scholarly journals The Extension of the Fourth Transmembrane Helix of the Sensor Kinase KdpD of Escherichia coli Is Involved in Sensing

2007 ◽  
Vol 189 (20) ◽  
pp. 7326-7334 ◽  
Author(s):  
Petra Zimmann ◽  
Anne Steinbrügge ◽  
Maren Schniederberend ◽  
Kirsten Jung ◽  
Karlheinz Altendorf
Keyword(s):  
Escherichia Coli ◽  
Response Regulator ◽  
Transmembrane Helix ◽  
Random Mutagenesis ◽  
Constitutive Expression ◽  
Single Amino Acid ◽  
Sensor Kinase ◽  
Fold Induction ◽  
Autokinase Activity

ABSTRACT The KdpD sensor kinase and the KdpE response regulator control expression of the kdpFABC operon coding for the KdpFABC high-affinity K+ transport system of Escherichia coli. In search of a distinct part of the input domain of KdpD which is solely responsible for K+ sensing, sequences of kdpD encoding the transmembrane region and adjacent N-terminal and C-terminal extensions were subjected to random mutagenesis. Nine KdpD derivatives were identified that had lost tight regulation of kdpFABC expression. They all carried single amino acid replacements located in a region encompassing the fourth transmembrane helix and the adjacent arginine cluster of KdpD. All mutants exhibited high levels of kdpFABC expression regardless of the external K+ concentration. However, 3- to 14-fold induction was observed under extreme K+-limiting conditions and in response to an osmotic upshift when sucrose was used as an osmolyte. These KdpD derivatives were characterized by a reduced phosphatase activity in comparison to the autokinase activity in vitro, which explains constitutive expression. Whereas for wild-type KdpD the autokinase activity and also, in turn, the phosphotransfer activity to KdpE were inhibited by increasing concentrations of K+, both activities were unaffected in the KdpD derivatives. These data clearly show that the extension of the fourth transmembrane helix encompassing the arginine cluster is mainly involved in sensing both K+ limitation and osmotic upshift, which may not be separated mechanistically.

scholarly journals Role of RcsF in Signaling to the Rcs Phosphorelay Pathway in Escherichia coli

2005 ◽  
Vol 187 (19) ◽  
pp. 6770-6778 ◽  
Author(s):  
Nadim Majdalani ◽  
Michael Heck ◽  
Valerie Stout ◽  
Susan Gottesman
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Small Rna ◽  
Kinase Activity ◽  
Response Regulator ◽  
Constitutive Expression ◽  
Activation Mechanism ◽  
Sensor Kinase ◽  
High Level

ABSTRACT The rcs phosphorelay pathway components were originally identified as regulators of capsule synthesis. In addition to the transmembrane sensor kinase RcsC, the RcsA coregulator, and the response regulator RcsB, two new components have been characterized, RcsD and RcsF. RcsD, the product of the yojN gene, now renamed rcsD, acts as a phosphorelay between RcsC and RcsB. Transcription of genes for capsule synthesis (cps) requires both RcsA and RcsB; transcription of other promoters, including that for the small RNA RprA, requires only RcsB. RcsF was described as an alternative sensor kinase for RcsB. We have examined the role of RcsF in the activation of both the rprA and cps promoters. We find that a number of signals that lead to activation of the phosphorelay require both RcsF and RcsC; epistasis experiments place RcsF upstream of RcsC. The RcsF sequence is characteristic of lipoproteins, consistent with a role in sensing cell surface perturbation and transmitting this signal to RcsC. Activation of RcsF does not require increased transcription of the gene, suggesting that modification of the RcsF protein may act as an activating signal. Signals from RcsC require RcsD to activate RcsB. Sequencing of an rcsC allele, rcsC137, that leads to high-level constitutive expression of both cps and rprA suggests that the response regulator domain of RcsC plays a role in negatively regulating the kinase activity of RcsC. The phosphorelay and the variation in the activation mechanism (dependent upon or independent of RcsA) provide multiple steps for modulating the output from this system.

scholarly journals Enhancement of the Efficiency of Secretion of Heterologous Lipase in Escherichia coli by Directed Evolution of the ABC Transporter System

2005 ◽  
Vol 71 (7) ◽  
pp. 3468-3474 ◽  
Author(s):  
Gyeong Tae Eom ◽  
Jae Kwang Song ◽  
Jung Hoon Ahn ◽  
Yeon Soo Seo ◽  
Joon Shick Rhee
Keyword(s):  
Escherichia Coli ◽  
Directed Evolution ◽  
Abc Transporter ◽  
Microtiter Plate ◽  
Single Amino Acid ◽  
Wild Type ◽  
E Coli ◽  
Single Amino Acid Change ◽  
Secretion Efficiency

ABSTRACT The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in Escherichia coli, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated tliD library with the wild-type tliA lipase in E. coli and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24, and T35, showed 3.2-, 2.6-, 2.9-, and 3.0-fold increases in the level of secretion of TliA lipase, respectively, but had almost the same level of expression of TliD in the membrane as the strain with the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA lipase was mediated by the transporter mutations. Each mutant had a single amino acid change in the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of the target protein. We therefore concluded that the efficiency of secretion of a heterologous protein in E. coli can be enhanced by in vitro engineering of the ABC transporter.

scholarly journals σE Regulates and Is Regulated by a Small RNA in Escherichia coli

2007 ◽  
Vol 189 (11) ◽  
pp. 4243-4256 ◽  
Author(s):  
Karl M. Thompson ◽  
Virgil A. Rhodius ◽  
Susan Gottesman
Keyword(s):  
Escherichia Coli ◽  
Small Rnas ◽  
Noncoding Rna ◽  
Response Regulator ◽  
Heterologous Promoter ◽  
Genomic Libraries ◽  
Multicopy Suppressors ◽  
Intergenic Regions ◽  
Transposon Mutants

ABSTRACT RybB is a small, Hfq-binding noncoding RNA originally identified in a screen of conserved intergenic regions in Escherichia coli. Fusions of the rybB promoter to lacZ were used to screen plasmid genomic libraries and genomic transposon mutants for regulators of rybB expression. A number of plasmids, including some carrying rybB, negatively regulated the fusion. An insertion in the rep helicase and one upstream of dnaK decreased expression of the fusion. Multicopy suppressors of these insertions led to identification of two plasmids that stimulated the fusion. One contained the gene for the response regulator OmpR; the second contained mipA, encoding a murein hydrolase. The involvement of MipA and OmpR in cell surface synthesis suggested that the rybB promoter might be dependent on σE. The sequence upstream of the +1 of rybB contains a consensus σE promoter. The activity of rybB-lacZ was increased in cells lacking the RseA anti-sigma factor and when σE was overproduced from a heterologous promoter. The activity of rybB-lacZ and the detection of RybB were totally abolished in an rpoE-null strain. In vitro, σE efficiently transcribes from this promoter. Both a rybB mutation and an hfq mutation significantly increased expression of both rybB-lacZ and rpoE-lacZ fusions, consistent with negative regulation of the σE response by RybB and other small RNAs. Based on the plasmid screens, NsrR, a repressor sensitive to nitric oxide, was also found to negatively regulate σE-dependent promoters in an RseA-independent fashion.

scholarly journals Investigation of the Role of Electrostatic Charge in Activation of the Escherichia coli Response Regulator CheY

2003 ◽  
Vol 185 (21) ◽  
pp. 6385-6391 ◽  
Author(s):  
Jenny G. Smith ◽  
Jamie A. Latiolais ◽  
Gerald P. Guanga ◽  
Sindhura Citineni ◽  
Ruth E. Silversmith ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Signal Transduction ◽  
Amino Acid ◽  
Active Site ◽  
Response Regulator ◽  
Residual Activity ◽  
Phosphoryl Group ◽  
Sensor Kinase ◽  
Amino Acid Substitutions ◽  
Flagellar Rotation

ABSTRACT In a two-component regulatory system, an important means of signal transduction in microorganisms, a sensor kinase phosphorylates a response regulator protein on an aspartyl residue, resulting in activation. The active site of the response regulator is highly charged (containing a lysine, the phosphorylatable aspartate, two additional aspartates involved in metal binding, and an Mg2+ ion), and introduction of the dianionic phosphoryl group results in the repositioning of charged moieties. Furthermore, substitution of one of the Mg2+-coordinating aspartates with lysine or arginine in the Escherichia coli chemotaxis response regulator CheY results in phosphorylation-independent activation. In order to examine the consequences of altered charge distribution for response regulator activity and to identify possible additional amino acid substitutions that result in phosphorylation-independent activation, we made 61 CheY mutants in which residues close to the site of phosphorylation (Asp57) were replaced by various charged amino acids. Most substitutions (47 of 61) resulted in the complete loss of CheY activity, as measured by the inability to support clockwise flagellar rotation. However, 10 substitutions, all introducing a new positive charge, resulted in the loss of chemotaxis but in the retention of some clockwise flagellar rotation. Of the mutants in this set, only the previously identified CheY13DK and CheY13DR mutants displayed clockwise activity in the absence of the CheA sensor kinase. The absence of negatively charged substitution mutants with residual activity suggests that the introduction of additional negative charges into the active site is particularly deleterious for CheY function. Finally, the spatial distribution of positions at which amino acid substitutions are functionally tolerated or not tolerated is consistent with the presently accepted mechanism of response regulator activation and further suggests a possible role for Met17 in signal transduction by CheY.

scholarly journals Regulation of fixLJ by Hfq Controls Symbiotically Important Genes in Sinorhizobium meliloti

2016 ◽  
Vol 29 (11) ◽  
pp. 844-853 ◽  
Author(s):  
Mengsheng Gao ◽  
Hahn Nguyen ◽  
Isai Salas González ◽  
Max Teplitski
Keyword(s):  
Sinorhizobium Meliloti ◽  
Rna Binding ◽  
Response Regulator ◽  
Sensor Kinase ◽  
In Planta ◽  
Symbiotic Efficiency ◽  
Posttranscriptional Control ◽  
Nitrogenase Genes ◽  
Regulatory Effects

The RNA-binding chaperone Hfq plays critical roles in the establishment and functionality of the symbiosis between Sinorhizobium meliloti and its legume hosts. A mutation in hfq reduces symbiotic efficiency resulting in a Fix− phenotype, characterized by the inability of the bacterium to fix nitrogen. At least in part, this is due to the ability of Hfq to regulate the fixLJ operon, which encodes a sensor kinase–response regulator pair that controls expression of the nitrogenase genes. The ability of Hfq to bind fixLJ in vitro and in planta was demonstrated with gel shift and coimmunoprecipitation experiments. Two (ARN)2 motifs in the fixLJ message were the likely sites through which Hfq exerted its posttranscriptional control. Consistent with the regulatory effects of Hfq, downstream genes controlled by FixLJ (such as nifK, noeB) were also subject to Hfq regulation in planta.

scholarly journals Interactions of the Antizyme AtoC with Regulatory Elements of the Escherichia coli atoDAEB Operon

2007 ◽  
Vol 189 (17) ◽  
pp. 6324-6332 ◽  
Author(s):  
Meropi K. Matta ◽  
Efthimia E. Lioliou ◽  
Cynthia H. Panagiotidis ◽  
Dimitrios A. Kyriakidis ◽  
Christos A. Panagiotidis
Keyword(s):  
Escherichia Coli ◽  
Transcription Initiation ◽  
Response Regulator ◽  
Regulatory Elements ◽  
Initiation Site ◽  
Integration Host Factor ◽  
Chemical Mutagenesis ◽  
Dual Function

ABSTRACT AtoC has a dual function as both an antizyme, the posttranslational inhibitor of polyamine biosynthetic enzymes, and the transcriptional regulator of genes involved in short-chain fatty acid catabolism (the atoDAEB operon). We have previously shown that AtoC is the response regulator of the AtoS-AtoC two-component signal transduction system that activates atoDAEB when Escherichia coli is exposed to acetoacetate. Here, we show that the same cis elements control both promoter inducibility and AtoC binding. Chromatin immunoprecipitation experiments confirmed the acetoacetate-inducible binding of AtoC to the predicted DNA region in vivo. DNase I protection footprinting analysis revealed that AtoC binds two 20-bp stretches, constituting an inverted palindrome, that are located at −146 to −107 relative to the transcription initiation site. Analyses of promoter mutants obtained by in vitro chemical mutagenesis of the atoDAEB promoter verified both the importance of AtoC binding for the inducibility of the promoter by acetoacetate and the σ54 dependence of atoDAEB expression. The integration host factor was also identified as a critical component of the AtoC-mediated induction of atoDAEB.

Phosphorylation and DNA binding of the regulator DcuR of the fumarate-responsive two-component system DcuSR of Escherichia coli

Microbiology ◽  
2004 ◽  
Vol 150 (4) ◽  
pp. 877-883 ◽  
Author(s):  
Ingo G. Janausch ◽  
Inma Garcia-Moreno ◽  
Daniela Lehnen ◽  
Yvonne Zeuner ◽  
Gottfried Unden
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Response Regulator ◽  
Sensory System ◽  
Phosphoryl Transfer ◽  
Affinity Binding ◽  
High Affinity ◽  
Two Component ◽  
Target Promoters

The function of the response regulator DcuR of the DcuSR fumarate two-component sensory system of Escherichia coli was analysed in vitro. Isolated DcuR protein was phosphorylated by the sensory histidine kinase, DcuS, and ATP, or by acetyl phosphate. In gel retardation assays with target promoters (frdA, dcuB, dctA), phosphoryl DcuR (DcuR-P) formed a high-affinity complex, with an apparent K D (app. K D) of 0·2–0·3 μM DcuR-P, and a low-affinity (app. K D 0·8–2 μM) complex. The high-affinity complex was formed only with promoters transcriptionally-regulated by DcuSR, whereas low-affinity binding was seen also with some DcuSR-independent promoters. The binding site of DcuR-P at the dcuB promoter was determined by DNase I footprinting. One binding site of 42–52 nt (position −359 to −400/−410 nt upstream of the transcriptional start) was identified in the presence of low and high concentrations of DcuR-P. Non-phosphorylated DcuR, or DcuR-D56N mutated in the phosphoryl-accepting Asp56 residue, showed low-affinity binding to target promoters. DcuR-D56N was still able to interact with DcuS. DcuR-D56N increased the phosphorylation of DcuS and competitively inhibited phosphoryl transfer to wild-type DcuR.

scholarly journals Mutational Analysis of RetS, an Unusual Sensor Kinase-Response Regulator Hybrid Required for Pseudomonas aeruginosa Virulence

2006 ◽  
Vol 74 (8) ◽  
pp. 4462-4473 ◽  
Author(s):  
Michelle A. Laskowski ◽  
Barbara I. Kazmierczak
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Mutational Analysis ◽  
Response Regulator ◽  
Acute Infection ◽  
Opportunistic Pathogen ◽  
Sensor Kinase ◽  
Chronic Infections ◽  
Reciprocal Regulation ◽  
Wide Range

ABSTRACT Pseudomonas aeruginosa is an opportunistic pathogen capable of causing both acute and chronic infections in a wide range of hosts. Expression of the type III secretion system (T3SS) proteins is correlated with virulence in models of acute infection, while downregulation of the T3SS and upregulation of genes important for biofilm formation are observed during chronic infections. RetS, a hybrid sensor kinase-response regulator protein of P. aeruginosa, plays a key role in the reciprocal regulation of virulence factors required for acute versus chronic infection and is postulated to act in concert with two other sensor kinase-response regulator hybrids, GacS and LadS. This work examines the roles of the putative sensing and signal transduction domains of RetS in induction of the T3SS in vitro and in a murine model of acute pneumonia. We identify distinct signaling roles for the tandem receiver domains of RetS and present evidence suggesting that RetS may serve as a substrate for another sensor kinase. Phenotypes associated with RetS alleles lacking periplasmic and/or transmembrane domains further indicate that the periplasmic domain of RetS may transmit a signal that inhibits RetS activity during acute infections.

scholarly journals The Type III Effector NleD from Enteropathogenic Escherichia coli Differentiates between Host Substrates p38 and JNK

2016 ◽  
Vol 85 (2) ◽  
Author(s):  
Kristina Creuzburg ◽  
Cristina Giogha ◽  
Tania Wong Fok Lung ◽  
Nichollas E. Scott ◽  
Sabrina Mühlen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Effector Proteins ◽  
Mitogen Activated Protein Kinase ◽  
Mass Spectrometry Analysis ◽  
Single Amino Acid ◽  
Luciferase Reporter ◽  
Type Iii Effector ◽  
Type Iii ◽  
Infected Cells

ABSTRACT Enteropathogenic Escherichia coli (EPEC) is a gastrointestinal pathogen that utilizes a type III secretion system (T3SS) to inject an array of virulence effector proteins into host enterocytes to subvert numerous cellular processes for successful colonization and dissemination. The T3SS effector NleD is a 26-kDa zinc metalloprotease that is translocated into host enterocytes, where it directly cleaves and inactivates the mitogen-activated protein kinase signaling proteins JNK and p38. Here a library of 91 random transposon-based, in-frame, linker insertion mutants of NleD were tested for their ability to cleave JNK and p38 during transient transfection of cultured epithelial cells. Immunoblot analysis of p38 and JNK cleavage showed that 7 mutant derivatives of NleD no longer cleaved p38 but maintained the ability to cleave JNK. Site-directed mutation of specific regions surrounding the insertion sites within NleD revealed that a single amino acid, R203, was essential for cleavage of p38 but not JNK in a direct in vitro cleavage assay, in transiently transfected cells, or in EPEC-infected cells. Mass spectrometry analysis narrowed the cleavage region to within residues 187 and 213 of p38. Mutation of residue R203 within NleD to a glutamate residue abolished the cleavage of p38 and impaired the ability of NleD to inhibit AP-1-dependent gene transcription of a luciferase reporter. Furthermore, the R203 mutation abrogated the ability of NleD to dampen interleukin-6 production in EPEC-infected cells. Overall, this work provides greater insight into substrate recognition and specificity by the type III effector NleD.

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.

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