Regulator ◽  
Phosphorylation Site ◽  
Regulatory Region ◽  
Repeat Sequence ◽  
Medium Composition ◽  
Cold Shock Protein ◽  
Mobility Shift

ABSTRACT The cold shock protein (CSP) family includes small polypeptides that are induced upon temperature downshift and stationary phase. The genome of the alphaproteobacterium Caulobacter crescentus encodes four CSPs, with two being induced by cold shock and two at the onset of stationary phase. In order to identify the environmental signals and cell factors that are involved in cspD expression at stationary phase, we have analyzed cspD transcription during growth under several nutrient conditions. The results showed that expression of cspD was affected by the medium composition and was inversely proportional to the growth rate. The maximum levels of expression were decreased in a spoT mutant, indicating that ppGpp may be involved in the signalization for carbon starvation induction of cspD. A Tn5 mutant library was screened for mutants with reduced cspD expression, and 10 clones that showed at least a 50% reduction in expression were identified. Among these, a strain with a transposon insertion into a response regulator of a two-component system showed no induction of cspD at stationary phase. This protein (SpdR) was able to acquire a phosphate group from its cognate histidine kinase, and gel mobility shift assay and DNase I footprinting experiments showed that it binds to an inverted repeat sequence of the cspD regulatory region. A mutated SpdR with a substitution of the conserved aspartyl residue that is the probable phosphorylation site is unable to bind to the cspD regulatory region and to complement the spdR mutant phenotype.

scholarly journals A Pair of Highly Conserved Two-Component Systems Participates in the Regulation of the Hypervariable FIR Proteins in Different Legionella Species

2007 ◽  
Vol 189 (9) ◽  
pp. 3382-3391 ◽  
Author(s):  
Michal Feldman ◽  
Gil Segal
Keyword(s):  
Binding Site ◽  
Legionella Pneumophila ◽  
Response Regulator ◽  
Regulatory Element ◽  
Expression System ◽  
Regulatory Region ◽  
Regulatory Elements ◽  
Mobility Shift ◽  
Type Iv ◽  
Essential Components

ABSTRACT Legionella pneumophila and other pathogenic Legionella species multiply inside protozoa and human macrophages by using the Icm/Dot type IV secretion system. The IcmQ protein, which possesses pore-forming activity, and IcmR, which functions as its chaperone, are two essential components of this system. It was previously shown that in 29 Legionella species, a large hypervariable-gene family (fir genes) is located upstream from a conserved icmQ gene, but although nonhomologous, the FIR proteins were found to function similarly together with their corresponding IcmQ proteins. Alignment of the regulatory regions of 29 fir genes revealed that they can be divided into three regulatory groups; the first group contains a binding site for the CpxR response regulator, which was previously shown to regulate the L. pneumophila fir gene (icmR); the second group, which includes most of the fir genes, contains the CpxR binding site and an additional regulatory element that was identified here as a PmrA binding site; and the third group contains only the PmrA binding site. Analysis of the regulatory region of two fir genes, which included substitutions in the CpxR and PmrA consensus sequences, a controlled expression system, as well as examination of direct binding with mobility shift assays, revealed that both CpxR and PmrA positively regulate the expression of the fir genes that contain both regulatory elements. The change in the regulation of the fir genes that occurred during the course of evolution might be required for the adaptation of the different Legionella species to their specific environmental hosts.

scholarly journals Microarray-Based Characterization of the Listeria monocytogenes Cold Regulon in Log- and Stationary-Phase Cells

2007 ◽  
Vol 73 (20) ◽  
pp. 6484-6498 ◽  
Author(s):  
Yvonne C. Chan ◽  
Sarita Raengpradub ◽  
Kathryn J. Boor ◽  
Martin Wiedmann
Keyword(s):  
Listeria Monocytogenes ◽  
Heat Shock ◽  
Stationary Phase ◽  
Response Regulator ◽  
Differentially Expressed ◽  
Cold Shock Protein ◽  
Rna Helicases ◽  
Transcript Levels ◽  
Number Of Genes ◽  
Genes Encoding

ABSTRACT Whole-genome microarray experiments were performed to define the Listeria monocytogenes cold growth regulon and to identify genes differentially expressed during growth at 4 and 37°C. Microarray analysis using a stringent cutoff (adjusted P < 0.001; ≥2.0-fold change) revealed 105 and 170 genes that showed higher transcript levels in logarithmic- and stationary-phase cells, respectively, at 4°C than in cells grown at 37°C. A total of 74 and 102 genes showed lower transcript levels in logarithmic- and stationary-phase cells, respectively, grown at 4°C. Genes with higher transcript levels at 4°C in both stationary- and log-phase cells included genes encoding a two-component response regulator (lmo0287), a cold shock protein (cspL), and two RNA helicases (lmo0866 and lmo1722), whereas a number of genes encoding virulence factors and heat shock proteins showed lower transcript levels at 4°C. Selected genes that showed higher transcript levels at 4°C during both stationary and log phases were confirmed by quantitative reverse transcriptase PCR. Our data show that (i) a large number of L. monocytogenes genes are differentially expressed at 4 and 37°C, with more genes showing higher transcript levels than lower transcript levels at 4°C, (ii) L. monocytogenes genes with higher transcript levels at 4°C include a number of genes and operons with previously reported or plausible roles in cold adaptation, and (iii) L. monocytogenes genes with lower transcript levels at 4°C include a number of virulence and virulence-associated genes as well as some heat shock genes.

scholarly journals Staphylococcus aureus AgrA Binding to the RNAIII-agr Regulatory Region

2004 ◽  
Vol 186 (22) ◽  
pp. 7549-7555 ◽  
Author(s):  
Robbin L. Koenig ◽  
Jessica L. Ray ◽  
Soheila J. Maleki ◽  
Mark S. Smeltzer ◽  
Barry K. Hurlburt
Keyword(s):  
Staphylococcus Aureus ◽  
Response Regulator ◽  
Osmotic Shock ◽  
Sequence Similarity ◽  
Regulatory Region ◽  
Amino Acid Sequence Similarity ◽  
Direct Repeats ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Partial Control

ABSTRACT The control of virulence gene expression in the human pathogen Staphylococcus aureus is under the partial control of the two-component quorum-sensing system encoded by genes of the agr locus. The product of the agrA gene has been shown by amino acid sequence similarity to be the putative response regulator; however, binding of AgrA to promoters under its control has not yet been demonstrated. In this study, we isolated and purified soluble AgrA by expression under osmotic shock conditions and ion-exchange chromatography. Purified AgrA showed high-affinity binding to the RNAIII-agr intergenic region by electrophoretic mobility shift assays. Binding was localized by DNase I protection assays to a pair of direct repeats in the P2 and P3 promoter regions of the agr locus. We found that this binding was enhanced by the addition of the small phosphoryl donor, acetyl phosphate. The difference in binding affinity between these two promoters was found to result from a 2-bp difference between the downstream direct repeats of the P2 and P3 sites. Mutation of these base pairs in the P3 site to match those found in the P2 site increased the affinity of AgrA for the P3 site relative to that for the P2 site. These results are consistent with the function of AgrA as a response regulator with recognition sites in the promoter regions of RNAIII and the agr locus.

scholarly journals Identification of CpxR as a Positive Regulator of icm and dot Virulence Genes of Legionella pneumophila

2003 ◽  
Vol 185 (16) ◽  
pp. 4908-4919 ◽  
Author(s):  
Ohad Gal-Mor ◽  
Gil Segal
Keyword(s):  
Legionella Pneumophila ◽  
Response Regulator ◽  
Regulatory Region ◽  
Acanthamoeba Castellanii ◽  
Virulence Gene ◽  
Growth Defect ◽  
Insertion Mutant ◽  
Mobility Shift ◽  
Successful Infection ◽  
Mobility Shift Assay

ABSTRACT To date, 24 Legionella pneumophila genes (icm and dot genes) have been shown to be required for intercellular growth and host cell killing. A previous report indicated that the regulation of these genes is complicated and probably involves several regulatory proteins. In this study, a genetic screen performed in Escherichia coli identified the CpxR response regulator as an activator of the L. pneumophila icmR gene. Construction of an L. pneumophila cpxR insertion mutant showed that the expression of icmR is regulated by CpxR. In addition, a conserved CpxR binding site (GTAAA) was identified in the icmR regulatory region and L. pneumophila His-tagged CpxR protein was shown to bind to the icmR regulatory region using a mobility shift assay. Besides its dramatic effect on the icmR level of expression, the CpxR regulator was also found to affect the expression of the icmV-dotA and icmW-icmX operons, but to a lesser extent. The role of CpxA, the cognate sensor kinase of CpxR, was also examined and its effect on the icmR level of expression was found to be less pronounced than the effect of CpxR. The RpoE sigma factor, which was shown to coregulate genes together with CpxR, was examined as well, but it did not influence icm and dot gene expression. In addition, when the cpxR mutant strain, in which the expression of the icmR gene was dramatically reduced, and the cpxA and rpoE mutant strains were examined for their ability to grow inside Acanthamoeba castellanii and HL-60-derived human macrophages, no intracellular growth defect was observed. This study presents the first evidence for a direct regulator (CpxR) of an icm-dot virulence gene (icmR). The CpxR regulator together with other regulatory factors probably concerts with the expression of icm and dot genes to result in successful infection.

scholarly journals Transcriptomic analysis of the stationary phase response regulator SpdR in Caulobacter crescentus

2016 ◽  
Vol 16 (1) ◽  
Author(s):  
Carolina A. P. T. da Silva ◽  
Rogério F. Lourenço ◽  
Ricardo R. Mazzon ◽  
Rodolfo A. Ribeiro ◽  
Marilis V. Marques
Keyword(s):  
Stationary Phase ◽  
Caulobacter Crescentus ◽  
Response Regulator ◽  
Phase Response ◽  
Transcriptomic Analysis

scholarly journals DivL Performs Critical Cell Cycle Functions in Caulobacter crescentus Independent of Kinase Activity

2007 ◽  
Vol 189 (22) ◽  
pp. 8308-8320 ◽  
Author(s):  
Sarah J. Reisinger ◽  
Sarah Huntwork ◽  
Patrick H. Viollier ◽  
Kathleen R. Ryan
Keyword(s):  
Cell Cycle ◽  
Caulobacter Crescentus ◽  
Essential Gene ◽  
Response Regulator ◽  
Phosphorylation Site ◽  
Point Mutations ◽  
Phosphoryl Transfer ◽  
Cell Cycle Regulators ◽  
Two Component Signal Transduction ◽  
Signal Transduction Proteins

ABSTRACT The Caulobacter cell cycle is regulated by a network of two-component signal transduction proteins. Phosphorylation and stability of the master transcriptional regulator CtrA are controlled by the CckA-ChpT phosphorelay, and CckA activity is modulated by another response regulator, DivK. In a screen to identify suppressors of the cold-sensitive divK341 mutant, we found point mutations in the essential gene divL. DivL is similar to histidine kinases but has a tyrosine instead of a histidine at the conserved phosphorylation site (Y550). Surprisingly, we found that the ATPase domain of DivL is not essential for Caulobacter viability. We show that DivL selectively affects CtrA phosphorylation but not CtrA proteolysis, indicating that DivL acts in a pathway independent of the CckA-ChpT phosphorelay. divL can be deleted in a strain overproducing the phosphomimetic protein CtrAD51E, but unlike ΔctrA cells expressing CtrAD51E, this strain is profoundly impaired in the control of chromosome replication and cell division. Thus, DivL performs a second function in addition to promoting CtrA phosphorylation. DivL is required for bipolar DivK localization and positively regulates DivK phosphorylation. Our results show that DivL controls two key cell cycle regulators, CtrA and DivK, and that phosphoryl transfer is not DivL's essential cellular activity.

scholarly journals CtrA, a Global Response Regulator, Uses a Distinct Second Category of Weak DNA Binding Sites for Cell Cycle Transcription Control in Caulobacter crescentus

2009 ◽  
Vol 191 (17) ◽  
pp. 5458-5470 ◽  
Author(s):  
William Spencer ◽  
Rania Siam ◽  
Marie-Claude Ouimet ◽  
D. Patrick Bastedo ◽  
Gregory T. Marczynski
Keyword(s):  
Cell Cycle ◽  
Binding Sites ◽  
Caulobacter Crescentus ◽  
Response Regulator ◽  
Cell Cycle Control ◽  
Transcription Control ◽  
Mobility Shift ◽  
Dna Binding Sites ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT CtrA controls cell cycle programs of chromosome replication and genetic transcription. Phosphorylated CtrA∼P exhibits high affinity (dissociation constant [Kd ], <10 nM) for consensus TTAA-N7-TTAA binding sites with “typical” (N = 7) spacing. We show here that ctrA promoters P1 and P2 use low-affinity (Kd , >500 nM) CtrA binding sites with “atypical” (N ≠ 7) spacing. Footprints demonstrated that phosphorylated CtrA∼P does not exhibit increased affinity for “atypical” sites, as it does for sites in the replication origin. Instead, high levels of CtrA (>10 μM) accumulate, which can drive CtrA binding to “atypical” sites. In vivo cross-linking showed that when the stable CtrAΔ3 protein persists during the cell cycle, the “atypical” sites at ctrA and motB are persistently bound. Interestingly, the cell cycle timing of ctrA P1 and P2 transcription is not altered by persistent CtrAΔ3 binding. Therefore, operator DNA occupancy is not sufficient for regulation, and it is the cell cycle variation of CtrA∼P phosphorylation that provides the dominant “activation” signal. Protein dimerization is one potential means of “activation.” The glutathione S-transferase (GST) protein dimerizes, and fusion with CtrA (GST-CtrA) creates a stable dimer with enhanced affinity for TTAA motifs. Electrophoretic mobility shift assays with GST-CtrA revealed cooperative modes of binding that further distinguish the “atypical” sites. GST-CtrA also binds a single TTAA motif in ctrA P1 aided by DNA in the extended TTAACCAT motif. We discuss how “atypical” sites are a common yet distinct category of CtrA regulatory sites and new implications for the working and evolution of cell cycle control networks.

scholarly journals Cooperative Function of TraJ and ArcA in Regulating the F PlasmidtraOperon

2018 ◽  
Vol 201 (1) ◽  
Author(s):  
Jun Lu ◽  
Yun Peng ◽  
Sereana Wan ◽  
Laura S. Frost ◽  
Tracy Raivio ◽  
...  
Keyword(s):  
Regulatory Region ◽  
Repeat Sequence ◽  
Bacterial Conjugation ◽  
Alternative Promoters ◽  
Mobility Shift ◽  
Bacterial Populations ◽  
Major Mechanism ◽  
Regulatory Circuit ◽  
Donor Ability ◽  
Electrophoretic Mobility Shift Assays

ABSTRACTThe F plasmidtraoperon encodes most of the proteins required for bacterial conjugation. TraJ and ArcA are known activators of thetraoperon promoter PY, which is subject to H-NS-mediated silencing. Donor ability and promoter activity assays indicated that PYis inactivated by silencers and requires both TraJ and ArcA for activation to support efficient F conjugation. The observed low-level, ArcA-independent F conjugation is caused bytraexpression from upstream alternative promoters. Electrophoretic mobility shift assays showed that TraJ alone weakly binds to PYregulatory DNA; however, TraJ binding is significantly enhanced by ArcA binding to the same DNA, indicating cooperativity of the two proteins. Analysis of binding affinities between ArcA and various DNA fragments in the PYregulatory region defined a 22-bp tandem repeat sequence (from −76 to −55 of PY) sufficient for optimal ArcA binding, which is immediately upstream of the predicted TraJ-binding site (from −54 to −34). Deletion analysis of the PYpromoter in strains deficient in TraJ, ArcA, and/or H-NS determined that sequences upstream of −103 are required by silencers including H-NS for PYsilencing, whereas sequences downstream of −77 are targeted by TraJ and ArcA for activation. TraJ and ArcA appear not only to counteract PYsilencers but also to directly activate PYin a cooperative manner. Our data reveal the cooperativity of TraJ and ArcA during PYactivation and provide insights into the regulatory circuit controlling F-family plasmid-mediated bacterial conjugation.IMPORTANCEConjugation is a major mechanism for dissemination of antibiotic resistance and virulence among bacterial populations. Thetraoperon in the F family of conjugative plasmids encodes most of the proteins involved in bacterial conjugation. This work reveals that activation oftraoperon transcription requires two proteins, TraJ and ArcA, to bind cooperatively to adjacent sites immediately upstream of the majortrapromoter PY. The interaction of TraJ and ArcA with thetraoperon not only relieves PYfrom silencers but also directly activates it. These findings provide insights into the regulatory circuit of the F-family plasmid-mediated bacterial conjugation.

scholarly journals Transcriptional regulation of the Aggregatibacter actinomycetemcomitans ygiW–qseBC operon by QseB and integration host factor proteins

Microbiology ◽  
2014 ◽  
Vol 160 (12) ◽  
pp. 2583-2594 ◽  
Author(s):  
María Dolores Juárez-Rodríguez ◽  
Ascención Torres-Escobar ◽  
Donald R. Demuth
Keyword(s):  
Response Regulator ◽  
Direct Repeat ◽  
Aggregatibacter Actinomycetemcomitans ◽  
Single Copy ◽  
Repeat Sequence ◽  
Integration Host Factor ◽  
Host Factor ◽  
Negative Regulator ◽  
Mobility Shift ◽  
Biofilm Growth

The QseBC two-component system plays a pivotal role in regulating virulence and biofilm growth of the oral pathogen Aggregatibacter actinomycetemcomitans. We previously showed that QseBC autoregulates the ygiW–qseBC operon. In this study, we characterized the promoter that drives ygiW–qseBC expression. Using lacZ transcriptional fusion constructs and 5′-rapid amplification of cDNA ends, we showed that ygiW–qseBC expression is driven by a promoter that initiates transcription 53 bases upstream of ygiW and identified putative cis-acting promoter elements, whose function was confirmed using site-specific mutagenesis. Using electrophoretic mobility shift assays, two trans-acting proteins were shown to interact with the ygiW–qseBC promoter. The QseB response regulator bound to probes containing the direct repeat sequence CTTAA-N6-CTTAA, where the CTTAA repeats flank the −35 element of the promoter. The ygiW–qseBC expression could not be detected in A. actinomycetemcomitans ΔqseB or ΔqseBC strains, but was restored to WT levels in the ΔqseBC mutant when complemented by single copy chromosomal insertion of qseBC. Interestingly, qseB partially complemented the ΔqseBC strain, suggesting that QseB could be activated in the absence of QseC. QseB activation required its phosphorylation since complementation did not occur using qseB pho−, encoding a protein with the active site aspartate substituted with alanine. These results suggest that QseB is a strong positive regulator of ygiW–qseBC expression. In addition, integration host factor (IHF) bound to two sites in the promoter region and an additional site near the 5′ end of the ygiW ORF. The expression of ygiW–qseBC was increased by twofold in ΔihfA and ΔihfB strains of A. actinomycetemcomitans, suggesting that IHF is a negative regulator of the ygiW–qseBC operon.

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