scholarly journals Elucidation of Regulatory Modes for Five Two-Component Systems in Escherichia coli Reveals Novel Relationships

mSystems ◽  
2020 ◽  
Vol 5 (6) ◽  
Author(s):  
Kumari Sonal Choudhary ◽  
Julia A. Kleinmanns ◽  
Katherine Decker ◽  
Anand V. Sastry ◽  
Ye Gao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Target Gene ◽  
Target Genes ◽  
Rna Seq ◽  
Content Type ◽  
E Coli ◽  
Component Systems ◽  
Two Component ◽  
Two Component Systems

ABSTRACT Escherichia coli uses two-component systems (TCSs) to respond to environmental signals. TCSs affect gene expression and are parts of E. coli’s global transcriptional regulatory network (TRN). Here, we identified the regulons of five TCSs in E. coli MG1655: BaeSR and CpxAR, which were stimulated by ethanol stress; KdpDE and PhoRB, induced by limiting potassium and phosphate, respectively; and ZraSR, stimulated by zinc. We analyzed RNA-seq data using independent component analysis (ICA). ChIP-exo data were used to validate condition-specific target gene binding sites. Based on these data, we do the following: (i) identify the target genes for each TCS; (ii) show how the target genes are transcribed in response to stimulus; and (iii) reveal novel relationships between TCSs, which indicate noncognate inducers for various response regulators, such as BaeR to iron starvation, CpxR to phosphate limitation, and PhoB and ZraR to cell envelope stress. Our understanding of the TRN in E. coli is thus notably expanded. IMPORTANCE E. coli is a common commensal microbe found in the human gut microenvironment; however, some strains cause diseases like diarrhea, urinary tract infections, and meningitis. E. coli’s two-component systems (TCSs) modulate target gene expression, especially related to virulence, pathogenesis, and antimicrobial peptides, in response to environmental stimuli. Thus, it is of utmost importance to understand the transcriptional regulation of TCSs to infer bacterial environmental adaptation and disease pathogenicity. Utilizing a combinatorial approach integrating RNA sequencing (RNA-seq), independent component analysis, chromatin immunoprecipitation coupled with exonuclease treatment (ChIP-exo), and data mining, we suggest five different modes of TCS transcriptional regulation. Our data further highlight noncognate inducers of TCSs, which emphasizes the cross-regulatory nature of TCSs in E. coli and suggests that TCSs may have a role beyond their cognate functionalities. In summary, these results can lead to an understanding of the metabolic capabilities of bacteria and correctly predict complex phenotype under diverse conditions, especially when further incorporated with genome-scale metabolic models.

scholarly journals Elucidation of regulatory modes for five two-component systems in Escherichia coli reveals novel relationships

2020 ◽  
Author(s):  
Kumari Sonal Choudhary ◽  
Julia A. Kleinmanns ◽  
Katherine Decker ◽  
Anand V Sastry ◽  
Ye Gao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Transcriptional Regulation ◽  
Independent Component Analysis ◽  
Target Gene ◽  
Target Genes ◽  
E Coli ◽  
Component Systems ◽  
Two Component ◽  
Two Component Systems

AbstractEscherichia coli uses two-component systems (TCSs) to respond to environmental signals. TCSs affect gene expression and are parts of E. coli’s global transcriptional regulatory network (TRN). Here, we identified the regulons of five TCSs in E. coli MG1655: BaeSR and CpxAR, which were stimulated by ethanol stress; KdpDE and PhoRB, induced by limiting potassium and phosphate, respectively; and ZraSR, stimulated by zinc. We analyzed RNA-seq data using independent component analysis (ICA). ChIP-exo data was used to validate condition-specific target gene binding sites. Based on this data we (1) identify the target genes for each TCS; (2) show how the target genes are transcribed in response to stimulus; and (3) reveal novel relationships between TCSs, which indicate non-cognate inducers for various response regulators, such as BaeR to iron starvation, CpxR to phosphate limitation, and PhoB and ZraR to cell envelope stress. Our understanding of the TRN in E. coli is thus notably expanded.ImportanceE. coli is a common commensal microbe found in human gut microenvironment; however, some strains cause diseases like diarrhea, urinary tract infections and meningitis. E. coli’s two-component system (TCS) modulates target gene expression, specially related to virulence, pathogenesis and anti-microbial peptides, in response to environmental stimuli. Thus, it is of utmost importance to understand the transcriptional regulation of the TCSs to infer its environmental adaptation and disease pathogenicity. Utilizing a combinatorial approach integrating RNAseq, independent component analysis, ChIP-exo and data mining, we show that TCSs have five different modes of transcriptional regulation. Our data further highlights non-cognate inducers of TCSs emphasizing cross-regulatory nature of TCSs in E. coli and suggests that TCSs may have a role beyond their cognate functionalities. In summary, these results when further incorporated with genome scale metabolic models can lead to understanding of metabolic capabilities of bacteria and correctly predict complex phenotype under diverse conditions.

scholarly journals Hybrid Two-Component Sensors for Identification of Bacterial Chemoreceptor Function

2019 ◽  
Vol 85 (22) ◽  
Author(s):  
Rita A. Luu ◽  
Rebecca A. Schomer ◽  
Ceanne N. Brunton ◽  
Richard Truong ◽  
Albert P. Ta ◽  
...  
Keyword(s):  
Gene Expression ◽  
Soil Bacteria ◽  
Tricarboxylic Acid Cycle ◽  
Chemical Stimulus ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Component Systems ◽  
Two Component ◽  
Two Component Systems

ABSTRACT Soil bacteria adapt to diverse and rapidly changing environmental conditions by sensing and responding to environmental cues using a variety of sensory systems. Two-component systems are a widespread type of signal transduction system present in all three domains of life and typically are comprised of a sensor kinase and a response regulator. Many two-component systems function by regulating gene expression in response to environmental stimuli. The bacterial chemotaxis system is a modified two-component system with additional protein components and a response that, rather than regulating gene expression, involves behavioral adaptation and results in net movement toward or away from a chemical stimulus. Soil bacteria generally have 20 to 40 or more chemoreceptors encoded in their genomes. To simplify the identification of chemoeffectors (ligands) sensed by bacterial chemoreceptors, we constructed hybrid sensor proteins by fusing the sensor domains of Pseudomonas putida chemoreceptors to the signaling domains of the Escherichia coli NarX/NarQ nitrate sensors. Responses to potential attractants were monitored by β-galactosidase assays using an E. coli reporter strain in which the nitrate-responsive narG promoter was fused to lacZ. Hybrid receptors constructed from PcaY, McfR, and NahY, which are chemoreceptors for aromatic acids, tricarboxylic acid cycle intermediates, and naphthalene, respectively, were sensitive and specific for detecting known attractants, and the β-galactosidase activities measured in E. coli correlated well with results of chemotaxis assays in the native P. putida strain. In addition, a screen of the hybrid receptors successfully identified new ligands for chemoreceptor proteins and resulted in the identification of six receptors that detect propionate. IMPORTANCE Relatively few of the thousands of chemoreceptors encoded in bacterial genomes have been functionally characterized. More importantly, although methyl-accepting chemotaxis proteins, the major type of chemoreceptors present in bacteria, are easily identified bioinformatically, it is not currently possible to predict what chemicals will bind to a particular chemoreceptor. Chemotaxis is known to play roles in biodegradation as well as in host-pathogen and host-symbiont interactions, but many studies are currently limited by the inability to identify relevant chemoreceptor ligands. The use of hybrid receptors and this simple E. coli reporter system allowed rapid and sensitive screening for potential chemoeffectors. The fusion site chosen for this study resulted in a high percentage of functional hybrids, indicating that it could be used to broadly test chemoreceptor responses from phylogenetically diverse samples. Considering the wide range of chemical attractants detected by soil bacteria, hybrid receptors may also be useful as sensitive biosensors.

scholarly journals Effects of Regulatory Network Organization and Environment on PmrD Connector Activity and Polymyxin Resistance in Klebsiella pneumoniae and Escherichia coli

2020 ◽  
Vol 65 (3) ◽  
Author(s):  
Annie I. Chen ◽  
Francisco Javier Albicoro ◽  
Jun Zhu ◽  
Mark Goulian
Keyword(s):  
Escherichia Coli ◽  
Klebsiella Pneumoniae ◽  
Critical Role ◽  
Negative Regulator ◽  
Network Organization ◽  
Content Type ◽  
Resistance Pathway ◽  
Component Systems ◽  
Two Component ◽  
Two Component Systems

ABSTRACT Polymyxins are a class of cyclic peptides with antimicrobial activity against Gram-negative bacteria. In Enterobacteriaceae, the PhoQ/PhoP and PmrB/PmrA two-component systems regulate many genes that confer resistance to both polymyxins and host antimicrobial peptides. The activities of these two-component systems are modulated by additional proteins that are conserved across Enterobacteriaceae, such as MgrB, a negative regulator of PhoQ, and PmrD, a “connector” protein that activates PmrB/PmrA in response to PhoQ/PhoP stimulation. Despite the conservation of many protein components of the PhoQ/PhoP-PmrD-PmrB/PmrA network, the specific molecular interactions and regulatory mechanisms vary across different genera. Here, we explore the role of PmrD in modulating this signaling network in Klebsiella pneumoniae and Escherichia coli. We show that in K. pneumoniae, PmrD is not required for polymyxin resistance arising from mutation of mgrB—the most common cause of spontaneous polymyxin resistance in this bacterium—suggesting that direct activation of polymyxin resistance genes by PhoQ/PhoP plays a critical role in this resistance pathway. However, for conditions of low pH or intermediate iron concentrations, both of which stimulate PmrB/PmrA, we find that PmrD does contribute to resistance. We further show that in E. coli, PmrD functions as a connector between PhoQ/PhoP and PmrB/PmrA, in contrast with previous reports. In this case, activity also depends on PmrB/PmrA stimulation, or on very high activation of PhoQ/PhoP. Our results indicate that the importance of the PmrD connector in modulating the polymyxin resistance network depends on both the network organization and on the environmental conditions associated with PmrB stimulation.

scholarly journals A transcriptome study of the QseEF two-component system and the QseG membrane protein in enterohaemorrhagic Escherichia coli O157 : H7

Microbiology ◽  
2010 ◽  
Vol 156 (4) ◽  
pp. 1167-1175 ◽  
Author(s):  
Nicola C. Reading ◽  
David Rasko ◽  
Alfredo G. Torres ◽  
Vanessa Sperandio
Keyword(s):  
Escherichia Coli ◽  
Membrane Protein ◽  
Response Regulator ◽  
Two Component System ◽  
E Coli ◽  
Enterohaemorrhagic Escherichia Coli ◽  
Component Systems ◽  
Two Component ◽  
Two Component Systems ◽  
Type Iii Secretion Effector

QseE is a sensor kinase that responds to epinephrine, sulfate and phosphate. QseE constitutes a two-component signalling system together with the QseF σ 54-dependent response regulator. Encoded within the same operon as qseEF is the qseG gene, which encodes a membrane protein involved in the translocation of a type III secretion effector protein of enterohaemorrhagic Escherichia coli (EHEC) into epithelial cells. The qseEGF genes also form an operon with the glnB gene, which encodes the E. coli nitrogen sensor PII protein. Here we report a transcriptome analysis comparing qseE, qseF andqseG single mutants with the wild-type strain. This study revealed that the proteins encoded by these genes play a modest but significant role in iron uptake. Although QseEFG regulate genes involved in nitrogen utilization, these proteins do not play a notable role in nitrogen metabolism. In addition, QseEFG regulate transcription of the rcsBC and phoPQ two-component systems, linking several signal transduction pathways. The similarity of the microarray profiles of these mutants also indicates that these proteins work together. These data indicate that QseEFG are involved in the regulation of virulence and metabolism in EHEC.

scholarly journals Regulation of Virulence by Two-Component Systems in Pathogenic Burkholderia

2020 ◽  
Vol 88 (7) ◽  
Author(s):  
Matthew M. Schaefers
Keyword(s):  
Histidine Kinase ◽  
Burkholderia Cepacia ◽  
Target Genes ◽  
Response Regulator ◽  
Pharmaceutical Products ◽  
Phosphoryl Group ◽  
Content Type ◽  
Component Systems ◽  
Two Component ◽  
Two Component Systems

ABSTRACT The regulation and timely expression of bacterial genes during infection is critical for a pathogen to cause an infection. Bacteria have multiple mechanisms to regulate gene expression in response to their environment, one of which is two-component systems (TCS). TCS have two components. One component is a sensory histidine kinase (HK) that autophosphorylates when activated by a signal. The activated sensory histidine kinase then transfers the phosphoryl group to the second component, the response regulator, which activates transcription of target genes. The genus Burkholderia contains members that cause human disease and are often extensively resistant to many antibiotics. The Burkholderia cepacia complex (BCC) can cause severe lung infections in patients with cystic fibrosis (CF) or chronic granulomatous disease (CGD). BCC members have also recently been associated with several outbreaks of bacteremia from contaminated pharmaceutical products. Separate from the BCC is Burkholderia pseudomallei, which is the causative agent of melioidosis, a serious disease that occurs in the tropics, and a potential bioterrorism weapon. Bioinformatic analysis of sequenced Burkholderia isolates predicts that most strains have at least 40 TCS. The vast majority of these TCS are uncharacterized both in terms of the signals that activate them and the genes that are regulated by them. This review will highlight TCS that have been described to play a role in virulence in either the BCC or B. pseudomallei. Since many of these TCS are involved in virulence, TCS are potential novel therapeutic targets, and elucidating their function is critical for understanding Burkholderia pathogenesis.

scholarly journals New Shuttle Vector-Based Expression System To Generate Polyhistidine-Tagged Fusion Proteins in Staphylococcus aureus and Escherichia coli

2015 ◽  
Vol 81 (9) ◽  
pp. 3243-3254 ◽  
Author(s):  
Sybille Schwendener ◽  
Vincent Perreten
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Fusion Proteins ◽  
Target Gene ◽  
Shuttle Vector ◽  
Expression System ◽  
Multiple Cloning Site ◽  
Content Type ◽  
E Coli

ABSTRACTFourStaphylococcus aureus-Escherichia colishuttle vectors were constructed for gene expression and production of tagged fusion proteins. Vectors pBUS1-HC and pTSSCm have no promoter upstream of the multiple cloning site (MCS), and this allows study of genes under the control of their native promoters, and pBUS1-Pcap-HC and pTSSCm-Pcapcontain the strong constitutive promoter ofS. aureustype 1 capsule gene 1A (Pcap) upstream of a novel MCS harboring codons for the peptide tag Arg-Gly-Ser-hexa-His (rgs-his6). All plasmids contained the backbone derived from pBUS1, including theE. coliorigin ColE1, five copies of terminatorrrnBT1, and tetracycline resistance markertet(L) forS. aureusandE. coli. The minimum pAMα1 replicon from pBUS1 was improved through either complementation with the single-strand originoriLfrom pUB110 (pBUS1-HC and pBUS1-Pcap-HC) or substitution with a pT181-family replicon (pTSSCm and pTSSCm-Pcap). The new constructs displayed increased plasmid yield and segregational stability inS. aureus. Furthermore, pBUS1-Pcap-HC and pTSSCm-Pcapoffer the potential to generate C-terminal RGS-His6translational fusions of cloned genes using simple molecular manipulation. BcgI-induced DNA excision followed by religation converts the TGA stop codon of the MCS into a TGC codon and links thergs-his6codons to the 3′ end of the target gene. The generation of thergs-his6codon-fusion, gene expression, and protein purification were demonstrated in bothS. aureusandE. coliusing the macrolide-lincosamide-streptogramin B resistance geneerm(44) inserted downstream of Pcap. The new His tag expression system represents a helpful tool for the direct analysis of target gene function in staphylococcal cells.

scholarly journals The Histidine Residue of QseC Is Required for Canonical Signaling between QseB and PmrB in Uropathogenic Escherichia coli

2017 ◽  
Vol 199 (18) ◽  
Author(s):  
Erin J. Breland ◽  
Ellisa W. Zhang ◽  
Tomas Bermudez ◽  
Charles R. Martinez ◽  
Maria Hadjifrangiskou
Keyword(s):  
Escherichia Coli ◽  
Response Regulator ◽  
Polymyxin B ◽  
Sensor Kinase ◽  
Histidine Residue ◽  
Content Type ◽  
Component Systems ◽  
Two Component ◽  
Two Component Systems ◽  
Tract Infections

ABSTRACT Two-component systems are prototypically comprised of a histidine kinase (sensor) and a response regulator (responder). The sensor kinases autophosphorylate at a conserved histidine residue, acting as a phosphodonor for subsequent phosphotransfer to and activation of a cognate response regulator. In rare cases, the histidine residue is also essential for response regulator dephosphorylation via a reverse-phosphotransfer reaction. In this work, we present an example of a kinase that relies on reverse phosphotransfer to catalyze the dephosphorylation of its cognate partner. The QseC sensor kinase is conserved across several Gram-negative pathogens; its interaction with its cognate partner QseB is critical for maintaining pathogenic potential. Here, we demonstrate that QseC-mediated dephosphorylation of QseB occurs via reverse phosphotransfer. In previous studies, we demonstrated that, in uropathogenic Escherichia coli, exposure to high concentrations of ferric iron (Fe3+) stimulates the PmrB sensor kinase. This stimulation, in turn, activates the cognate partner, PmrA, and noncognate QseB to enhance tolerance to polymyxin B. We demonstrate that in the absence of signal, kinase-inactive QseC variants, in which the H246 residue was changed to alanine (A) aspartate (D) or leucine (L), rescued a ΔqseC deletion mutant, suggesting that QseC can control QseB activation via a mechanism that is independent of reverse phosphotransfer. However, in the presence of Fe3+, the same QseC variants were unable to mediate a wild-type stimulus response, indicating that QseC-mediated dephosphorylation is required for maintaining proper QseB-PmrB-PmrA interactions. IMPORTANCE Two-component signaling networks constitute one of the predominant methods by which bacteria sense and respond to their changing environments. Two-component systems allow bacteria to thrive and survive in a number of different environments, including within a human host. Uropathogenic Escherichia coli, the causative agent of urinary tract infections, rely on two interacting two-component systems, QseBC and PmrAB, to induce intrinsic resistance to the colistin antibiotic polymyxin B, which is a last line of defense drug. The presence of one sensor kinase, QseC, is required to regulate the interaction between the other sensor kinase, PmrB and the response regulators from both systems, QseB and PmrA, effectively creating a “four-component” system required for virulence. Understanding the important role of the sensor kinase QseC will provide insight into additional ways to therapeutically target uropathogens that harbor these signaling systems.

scholarly journals HypVW is an HOCl-Sensing Two Component System in Escherichia coli

2021 ◽  
Author(s):  
Sara El Hajj ◽  
Camille Henry ◽  
Alexandra Vergnes ◽  
Laurent Loiseau ◽  
Brasseur Gael ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Hypochlorous Acid ◽  
Methionine Sulfoxide ◽  
Methionine Sulfoxide Reductase ◽  
Bacterial Cells ◽  
E Coli ◽  
Two Component ◽  
Redox Switch ◽  
Two Component Systems ◽  
Methionine Residues

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, YedVW has been recently showed to be involved in the regulation of msrPQ, encoding for the periplasmic methionine sulfoxide reductase system. In this study, we demonstrate that hypochlorous acid (HOCl) induces the expression of msrPQ in a YedVW dependant manner, whereas H2O2, NO and paraquat (a superoxide generator) do not. Therefore, YedV appears to be an HOCl-sensing histidine kinase. Based on this finding, we proposed to rename this system HypVW.  Moreover, using a directed mutagenesis approach, we show that Met residues located in the periplasmic loop of HypV (formerly YedV) are important for its activity. Given that HOCl oxidizes preferentially Met residues, we bring evidences that HypV could be activated via the reversible oxidation of its methionine residues, thus conferring to MsrPQ a role in switching HypVW off. Based on these results, we propose that the activation of HypV by HOCl could occur through a Met redox switch. HypVW appears to be the first characterized TCS able to detect HOCl in E. coli. This study represents an important step in understanding the mechanisms of reactive chlorine species resistance in prokaryotes.

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