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

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.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01252-10 ◽

2010 ◽

Vol 55 (3) ◽

pp. 1211-1221 ◽

Cited By ~ 118

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).

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

Microbiology ◽

10.1099/mic.0.26882-0 ◽

2004 ◽

Vol 150 (6) ◽

pp. 1851-1857 ◽

Cited By ~ 21

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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Progress Overview of Bacterial Two-Component Regulatory Systems as Potential Targets for Antimicrobial Chemotherapy

Antibiotics ◽

10.3390/antibiotics9100635 ◽

2020 ◽

Vol 9 (10) ◽

pp. 635

Author(s):

Hidetada Hirakawa ◽

Jun Kurushima ◽

Yusuke Hashimoto ◽

Haruyoshi Tomita

Keyword(s):

Drug Resistance ◽

Antimicrobial Chemotherapy ◽

Response Regulator ◽

Regulatory System ◽

Laboratory Strain ◽

Regulatory Systems ◽

Two Component ◽

Bacterial Genes ◽

Conventional Antibiotics ◽

External Stimuli

Bacteria adapt to changes in their environment using a mechanism known as the two-component regulatory system (TCS) (also called “two-component signal transduction system” or “two-component system”). It comprises a pair of at least two proteins, namely the sensor kinase and the response regulator. The former senses external stimuli while the latter alters the expression profile of bacterial genes for survival and adaptation. Although the first TCS was discovered and characterized in a non-pathogenic laboratory strain of Escherichia coli, it has been recognized that all bacteria, including pathogens, use this mechanism. Some TCSs are essential for cell growth and fitness, while others are associated with the induction of virulence and drug resistance/tolerance. Therefore, the TCS is proposed as a potential target for antimicrobial chemotherapy. This concept is based on the inhibition of bacterial growth with the substances acting like conventional antibiotics in some cases. Alternatively, TCS targeting may reduce the burden of bacterial virulence and drug resistance/tolerance, without causing cell death. Therefore, this approach may aid in the development of antimicrobial therapeutic strategies for refractory infections caused by multi-drug resistant (MDR) pathogens. Herein, we review the progress of TCS inhibitors based on natural and synthetic compounds.

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Function of the bacteriophytochrome BphP in the RpoS/Las quorum-sensing network of Pseudomonas aeruginosa

Microbiology ◽

10.1099/mic.0.049007-0 ◽

2011 ◽

Vol 157 (6) ◽

pp. 1651-1664 ◽

Cited By ~ 15

Author(s):

Katalin Barkovits ◽

Britta Schubert ◽

Sabrina Heine ◽

Maurice Scheer ◽

Nicole Frankenberg-Dinkel

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Response Regulator ◽

Red Light ◽

Regulatory System ◽

Global Gene Expression ◽

Opportunistic Pathogen ◽

Pleiotropic Effect ◽

Genes Encoding

The bacterial phytochrome of Pseudomonas aeruginosa (PaBphP) is an in vitro-active red/far-red light sensor histidine kinase of a two-component regulatory system. Despite solid biochemical data, its function in this heterotrophic, opportunistic pathogen is still unknown. Previous studies established that the genes encoding the two necessary phytochrome components BphO, a chromophore-producing haem oxygenase, and BphP, the apo-phytochrome, are co-transcribed in a bicistronic operon. Transcription has been shown to be induced in the stationary phase and to be dependent on the alternative sigma factor RpoS. Here we show an additional regulation of bphP expression through the quorum-sensing (QS) regulator LasR. This regulation is also reflected in a combination of expression profile experiments and proteome analyses of wild-type and phytochrome-deficient strains. While PaBphP has a pleiotropic effect on global gene expression, 66 % of the downregulated genes in the phytochrome mutant display a link to the Las QS system. Most of these genes seem to be indirectly regulated by LasR through BphP and the unknown response regulator BphR. A model of phytochrome function within the Las QS network is presented.

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

Journal of Bacteriology ◽

10.1128/jb.180.14.3522-3528.1998 ◽

1998 ◽

Vol 180 (14) ◽

pp. 3522-3528 ◽

Cited By ~ 70

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.

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

Journal of Bacteriology ◽

10.1128/jb.00574-08 ◽

2008 ◽

Vol 190 (20) ◽

pp. 6559-6567 ◽

Cited By ~ 11

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.

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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

Journal of Bacteriology ◽

10.1128/jb.184.15.4168-4176.2002 ◽

2002 ◽

Vol 184 (15) ◽

pp. 4168-4176 ◽

Cited By ~ 174

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.

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

Applied and Environmental Microbiology ◽

10.1128/aem.01776-12 ◽

2012 ◽

Vol 78 (19) ◽

pp. 7032-7041 ◽

Cited By ~ 19

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.

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Molecular Mechanisms of Phosphate Stress Activation of Pseudomonas aeruginosa Quorum Sensing Systems

mSphere ◽

10.1128/msphere.00119-20 ◽

2020 ◽

Vol 5 (2) ◽

Cited By ~ 6

Author(s):

Xianfa Meng ◽

Stephen Dela Ahator ◽

Lian-Hui Zhang

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Response Regulator ◽

Regulatory System ◽

Regulatory Genes ◽

Phosphate Deficiency ◽

Content Type ◽

Sensing Systems ◽

Two Component ◽

Phosphate Depletion

ABSTRACT The hierarchical quorum sensing (QS) systems of Pseudomonas aeruginosa, consisting of las, pqs, and rhl, coordinate the expression of bacterial virulence genes. Previous studies showed that under phosphate deficiency conditions, two-component regulatory system PhoRB could activate various genes involved in cytotoxicity through modulation of QS systems, but the mechanism by which PhoR/PhoB influences QS remains largely unknown. Here, we provide evidence that among the key QS regulatory genes in P. aeruginosa, rhlR, pqsA, mvfR, and lasI were activated by the response regulator PhoB under phosphate-depleted conditions. We show that PhoB is a strong competitor against LasR and RsaL for binding to the promoter of lasI and induces significant expression of lasI, rhlR, and mvfR. However, expression of lasI, encoding the signal 3-oxo-C12-HSL, was increased only marginally under the same phosphate-depleted conditions. This seeming inconsistency was attributed to the induction of pvdQ, which encodes an enzyme for degradation of 3-oxo-C12-HSL signal molecules. Taken together, the results from this study demonstrate that through the two-component regulatory system PhoR/PhoB, phosphate depletion stress could influence the QS network by modulating several key regulators, including lasI, rhlR, mvfR, and pvdQ. The findings highlight not only the potency of the PhoR/PhoB-mediated bacterial stress response mechanism but also the plasticity of the P. aeruginosa QS systems in coping with the changed environmental conditions. IMPORTANCE It is not fully understood how phosphate deficiency could influence the virulence of Pseudomonas aeruginosa through modulation of the bacterial QS systems. This report presents a systemic investigation on the impact of phosphate depletion on the hierarchy of quorum sensing systems of P. aeruginosa. The results showed that phosphate stress could have an extensive impact on the QS networks of this bacterial pathogen. Among the 7 QS regulatory genes representing the 3 sets of QS systems tested, 4 were significantly upregulated by phosphate depletion stress through the PhoR/PhoB two-component regulatory system, especially the upstream QS regulatory gene lasI. We also present evidence that the response regulator PhoB was a strong competitor against the las regulators LasR and RsaL for the lasI promoter, unveiling the mechanistic basis of the process by which phosphate stress could modulate the bacterial QS systems.

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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

Journal of Bacteriology ◽

10.1128/jb.181.17.5309-5316.1999 ◽

1999 ◽

Vol 181 (17) ◽

pp. 5309-5316 ◽

Cited By ~ 48

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.

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