scholarly journals The Two-Component System PhoPR of Clostridium acetobutylicum Is Involved in Phosphate-Dependent Gene Regulation

2008 ◽  
Vol 190 (20) ◽  
pp. 6559-6567 ◽  
Author(s):  
Tomas Fiedler ◽  
Maren Mix ◽  
Uta Meyer ◽  
Stefan Mikkat ◽  
Michael O. Glocker ◽  
...  
Keyword(s):  
Clostridium Acetobutylicum ◽  
Response Regulator ◽  
Specific Binding ◽  
Expression Patterns ◽  
Regulatory System ◽  
Fold Increase ◽  
Growth Conditions ◽  
Two Dimensional Gel Electrophoresis ◽  
Two Component

ABSTRACT The phoPR gene locus of Clostridium acetobutylicum ATCC 824 comprises two genes, phoP and phoR. Deduced proteins are predicted to represent a response regulator and sensor kinase of a phosphate-dependent two-component regulatory system. We analyzed the expression patterns of phoPR in Pi-limited chemostat cultures and in response to Pi pulses. A basic transcription level under high-phosphate conditions was shown, and a significant increase in mRNA transcript levels was found when external Pi concentrations dropped below 0.3 mM. In two-dimensional gel electrophoresis experiments, a 2.5-fold increase in PhoP was observed under Pi-limiting growth conditions compared to growth with an excess of Pi. At least three different transcription start points for phoP were determined by primer extension analyses. Proteins PhoP and an N-terminally truncated *PhoR were individually expressed heterologously in Escherichia coli and purified. Autophosphorylation of *PhoR and phosphorylation of PhoP were shown in vitro. Electromobility shift assays proved that there was a specific binding of PhoP to the promoter region of the phosphate-regulated pst operon of C. acetobutylicum.

scholarly journals Requirement of the Receiver and Phosphotransfer Domains of ArcB for Efficient Dephosphorylation of Phosphorylated ArcA In Vivo

2005 ◽  
Vol 187 (9) ◽  
pp. 3267-3272 ◽  
Author(s):  
Gabriela R. Peña-Sandoval ◽  
Ohsuk Kwon ◽  
Dimitris Georgellis
Keyword(s):  
Response Regulator ◽  
Growth Conditions ◽  
Sensor Kinase ◽  
Aerobic Growth ◽  
Two Component System ◽  
Two Component ◽  
Necessary And Sufficient ◽  
Respiratory Conditions

ABSTRACT The Arc two-component system, comprising the ArcB sensor kinase and the ArcA response regulator, modulates the expression of numerous genes in response to the respiratory conditions of growth. Under anoxic growth conditions, ArcB autophosphorylates and transphosphorylates ArcA, which in turn represses or activates its target operons. Under aerobic growth conditions, phosphorylated ArcA (ArcA-P) dephosphorylates and its transcriptional regulation is released. The dephosphorylation of ArcA-P has been shown to occur, at least in vitro, via an ArcAAsp54-P → ArcBHis717-P → ArcBAsp576-P → Pi reverse phosphorelay. In this study, the physiological significance of this pathway was assessed. The results demonstrate that the receiver and phosphotransfer domains of the tripartite sensor kinase ArcB are necessary and sufficient for efficient ArcA-P dephosphorylation in vivo.

scholarly journals Effect of d-Lactate on the Physiological Activity of the ArcB Sensor Kinase in Escherichia coli

2004 ◽  
Vol 186 (7) ◽  
pp. 2085-2090 ◽  
Author(s):  
Claudia Rodriguez ◽  
Ohsuk Kwon ◽  
Dimitris Georgellis
Keyword(s):  
Kinase Activity ◽  
Response Regulator ◽  
Physiological Activity ◽  
Growth Conditions ◽  
Sensor Kinase ◽  
Two Component System ◽  
Direct Signal ◽  
Two Component

ABSTRACT The Arc two-component system, comprising the ArcB sensor kinase and the ArcA response regulator, modulates the expression of numerous genes in response to the respiratory growth conditions. Under anoxic growth conditions ArcB autophosphorylates and transphosphorylates ArcA, which in turn represses or activates its target operons. The anaerobic metabolite d-lactate has been shown to stimulate the in vitro autophosphorylating activity of ArcB. In this study, the in vivo effect of d-lactate on the kinase activity of ArcB was assessed. The results demonstrate that d-lactate does not act as a direct signal for activation of ArcB, as previously proposed, but acts as a physiologically significant effector that amplifies ArcB kinase activity.

scholarly journals Identification of cpxR as a Positive Regulator Essential for Expression of the Shigella sonnei virF Gene

1998 ◽  
Vol 180 (14) ◽  
pp. 3522-3528 ◽  
Author(s):  
Shu-ichi Nakayama ◽  
Haruo Watanabe
Keyword(s):  
Response Regulator ◽  
Binding Capacity ◽  
Regulatory System ◽  
Upstream Region ◽  
Dependent Manner ◽  
Acetyl Phosphate ◽  
Two Component ◽  
Transcription In Vitro ◽  
Cognate Response Regulator

ABSTRACT virF is the master regulator which activates the virulence determinant genes of Shigella spp. such asipaBCD and virG. We previously reported that expression of virF itself is regulated in a pH-dependent manner and that cpxA, a sensor of a two-component regulatory system, is involved in this regulation (S. Nakayama and H. Watanabe, J. Bacteriol. 177:5062–5069, 1995). Disruption of cpxR, which has been thought to be the cognate response regulator of cpxA (J. Dong, S. Iuchi, H.-S. Kwan, Z. Lue, and E. C. C. Lin, Gene 136:227–230, 1993), abolishedvirF expression almost completely. Purified CpxR bound directly to the upstream region of virF. Binding capacity was enhanced when CpxR was phosphorylated by coincubation with acetyl phosphate in vitro. Furthermore, we observed that phosphorylated CpxR could activate virF transcription in vitro. These results clearly indicated that CpxR was an essential activator for virF expression and strongly suggested that the binding of phosphorylated CpxR to the target site upstream of the virF gene induced a direct activation of virF transcription.

scholarly journals The BaeSR Two-Component Regulatory System Activates Transcription of the yegMNOB (mdtABCD) Transporter Gene Cluster in Escherichia coli and Increases Its Resistance to Novobiocin and Deoxycholate

2002 ◽  
Vol 184 (15) ◽  
pp. 4168-4176 ◽  
Author(s):  
Natalya Baranova ◽  
Hiroshi Nikaido
Keyword(s):  
Escherichia Coli ◽  
Response Regulator ◽  
Regulatory System ◽  
Major Facilitator Superfamily ◽  
Multicopy Plasmid ◽  
Active Efflux ◽  
Membrane Fusion Protein ◽  
Two Component ◽  
Major Facilitator

ABSTRACT Screening of random fragments of Escherichia coli genomic DNA for their ability to increase the novobiocin resistance of a hypersusceptible ΔacrAB mutant resulted in the isolation of a plasmid containing baeR, which codes for the response regulator of the two-component regulatory system BaeSR. When induced for expression, baeR cloned in multicopy plasmid pTrc99A significantly increased the resistance of the ΔacrAB host strain to novobiocin (16-fold) and to deoxycholate (8-fold). Incubation of cells with novobiocin followed by a chromatographic assay for intracellular drug showed that overproduced BaeR decreased drastically the drug accumulation, presumably via increased active efflux. The genes baeSR are part of a putative operon, yegMNOB baeSR. Direct binding of BaeR to the yegM promoter was demonstrated in vitro by gel retardation assay. The gene yegB, which codes for a major facilitator superfamily transporter, was not necessary for increased resistance, but deletion of yegO or an in-frame deletion of yegN, both of which code for resistance-nodulation-cell division-type multidrug transporters, abolished the BaeR-induced increase in resistance. It is likely that both YegN and YegO produce a complex(es) with the membrane fusion protein family member YegM and pump out novobiocin and deoxycholate. We accordingly propose to rename yegMNOB as mdtABCD (mdt for multidrug transporter). Finally, the expression of two other genes, yicO and ygcL, was shown to be regulated by BaeR, but it is not known if they play any roles in resistance.

scholarly journals A Two-Component Regulatory System Controls Autoregulated Serpin Expression in Bifidobacterium breve UCC2003

2012 ◽  
Vol 78 (19) ◽  
pp. 7032-7041 ◽  
Author(s):  
Pablo Alvarez-Martin ◽  
Mary O'Connell Motherway ◽  
Francesca Turroni ◽  
Elena Foroni ◽  
Marco Ventura ◽  
...  
Keyword(s):  
Response Regulator ◽  
Regulatory System ◽  
Site Directed Mutagenesis ◽  
Insertion Mutant ◽  
Recognition Sequence ◽  
Mobility Shift ◽  
Content Type ◽  
Bifidobacterium Breve ◽  
Two Component

ABSTRACTThis work reports on the identification and molecular characterization of a two-component regulatory system (2CRS), encoded byserRK, which is believed to control the expression of theser2003locus inBifidobacterium breveUCC2003. Theser2003locus consists of two genes, Bbr_1319 (sagA) and Bbr_1320 (serU), which are predicted to encode a hypothetical membrane-associated protein and a serpin-like protein, respectively. The response regulator SerR was shown to bind to the promoter region ofser2003, and the probable recognition sequence of SerR was determined by a combinatorial approach ofin vitrosite-directed mutagenesis coupled to transcriptional fusion and electrophoretic mobility shift assays (EMSAs). The importance of theserRK2CRS in the response ofB. breveto protease-mediated induction was confirmed by generating aB. breve serRinsertion mutant, which was shown to exhibit alteredser2003transcriptional induction patterns compared to the parent strain, UCC2003. Interestingly, the analysis of aB. breve serUmutant revealed that the SerRK signaling pathway appears to include a SerU-dependent autoregulatory loop.

scholarly journals A Two-Component Regulatory System Interconnects Resistance to Polymyxins, Aminoglycosides, Fluoroquinolones, and β-Lactams inPseudomonas aeruginosa

2010 ◽  
Vol 55 (3) ◽  
pp. 1211-1221 ◽  
Author(s):  
Cédric Muller ◽  
Patrick Plésiat ◽  
Katy Jeannot
Keyword(s):  
Multidrug Resistance ◽  
Response Regulator ◽  
Regulatory System ◽  
Single Mutation ◽  
Active Efflux ◽  
Resistance Phenotype ◽  
Clinical Strains ◽  
Adaptive Resistance ◽  
Two Component

ABSTRACTConstitutive overexpression of the active efflux system MexXY/OprM is a major cause of resistance to aminoglycosides, fluoroquinolones, and cefepime in clinical strains ofPseudomonas aeruginosa. Upregulation of this pump often results from mutations occurring inmexZ, the local repressor gene of themexXYoperon. In this study, analysis of MexXY-overproducing mutants selectedin vitrofrom reference strain PAO1Bes on amikacin (at a concentration 1.5-fold higher than the MIC) led to identification of a new class of mutants harboring an intactmexZgene and exhibiting increased resistance to colistin and imipenem in addition to aminoglycosides, fluoroquinolones, and cefepime. Reverse transcription-quantitative PCR (RT-qPCR) experiments on a selected clone named PAOW2 demonstrated thatmexXYoverexpression was independent ofmexZand the PA5471 gene, which is required for drug-dependent induction ofmexXY. Furthermore, the transcript levels of theoprDgene, which encodes the carbapenem-selective porin OprD, were found to be reduced drastically in PAOW2. Whole-genome sequencing revealed a single mutation resulting in an M59I substitution in the ParR protein, the response regulator of the ParRS two-component regulatory system (with ParS being the sensor kinase), which is required for adaptive resistance ofP. aeruginosato polycationic peptides such as colistin. The multidrug resistance phenotype was suppressed in PAOW2 by deletion of theparSandparRSgenes and conferred to PAO1Bes by chromosomal insertion of the mutatedparRSlocus from PAOW2. As shown by transcriptomic analysis, only a very small number of genes were expressed differentially between PAOW2 and PAO1Bes, including the lipopolysaccharide (LPS) modification operonarnBCADTEF-ugd, responsible for resistance to polycationic agents. Exposure of wild-type PAO1Bes to different polycationic peptides, including colistin, was shown to result in increasedmexYand repressedoprDexpression via ParRS, independent of PA5471. In agreement with these results, colistin antagonized activity of the MexXY/OprM substrates in PAO1Bes but not in a ΔparRSderivative. Finally, screening of clinical strains exhibiting the PAOW2 resistance phenotype allowed the identification of additional alterations in ParRS. Collectively, our data indicate that ParRS may promote either induced or constitutive multidrug resistance to four different classes of antibiotics through the activation of three distinct mechanisms (efflux, porin loss, and LPS modification).

scholarly journals Bordetella pertussis TonB, a Bvg-Independent Virulence Determinant

2000 ◽  
Vol 68 (4) ◽  
pp. 1919-1927 ◽  
Author(s):  
Elizabeth Pradel ◽  
Nicole Guiso ◽  
Franco D. Menozzi ◽  
Camille Locht
Keyword(s):  
Bordetella Pertussis ◽  
Regulatory System ◽  
Growth Conditions ◽  
Membrane Receptors ◽  
Infection Model ◽  
Two Component ◽  
Colonization Ability ◽  
Titration Assay

ABSTRACT In gram-negative bacteria, high-affinity iron uptake requires the TonB/ExbB/ExbD envelope complex to release iron chelates from their specific outer membrane receptors into the periplasm. Based on sequence similarities, the Bordetella pertussis tonB exbB exbD locus was identified on a cloned DNA fragment. The tight organization of the three genes suggests that they are cotranscribed. A putative Fur-binding sequence located upstream from tonB was detected in a Fur titration assay, indicating that the tonB exbB exbD operon may be Fur-repressed in high-iron growth conditions. Putative structural genes of the β-subunit of the histone-like protein HU and of a new two-component regulatory system were identified upstream from tonB and downstream from exbD, respectively. A B. pertussis ΔtonB exbB::Kmr mutant was constructed by allelic exchange and characterized. The mutant was impaired for growth in low-iron medium in vitro and could not use ferrichrome, desferal, or hemin as iron sources. Levels of production of the major bacterial toxins and adhesins were similar in the TonB+/TonB− pair. The ΔtonB exbBmutant was still responsive to chemical modulators of virulence; thus, the BvgA/BvgS two-component system is not TonB dependent. Nevertheless, in vivo in the mouse respiratory infection model, the colonization ability of the mutant was reduced compared to the parental strain.

scholarly journals Signal-Dependent Phosphorylation of the Membrane-Bound NarX Two-Component Sensor-Transmitter Protein ofEscherichia coli: Nitrate Elicits a Superior Anion Ligand Response Compared to Nitrite

1999 ◽  
Vol 181 (17) ◽  
pp. 5309-5316 ◽  
Author(s):  
Angela I. Lee ◽  
Asunción Delgado ◽  
Robert P. Gunsalus
Keyword(s):  
Kinase Activity ◽  
Response Regulator ◽  
Regulatory System ◽  
Respiratory Pathway ◽  
Vitro Assay ◽  
Environmental Signals ◽  
Dual Sensor ◽  
Two Component ◽  
Nitrate And Nitrite

ABSTRACT The Nar two-component regulatory system, consisting of the dual sensor-transmitters NarX and NarQ and the dual response regulators NarL and NarP, controls the expression of various anaerobic respiratory pathway genes and fermentation pathway genes. Although both NarX and NarQ are known to detect the two environmental signals nitrate and nitrite, little is known regarding the sensitivity and selectivity of ligand for detection or activation of the sensor-transmitters. In this study, we have developed a sensitive anion-specific in vitro assay for NarX autophosphorylation by using Escherichia colimembranes highly enriched in the full-length NarX protein. In this ATP- and magnesium-dependent reaction, nitrate elicited a greater signal output (i.e., NarX autophosphorylation) than did nitrite. Nitrate stimulation occurred at concentrations as low as 5 μM, and the half-maximal level of NarX autophosphorylation occurred at approximately 35 μM nitrate. In contrast, nitrite-dependent stimulation was detected only at 500 μM, while 3.5 mM nitrite was needed to achieve half-maximal NarX autophosphorylation. Maximal nitrate- and nitrite-stimulated levels of NarX phosphorylation were five and two times, respectively, over the basal level of NarX autophosphorylation. The presence of Triton X-100 eliminated the nitrate-stimulated kinase activity and lowered the basal level of activity, suggesting that the membrane environment plays a crucial role in nitrate detection and/or regulation of kinase activity. These results provide in vitro evidence for the differential detection of dual signaling ligands by the NarX sensor-transmitter protein, which modulates the cytoplasmic NarX autokinase activity and phosphotransfer to NarL, the cognate response regulator.

scholarly journals Regulation of Membrane Permeability by a Two-Component Regulatory System in Pseudomonas aeruginosa

2003 ◽  
Vol 47 (1) ◽  
pp. 95-101 ◽  
Author(s):  
Yanping Wang ◽  
Unhwan Ha ◽  
Lin Zeng ◽  
Shouguang Jin
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Multidrug Resistance ◽  
Membrane Permeability ◽  
Response Regulator ◽  
Regulatory System ◽  
Laboratory Strain ◽  
Clinical Isolates ◽  
Two Component ◽  
Increased Sensitivity

ABSTRACT Membrane impermeability is the major contributing factor to multidrug resistance in clinical isolates of Pseudomonas aeruginosa. By using laboratory strain PAK, a spontaneous P. aeruginosa mutant (mutant PAK1-3) whose membrane had reduced permeability and which displayed increased levels of resistance to various antibiotics, especially aminoglycosides, was isolated. By complementation of the mutant with a genomic clone library derived from wild-type strain PAK, a novel two-component regulatory system (PprA and PprB) was identified and was found to be able to increase the permeability of the bacterial membrane and render PAK1-3 sensitive to antibiotics. Furthermore, specific phosphorylation of the response regulator (PprB) by histidine kinase (PprA) was observed in vitro, demonstrating that they are cognate two-component regulatory genes. Introduction of a plasmid expressing the pprB gene into randomly chosen clinical isolates (n = 17) resulted in increased sensitivity to aminoglycosides in the majority of isolates (n = 13) tested. This is the first demonstration that P. aeruginosa membrane permeability can be regulated, providing an important clue in the understanding of the mechanism of membrane impermeability-mediated multidrug resistance in P. aeruginosa.

AgmR controls transcription of a regulon with several operons essential for ethanol oxidation in Pseudomonas aeruginosa ATCC 17933

Microbiology ◽  
2004 ◽  
Vol 150 (6) ◽  
pp. 1851-1857 ◽  
Author(s):  
Nicole Gliese ◽  
Viola Khodaverdi ◽  
Max Schobert ◽  
Helmut Görisch
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Ethanol Oxidation ◽  
Response Regulator ◽  
Regulatory System ◽  
Pyrroloquinoline Quinone ◽  
Kanamycin Resistance ◽  
Kanamycin Resistance Cassette ◽  
Two Component ◽  
Ethanol Dehydrogenase ◽  
Oxidation System

The response regulator AgmR was identified to be involved in the regulation of the quinoprotein ethanol oxidation system of Pseudomonas aeruginosa ATCC 17933. Interruption of the agmR gene by insertion of a kanamycin-resistance cassette resulted in mutant NG3, unable to grow on ethanol. After complementation with the intact agmR gene, growth on ethanol was restored. Transcriptional lacZ fusions were used to identify four operons which are regulated by the AgmR protein: the exaA operon encodes the pyrroloquinoline quinone (PQQ)-dependent ethanol dehydrogenase, the exaBC operon encodes a soluble cytochrome c 550 and an aldehyde dehydrogenase, the pqqABCDE operon carries the PQQ biosynthetic genes, and operon exaDE encodes a two-component regulatory system which controls transcription of the exaA operon. Transcription of exaA was restored by transformation of NG3 with a pUCP20T derivative carrying the exaDE genes under lac-promoter control. These data indicate that the AgmR response regulator and the exaDE two-component regulatory system are organized in a hierarchical manner. Gene PA1977, which appears to form an operon with the agmR gene, was found to be non-essential for growth on ethanol.

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