Engineering Escherichia coli to sense acidic amino acids by introduction of a chimeric two-component system

2015 ◽  
Vol 32 (10) ◽  
pp. 2073-2077 ◽  
Author(s):  
Sambandam Ravikumar ◽  
Irisappan Ganesh ◽  
Murali Kannan Maruthamuthu ◽  
Soon Ho Hong
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Two Component System ◽  
Component System ◽  
Acidic Amino Acids ◽  
Two Component

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

2006 ◽  
Vol 72 (10) ◽  
pp. 6569-6577 ◽  
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.

scholarly journals Kinetic Characterization of the WalRKSpn (VicRK) Two-Component System of Streptococcus pneumoniae: Dependence of WalKSpn (VicK) Phosphatase Activity on Its PAS Domain

2010 ◽  
Vol 192 (9) ◽  
pp. 2346-2358 ◽  
Author(s):  
Alina D. Gutu ◽  
Kyle J. Wayne ◽  
Lok-To Sham ◽  
Malcolm E. Winkler
Keyword(s):  
Amino Acids ◽  
Streptococcus Pneumoniae ◽  
Phosphatase Activity ◽  
Pas Domain ◽  
Kinetic Characterization ◽  
Two Component System ◽  
Histidine Kinases ◽  
Component System ◽  
Two Component

ABSTRACT The WalRK two-component system plays important roles in maintaining cell wall homeostasis and responding to antibiotic stress in low-GC Gram-positive bacteria. In the major human pathogen, Streptococcus pneumoniae, phosphorylated WalR Spn (VicR) response regulator positively controls the transcription of genes encoding the essential PcsB division protein and surface virulence factors. WalR Spn is phosphorylated by the WalK Spn (VicK) histidine kinase. Little is known about the signals sensed by WalK histidine kinases. To gain information about WalK Spn signal transduction, we performed a kinetic characterization of the WalRK Spn autophosphorylation, phosphoryltransferase, and phosphatase reactions. We were unable to purify soluble full-length WalK Spn . Consequently, these analyses were performed using two truncated versions of WalK Spn lacking its single transmembrane domain. The longer version (Δ35 amino acids) contained most of the HAMP domain and the PAS, DHp, and CA domains, whereas the shorter version (Δ195 amino acids) contained only the DHp and CA domains. The autophosphorylation kinetic parameters of Δ35 and Δ195 WalK Spn were similar [Km (ATP) ≈ 37 μM; k cat ≈ 0.10 min−1] and typical of those of other histidine kinases. The catalytic efficiency of the two versions of WalK Spn ∼P were also similar in the phosphoryltransfer reaction to full-length WalR Spn . In contrast, absence of the HAMP-PAS domains significantly diminished the phosphatase activity of WalK Spn for WalR Spn ∼P. Deletion and point mutations confirmed that optimal WalK Spn phosphatase activity depended on the PAS domain as well as residues in the DHp domain. In addition, these WalK Spn DHp domain and ΔPAS mutations led to attenuation of virulence in a murine pneumonia model.

HprSR is a Reactive Chlorine Species-Sensing, Two-Component System in Escherichia coli

2021 ◽  
Author(s):  
Sara El Hajj ◽  
Camille Henry ◽  
Camille Andrieu ◽  
Alexandra Vergnes ◽  
Laurent Loiseau ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Hypochlorous Acid ◽  
Methionine Sulfoxide ◽  
Methionine Sulfoxide Reductase ◽  
Repair System ◽  
Bacterial Cells ◽  
Two Component System ◽  
Component System ◽  
Two Component ◽  
Redox Switch

Two-component systems (TCS) are signalling pathways that allow bacterial cells to sense, respond and adapt to fluctuating environments. Among the classical TCS of Escherichia coli , HprSR has recently been shown to be involved in the regulation of msrPQ , which encodes the periplasmic methionine sulfoxide reductase system. In this study, we demonstrate that hypochlorous acid (HOCl) induces the expression of msrPQ in an HprSR-dependant manner, whereas H 2 O 2 , NO and paraquat (a superoxide generator) do not. Therefore, HprS appears to be an HOCl-sensing histidine kinase. Using a directed mutagenesis approach, we show that Met residues located in the periplasmic loop of HprS are important for its activity: as HOCl preferentially oxidizes Met residues, we provide evidence that HprS could be activated via the reversible oxidation of its methionine residues, meaning that MsrPQ plays a role in switching HprSR off. We propose that the activation of HprS by HOCl could occur through a Met redox switch. HprSR appears to be the first characterized TCS able to detect reactive chlorine species (RCS) in E. coli . This study represents an important step towards understanding the mechanisms of RCS resistance in prokaryotes. IMPORTANCE Understanding how bacteria respond to oxidative stress at the molecular level is crucial in the fight against pathogens. HOCl is one of the most potent industrial and physiological microbiocidal oxidants. Therefore bacteria have developed counterstrategies to survive HOCl-induced stress. Over the last decade, important insights into these bacterial protection factors have been obtained. Our work establishes HprSR as a reactive chlorine species-sensing, two-component system in Escherichia coli MG1655, which regulates the expression of MsrPQ, a repair system for HOCl-oxidized proteins. Moreover we provide evidence suggesting that HOCl could activate HprS through a methionine redox switch.

Involvement of AtoSC two-component system in Escherichia coli flagellar regulon

Amino Acids ◽  
2011 ◽  
Vol 43 (2) ◽  
pp. 833-844 ◽  
Author(s):  
Marina C. Theodorou ◽  
Evaggelos C. Theodorou ◽  
Dimitrios A. Kyriakidis
Keyword(s):  
Escherichia Coli ◽  
Two Component System ◽  
Component System ◽  
Two Component ◽  
Flagellar Regulon

scholarly journals Polar Localization of a Tripartite Complex of the Two-Component System DcuS/DcuR and the Transporter DctA in Escherichia coli Depends on the Sensor Kinase DcuS

PLoS ONE ◽  
2014 ◽  
Vol 9 (12) ◽  
pp. e115534 ◽  
Author(s):  
Patrick D. Scheu ◽  
Philipp A. Steinmetz ◽  
Felix Dempwolff ◽  
Peter L. Graumann ◽  
Gottfried Unden
Keyword(s):  
Escherichia Coli ◽  
Sensor Kinase ◽  
Two Component System ◽  
Component System ◽  
Polar Localization ◽  
Two Component

scholarly journals First Insights into the Unexplored Two-Component System YehU/YehT in Escherichia coli

2012 ◽  
Vol 194 (16) ◽  
pp. 4272-4284 ◽  
Author(s):  
T. Kraxenberger ◽  
L. Fried ◽  
S. Behr ◽  
K. Jung
Keyword(s):  
Escherichia Coli ◽  
Two Component System ◽  
Component System ◽  
Two Component

Extracellular Ca2+ transients affect poly-(R)-3-hydroxybutyrate regulation by the AtoS-AtoC system in Escherichia coli

2009 ◽  
Vol 417 (3) ◽  
pp. 667-672 ◽  
Author(s):  
Marina C. Theodorou ◽  
Ekaterini Tiligada ◽  
Dimitrios A. Kyriakidis
Keyword(s):  
Escherichia Coli ◽  
Channel Blocker ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Signal Transduction System ◽  
Two Component System ◽  
Component System ◽  
E Coli ◽  
Two Component ◽  
Membrane Bound

Escherichia coli is exposed to wide extracellular concentrations of Ca2+, whereas the cytosolic levels of the ion are subject to stringent control and are implicated in many physiological functions. The present study shows that extracellular Ca2+ controls cPHB [complexed poly-(R)-3-hydroxybutyrate] biosynthesis through the AtoS-AtoC two-component system. Maximal cPHB accumulation was observed at higher [Ca2+]e (extracellular Ca2+ concentration) in AtoS-AtoC-expressing E. coli compared with their ΔatoSC counterparts, in both cytosolic and membrane fractions. The reversal of EGTA-mediated down-regulation of cPHB biosynthesis by the addition of Ca2+ and Mg2+ was under the control of the AtoS-AtoC system. Moreover, the Ca2+-channel blocker verapamil reduced total and membrane-bound cPHB levels, the inhibitory effect being circumvented by Ca2+ addition only in atoSC+ bacteria. Histamine and compound 48/80 affected cPHB accumulation in a [Ca2+]e-dependent manner directed by the AtoS-AtoC system. In conclusion, these data provide evidence for the involvement of external Ca2+ on cPHB synthesis regulated by the AtoS-AtoC two-component system, thus linking Ca2+ with a signal transduction system, most probably through a transporter.

Modulation of virulence genes by the two-component system PhoP-PhoQ in avian pathogenic Escherichia coli

2016 ◽  
Vol 19 (1) ◽  
pp. 31-40 ◽  
Author(s):  
Jian Tu ◽  
Boyan Huang ◽  
Yu Zhang ◽  
Yuxi Zhang ◽  
Ting Xue ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mutant Strain ◽  
Virulence Genes ◽  
Avian Pathogenic Escherichia Coli ◽  
Two Component System ◽  
Component System ◽  
Transcript Levels ◽  
Pathogenic Escherichia Coli ◽  
Two Component ◽  
Viable Bacteria

Abstract Avian pathogenic Escherichia coli (APEC) infections are a very important problem in the poultry industry. PhoP-PhoQ is a two-component system that regulates virulence genes in APEC. In this study, we constructed strains that lacked the PhoP or PhoQ genes to assess regulation of APEC pathogenicity by the PhoP-PhoQ two-component system. The PhoP mutant strain AE18, PhoQ mutant strain AE19, and PhoP/PhoQ mutant strain AE20 were constructed by the Red homologous recombination method. Swim plates were used to evaluate the motility of the APEC strains, viable bacteria counting was used to assess adhesion and invasion of chick embryo fibroblasts, and Real-Time PCR was used to measure mRNA expression of virulence genes. We first confirmed that AE18, AE19, and AE20 were successfully constructed from the wild-type AE17 strain. AE18, AE19, and AE20 showed significant decreases in motility of 70.97%, 83.87%, and 37.1%, respectively, in comparison with AE17. Moreover, in comparison with AE17, AE18, AE19, and AE20 showed significant decreases of 63.11%, 65.42%, and 30.26%, respectively, in CEF cell adhesion, and significant decreases of 59.83%, 57.82%, and 37.90%, respectively, in CEF cell invasion. In comparison with AE17, transcript levels of sodA, polA, and iss were significantly decreased in AE18, while transcript levels of fimC and iss were significantly decreased in AE19. Our results demonstrate that deletion of PhoP or PhoQ inhibits invasion and adhesion of APEC to CEF cells and significantly reduces APEC virulence by regulating transcription of virulence genes.

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