The impact of ColRS two-component system and TtgABC efflux pump on phenol tolerance of Pseudomonas putida becomes evident only in growing bacteria

ABSTRACT As reported, the two-component system ColRS is involved in two completely different processes. It facilitates the root colonization ability of Pseudomonas fluorescens and is necessary for the Tn4652 transposition-dependent accumulation of phenol-utilizing mutants in Pseudomonas putida. To determine the role of the ColRS system in P. putida, we searched for target genes of response regulator ColR by use of a promoter library. Promoter screening was performed on phenol plates to mimic the conditions under which the effect of ColR on transposition was detected. The library screen revealed the porin-encoding gene oprQ and the alginate biosynthesis gene algD occurring under negative control of ColR. Binding of ColR to the promoter regions of oprQ and algD in vitro confirmed its direct involvement in regulation of these genes. Additionally, the porin-encoding gene ompA PP0773 and the type I pilus gene csuB were also identified in the promoter screen. However, it turned out that ompA PP0773 and csuB were actually affected by phenol and that the influence of ColR on these promoters was indirect. Namely, our results show that ColR is involved in phenol tolerance of P. putida. Phenol MIC measurement demonstrated that a colR mutant strain did not tolerate elevated phenol concentrations. Our data suggest that increased phenol susceptibility is also the reason for inhibition of transposition of Tn4652 in phenol-starving colR mutant bacteria. Thus, the current study revealed the role of the ColRS two-component system in regulation of membrane functionality, particularly in phenol tolerance of P. putida.

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Involvement of the MxtR/ErdR (CrbS/CrbR) Two-Component System in Acetate Metabolism in Pseudomonas putida KT2440

Microorganisms ◽

10.3390/microorganisms9081558 ◽

2021 ◽

Vol 9 (8) ◽

pp. 1558

Author(s):

Tania Henriquez ◽

Heinrich Jung

Keyword(s):

Pseudomonas Putida ◽

Physiological Role ◽

Siderophore Production ◽

Acetate Metabolism ◽

Two Component System ◽

Promoter Regions ◽

Pseudomonas Putida Kt2440 ◽

Component System ◽

Two Component ◽

The Impact

MxtR/ErdR (also called CrbS/CrbR) is a two-component system previously identified as important for the utilization of acetate in Vibrio cholerae and some Pseudomonas species. In addition, evidence has been found in Pseudomonas aeruginosa for a role in regulating the synthesis and expression, respectively, of virulence factors such as siderophores and RND transporters. In this context, we investigated the physiological role of the MxtR/ErdR system in the soil bacterium Pseudomonas putida KT2440. To that end, mxtR and erdR were individually deleted and the ability of the resulting mutants to metabolize different carbon sources was analyzed in comparison to wild type. We also assessed the impact of the deletions on siderophore production, expression of mexEF-oprN (RND transporter), and the biocontrol properties of the strain. Furthermore, the MxtR/ErdR-dependent expression of putative target genes and binding of ErdR to respective promoter regions were analyzed. Our results indicated that the MxtR/ErdR system is active and essential for acetate utilization in P. putida KT2440. Expression of scpC, pp_0354, and acsA-I was stimulated by acetate, while direct interactions of ErdR with the promoter regions of the genes scpC, pp_0354, and actP-I were demonstrated by an electromobility shift assay. Finally, our results suggested that MxtR/ErdR is neither involved in regulating siderophore production nor the expression of mexEF-oprN in P. putida KT2440 under the conditions tested.

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AdeABC Efflux Pump Controlled by AdeRS Two Component System Conferring Resistance to Tigecycline, Omadacycline and Eravacycline in Clinical Carbapenem Resistant Acinetobacter nosocomialis

Frontiers in Microbiology ◽

10.3389/fmicb.2020.584789 ◽

2020 ◽

Vol 11 ◽

Author(s):

Yi-Tzu Lee ◽

Hsing-Yu Chen ◽

Ya-Sung Yang ◽

Yu-Ching Chou ◽

Tein-Yao Chang ◽

...

Keyword(s):

Efflux Pump ◽

Two Component System ◽

Component System ◽

Carbapenem Resistant ◽

Two Component

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Site-Directed Mutagenesis Identifies a Molecular Switch Involved in Copper Sensing by the Histidine Kinase CinS in Pseudomonas putida KT2440

Journal of Bacteriology ◽

10.1128/jb.00551-09 ◽

2009 ◽

Vol 191 (16) ◽

pp. 5304-5311 ◽

Cited By ~ 13

Author(s):

Davide Quaranta ◽

Megan M. McEvoy ◽

Christopher Rensing

Keyword(s):

Pseudomonas Putida ◽

Histidine Kinase ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Copper Binding ◽

Wild Type ◽

Two Component System ◽

Pseudomonas Putida Kt2440 ◽

Component System ◽

Two Component

ABSTRACT In the presence of copper, Pseudomonas putida activates transcription of cinAQ via the two-component system CinS-CinR. The CinS-CinR TCS was responsive to 0.5 μM copper and was specifically activated only by copper and silver. Modeling studies of CinS identified a potential copper binding site containing H37 and H147. CinS mutants with H37R and H147R mutations had an almost 10-fold reduced copper-dependent induction of cinAQ compared to the wild type.

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Quantitative Kinetic Analyses of Shutting Off a Two-Component System

mBio ◽

10.1128/mbio.00412-17 ◽

2017 ◽

Vol 8 (3) ◽

Cited By ~ 13

Author(s):

Rong Gao ◽

Ann M. Stock

Keyword(s):

Phosphatase Activity ◽

Response Regulator ◽

Two Component System ◽

Component System ◽

Signaling Systems ◽

Cellular Environment ◽

Two Component ◽

The Impact

ABSTRACT Cells rely on accurate control of signaling systems to adapt to environmental perturbations. System deactivation upon stimulus removal is as important as activation of signaling pathways. The two-component system (TCS) is one of the major bacterial signaling schemes. In many TCSs, phosphatase activity of the histidine kinase (HK) is believed to play an essential role in shutting off the pathway and resetting the system to the prestimulus state. Two basic challenges are to understand the dynamic behavior of system deactivation and to quantitatively evaluate the role of phosphatase activity under natural cellular conditions. Here we report a kinetic analysis of the response to shutting off the archetype Escherichia coli PhoR-PhoB TCS pathway using both transcription reporter assays and in vivo phosphorylation analyses. Upon removal of the stimulus, the pathway is shut off by rapid dephosphorylation of the PhoB response regulator (RR) while PhoB-regulated gene products gradually reset to prestimulus levels through growth dilution. We developed an approach combining experimentation and modeling to assess in vivo kinetic parameters of the phosphatase activity with kinetic data from multiple phosphatase-diminished mutants. This enabled an estimation of the PhoR phosphatase activity in vivo , which is much stronger than the phosphatase activity of PhoR cytoplasmic domains analyzed in vitro . We quantitatively modeled how strong the phosphatase activity needs to be to suppress nonspecific phosphorylation in TCSs and discovered that strong phosphatase activity of PhoR is required for cross-phosphorylation suppression. IMPORTANCE Activation of TCSs has been extensively studied; however, the kinetics of shutting off TCS pathways is not well characterized. We present comprehensive analyses of the shutoff response for the PhoR-PhoB system that reveal the impact of phosphatase activity on shutoff kinetics. This allows development of a quantitative framework not only to characterize the phosphatase activity in the natural cellular environment but also to understand the requirement for specific strengths of phosphatase activity to suppress nonspecific phosphorylation. Our model suggests that the ratio of the phosphatase rate to the nonspecific phosphorylation rate correlates with TCS expression levels and the ratio of the RR to HK, which may contribute to the great diversity of enzyme levels and activities observed in different TCSs.

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The WalKR System Controls Major Staphylococcal Virulence Genes and Is Involved in Triggering the Host Inflammatory Response

Infection and Immunity ◽

10.1128/iai.00195-12 ◽

2012 ◽

Vol 80 (10) ◽

pp. 3438-3453 ◽

Cited By ~ 76

Author(s):

Aurélia Delauné ◽

Sarah Dubrac ◽

Charlène Blanchet ◽

Olivier Poupel ◽

Ulrike Mäder ◽

...

Keyword(s):

Inflammatory Response ◽

Virulence Genes ◽

Immune Defense ◽

Two Component System ◽

Component System ◽

Content Type ◽

Two Component ◽

The Impact ◽

Host Inflammatory Response ◽

Constitutively Active

ABSTRACTThe WalKR two-component system is essential for the viability ofStaphylococcus aureus, playing a central role in controlling cell wall metabolism. We produced a constitutively active form of WalR inS. aureusthrough a phosphomimetic amino acid replacement (WalRc, D55E). The strain displayed significantly increased biofilm formation and alpha-hemolytic activity. Transcriptome analysis was used to determine the full extent of the WalKR regulon, revealing positive regulation of major virulence genes involved in host matrix interactions (efb,emp,fnbA, andfnbB), cytolysis (hlgACB,hla, andhlb), and innate immune defense evasion (scn,chp, andsbi), through activation of the SaeSR two-component system. The impact on pathogenesis of varying cell envelope dynamics was studied using a murine infection model, showing that strains producing constitutively active WalRcare strongly diminished in their virulence due to early triggering of the host inflammatory response associated with higher levels of released peptidoglycan fragments. Indeed, neutrophil recruitment and proinflammatory cytokine production were significantly increased when the constitutively activewalRcallele was expressed, leading to enhanced bacterial clearance. Taken together, our results indicate that WalKR play an important role in virulence and eliciting the host inflammatory response by controlling autolytic activity.

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The PedS2/PedR2 two-component system is crucial for the rare earth element switch in Pseudomonas putida KT2440

10.1101/364661 ◽

2018 ◽

Author(s):

Matthias Wehrmann ◽

Charlotte Berthelot ◽

Patrick Billard ◽

Janosch Klebensberger

Keyword(s):

Rare Earth ◽

Rare Earth Elements ◽

Pseudomonas Putida ◽

Response Regulator ◽

Regulatory Function ◽

Methylotrophic Bacteria ◽

Two Component System ◽

Component System ◽

Two Component ◽

Type Response

ABSTRACTIn Pseudomonas putida KT2440, two pyrroloquinoline quinone-dependent ethanol dehydrogenases (PQQ-EDHs) are responsible for the periplasmic oxidation of a broad variety of volatile organic compounds (VOCs). Depending on the availability of rare earth elements (REEs) of the lanthanide series (Ln3+), we have recently described that the transcription of the genes encoding the Ca2+-utilizing enzyme PedE and the Ln3+-utilizing enzyme PedH are inversely regulated. With adaptive evolution experiments, site-specific mutations, transcriptional reporter fusions, and complementation approaches, we herein demonstrate that the PedS2/PedR2 (PP_2671/PP_2672) two-component system (TCS) plays a central role in the observed REE-mediated switch of PQQ-EDHs in P. putida. We provide evidence that in the absence of lanthanum (La3+), the sensor histidine kinase PedS2 phosphorylates its cognate LuxR-type response regulator PedR2, which in turn not only activates pedE gene transcription but is also involved in repression of pedH. Our data further suggests that the presence of La3+ lowers kinase activity of PedS2, either by the direct binding of the metal ions to the periplasmic region of PedS2 or by an uncharacterized indirect interaction, leading to reduced levels of phosphorylated PedR2. Consequently, the fading pedE expression and concomitant alleviation of pedH repression causes – in conjunction with the transcriptional activation of the pedH gene by a yet unknown regulatory module – the Ln3+-dependent transition from PedE to PedH catalysed oxidation of alcoholic VOCs.IMPORTANCEThe function of lanthanides for methano- and methylotrophic bacteria is gaining increasing attention, while knowledge about the role of rare earth elements (REEs) in non-methylotrophic bacteria is still limited. The present study investigates the recently described differential expression of the two PQQ-EDHs of P. putida in response to lanthanides. We demonstrate that a specific TCS is crucial for their inverse regulation and provide evidence for a dual regulatory function of the LuxR-type response regulator involved. Thus, our study represents the first detailed characterization of the molecular mechanism underlying the REE switch of PQQ-EDHs in a non-methylotrophic bacterium and stimulates subsequent investigations for the identification of additional genes or phenotypic traits that might be co-regulated during REE-dependent niche adaptation.

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Expression of styAB is regulated by a two-component system during indigo biosynthesis in Pseudomonas putida

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2019.08.142 ◽

2019 ◽

Vol 519 (1) ◽

pp. 198-203

Author(s):

Lei Cheng ◽

Jianming Yue ◽

Sheng Yin ◽

Mingjing Ren ◽

Chengtao Wang

Keyword(s):

Pseudomonas Putida ◽

Two Component System ◽

Component System ◽

Two Component

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The AauR-AauS Two-Component System Regulates Uptake and Metabolism of Acidic Amino Acids in Pseudomonas putida

Applied and Environmental Microbiology ◽

10.1128/aem.00830-06 ◽

2006 ◽

Vol 72 (10) ◽

pp. 6569-6577 ◽

Cited By ~ 14

Author(s):

Avinash M. Sonawane ◽

Birendra Singh ◽

Klaus-Heinrich Röhm

Keyword(s):

Amino Acids ◽

Pseudomonas Putida ◽

Transcriptional Analysis ◽

Two Component System ◽

Component System ◽

Carbon And Nitrogen ◽

Acidic Amino Acids ◽

Wide Range ◽

Two Component ◽

Uptake And Metabolism

ABSTRACT Pseudomonas putida KT2440 metabolizes a wide range of carbon and nitrogen sources, including many amino acids. In this study, a σ54-dependent two-component system that controls the uptake and metabolism of acidic amino acids was identified. The system (designated aau, for acidic amino acid utilization) involves a sensor histidine kinase, AauS, encoded by PP1067, and a response regulator, AauR, encoded by PP1066. aauR and aauS deletion mutants were unable to efficiently utilize aspartate (Asp), glutamate (Glu), and glutamine (Gln) as sole sources of carbon and nitrogen. Growth of the mutants was partially restored when the above-mentioned amino acids were supplemented with glucose or succinate as an additional carbon source. Uptake of Gln, Asp, and asparagine (Asn) by the aauR mutant was moderately reduced, while Glu uptake was severely impaired. In the absence of glucose, the aauR mutant even secreted Glu into the medium. Furthermore, disruption of aauR affected the activities of several key enzymes of Glu and Asp metabolism, leading to the intracellular accumulation of Glu and greatly reduced survival times under conditions of nitrogen starvation. By a proteomics approach, four major proteins were identified that are downregulated during growth of the aauR mutant on Glu. Two of these were identified as periplasmic glutaminase/asparaginase and the solute-binding protein of a Glu/Asp transporter. Transcriptional analysis of lacZ fusions containing the putative promoter regions of these genes confirmed that their expression is indeed affected by the aau system. Three further periplasmic solute-binding proteins were strongly expressed during growth of the aauR deletion mutant on Glu but downregulated during cultivation on glucose/NH4 +. These systems may be involved in amino acid efflux.

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Expression of the RND-Type Efflux Pump AdeABC in Acinetobacter baumannii Is Regulated by the AdeRS Two-Component System

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.9.3298-3304.2004 ◽

2004 ◽

Vol 48 (9) ◽

pp. 3298-3304 ◽

Cited By ~ 225

Author(s):

Isabelle Marchand ◽

Laurence Damier-Piolle ◽

Patrice Courvalin ◽

Thierry Lambert

Keyword(s):

Acinetobacter Baumannii ◽

Efflux Pump ◽

Constitutive Expression ◽

Northern Hybridization ◽

Two Component System ◽

Component System ◽

Reverse Transcription Pcr ◽

Two Component ◽

Vitro Analysis

ABSTRACT The AdeABC pump of Acinetobacter baumannii BM4454, which confers resistance to various antibiotic classes including aminoglycosides, is composed of the AdeA, AdeB, and AdeC proteins; AdeB is a member of the RND superfamily. The adeA, adeB, and adeC genes are contiguous and adjacent to adeS and adeR, which are transcribed in the opposite direction and which specify proteins homologous to sensors and regulators of two-component systems, respectively (S. Magnet, P. Courvalin, and T. Lambert, Antimicrob. Agents Chemother. 45:3375-3380, 2001). Analysis by Northern hybridization indicated that the three genes were cotranscribed, although mRNAs corresponding to adeAB and adeC were also present. Cotranscription of the two regulatory genes was demonstrated by reverse transcription-PCR. Inactivation of adeS led to aminoglycoside susceptibility. Transcripts corresponding to adeAB were not detected in susceptible A. baumannii CIP 70-10 but were present in spontaneous gentamicin-resistant mutants obtained in vitro. Analysis of these mutants revealed the substitutions Thr153→Met in AdeS downstream from the putative His-149 site of autophosphorylation, which is presumably responsible for the loss of phosphorylase activity by the sensor, and Pro116→Leu in AdeR at the first residue of the α5 helix of the receiver domain, which is involved in interactions that control the output domain of response regulators. These mutations led to constitutive expression of the pump and, thus, to antibiotic resistance. These data indicate that the AdeABC pump is cryptic in wild A. baumannii due to stringent control by the AdeRS two-component system.

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