scholarly journals Roles of NhaA, NhaB, and NhaD Na+/H+ Antiporters in Survival of Vibrio cholerae in a Saline Environment

2003 ◽  
Vol 185 (4) ◽  
pp. 1236-1244 ◽  
Author(s):  
Katia Herz ◽  
Sophie Vimont ◽  
Etana Padan ◽  
Patrick Berche
Keyword(s):  
Stationary Phase ◽  
Vibrio Cholerae ◽  
Ph Regulation ◽  
Molecular System ◽  
Specific Inhibitor ◽  
Ion Pump ◽  
Quinone Oxidoreductase ◽  
Saline Environment ◽  
E Coli ◽  
Wide Range

ABSTRACT Vibrio cholerae, the causative agent of cholera, is a normal inhabitant of aquatic environments, where it survives in a wide range of conditions of pH and salinity. In this work, we investigated the role of three Na+/H+ antiporters on the survival of V. cholerae in a saline environment. We have previously cloned the Vc-nhaA gene encoding the V. cholerae homolog of Escherichia coli. Here we identified two additional antiporter genes, designated Vc-nhaB and Vc-nhaD, encoding two putative proteins of 530 and 477 residues, respectively, highly homologous to the respective antiporters of Vibrio species and E. coli. We showed that both Vc-NhaA and Vc-NhaB confer Na+ resistance and that Vc-NhaA displays an antiport activity in E. coli, which is similar in magnitude, kinetic parameters, and pH regulation to that of E. coli NhaA. To determine the roles of the Na+/H+ antiporters in V. cholerae, we constructed nhaA, nhaB, and nhaD mutants (single, double, and triple mutants). In contrast to E. coli, the inactivation of the three putative antiporter genes (Vc-nhaABD) in V. cholerae did not alter the bacterial exponential growth in the presence of high Na+ concentrations and had only a slight effect in the stationary phase. In contrast, a pronounced and similar Li+-sensitive phenotype was found with all mutants lacking Vc-nhaA during the exponential phase of growth and also with the triple mutant in the stationary phase of growth. By using 2-n-nonyl-4-hydroxyquinoline N-oxide, a specific inhibitor of the electron-transport-linked Na+ pump NADH-quinone oxidoreductase (NQR), we determined that in the absence of NQR activity, the Vc-NhaA Na+/H+ antiporter activity becomes essential for the resistance of V. cholerae to Na+ at alkaline pH. Since the ion pump NQR is Na+ specific, we suggest that its activity masks the Na+/H+ but not the Li+/H+ antiporter activities. Our results indicate that the Na+ resistance of the human pathogen V. cholerae requires a complex molecular system involving multiple antiporters and the NQR pump.

scholarly journals Vibrio cholerae Triggers SOS and Mutagenesis in Response to a Wide Range of Antibiotics: a Route towards Multiresistance

2011 ◽  
Vol 55 (5) ◽  
pp. 2438-2441 ◽  
Author(s):  
Zeynep Baharoglu ◽  
Didier Mazel
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Vibrio Cholerae ◽  
Fluorescent Protein ◽  
Resistance Development ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Green Fluorescent ◽  
Regulated Promoters

ABSTRACTAntibiotic resistance development has been linked to the bacterial SOS stress response. InEscherichia coli, fluoroquinolones are known to induce SOS, whereas other antibiotics, such as aminoglycosides, tetracycline, and chloramphenicol, do not. Here we address whether various antibiotics induce SOS inVibrio cholerae. Reporter green fluorescent protein (GFP) fusions were used to measure the response of SOS-regulated promoters to subinhibitory concentrations of antibiotics. We show that unlike the situation withE. coli, all these antibiotics induce SOS inV. cholerae.

scholarly journals In Vivo Modulation of a DnaJ Homolog, CbpA, by CbpM

2007 ◽  
Vol 189 (9) ◽  
pp. 3635-3638 ◽  
Author(s):  
Matthew R. Chenoweth ◽  
Nancy Trun ◽  
Sue Wickner
Keyword(s):  
Escherichia Coli ◽  
Cell Growth ◽  
Stationary Phase ◽  
Specific Inhibitor ◽  
Vitro Activity ◽  
In Vitro Activity ◽  
E Coli ◽  
Hsp70 Chaperone

ABSTRACT CbpA, an Escherichia coli DnaJ homolog, can function as a cochaperone for the DnaK/Hsp70 chaperone system, and its in vitro activity can be modulated by CbpM. We discovered that CbpM specifically inhibits the in vivo activity of CbpA, preventing it from functioning in cell growth and division. Furthermore, we have shown that CbpM interacts with CbpA in vivo during stationary phase, suggesting that the inhibition of activity is a result of the interaction. These results reveal that the activity of the E. coli DnaK system can be regulated in vivo by a specific inhibitor.

scholarly journals NhaA, an Na+/H+ Antiporter Involved in Environmental Survival of Vibrio cholerae

2000 ◽  
Vol 182 (10) ◽  
pp. 2937-2944 ◽  
Author(s):  
Sophie Vimont ◽  
Patrick Berche
Keyword(s):  
Vibrio Cholerae ◽  
Vibrio Parahaemolyticus ◽  
Natural Habitat ◽  
Aquatic Environments ◽  
Wild Type ◽  
Transcriptional Fusion ◽  
Free Living ◽  
E Coli ◽  
Functional Homology ◽  
Wide Range

ABSTRACT Vibrio cholerae, the agent of cholera, is a normal inhabitant of aquatic environments, in which it survives under a wide range of conditions of pH and salinity. In this work, we identified thenhaA gene in a wild-type epidemic strain of V. cholerae O1. nhaA encodes a protein of 382 amino acids that is very similar to the proteins NhaA of Vibrio parahaemolyticus, Vibrio alginolyticus (∼87% identity), and Escherichia coli (56% identity). V. cholerae NhaA complements an E. coli nhaA mutant, enabling it to grow in 700 mM NaCl, pH 7.5, indicating functional homology to E. coli NhaA. However, unlike E. coli, the growth of a nhaA-inactivated mutant ofV. cholerae was not restricted at various pH and NaCl concentrations, although it was inhibited in the presence of 120 mM LiCl at pH 8.5. Nevertheless, using a nhaA′-lacZtranscriptional fusion, we observed induction of nhaAtranscription by Na+, Li+, and K+. These results strongly suggest that NhaA is an Na+/H+ antiporter contributing to the Na+/H+ homeostasis of V. cholerae. nhaA-related sequences were detected in all strains ofV. cholerae from the various serogroups. This gene is presumably involved in the survival and persistence of free-living bacteria in their natural habitat.

scholarly journals Requirements for Conversion of the Na+-Driven Flagellar Motor of Vibrio cholerae to the H+-Driven Motor of Escherichia coli

2000 ◽  
Vol 182 (15) ◽  
pp. 4234-4240 ◽  
Author(s):  
Khoosheh K. Gosink ◽  
Claudia C. Häse
Keyword(s):  
Escherichia Coli ◽  
Vibrio Cholerae ◽  
Proton Translocation ◽  
Specific Inhibitor ◽  
The Other ◽  
E Coli ◽  
Alkaline Conditions ◽  
Flagellar Assembly ◽  
And Function ◽  
Hybrid Motor

ABSTRACT Bacterial flagella are powered by a motor that converts a transmembrane electrochemical potential of either H+ or Na+ into mechanical work. In Escherichia coli, the MotA and MotB proteins form the stator and function in proton translocation, whereas the FliG protein is located on the rotor and is involved in flagellar assembly and torque generation. The sodium-driven polar flagella of Vibrio species contain homologs of MotA and MotB, called PomA and PomB, and also contain two other membrane proteins called MotX and MotY, which are essential for motor rotation and that might also function in ion conduction. Deletions inpomA, pomB, motX, ormotY in Vibrio cholerae resulted in a nonmotile phenotype, whereas deletion of fliG gave a nonflagellate phenotype. fliG genes on plasmids complementedfliG-null strains of the parent species but notfliG-null strains of the other species. FliG-null strains were complemented by chimeric FliG proteins in which the C-terminal domain came from the other species, however, implying that the C-terminal part of FliG can function in conjunction with the ion-translocating components of either species. A V. cholerae strain deleted of pomA, pomB,motX, and motY became weakly motile when theE. coli motA and motB genes were introduced on a plasmid. Like E. coli, but unlike wild-type V. cholerae, motility of some V. cholerae strains containing the hybrid motor was inhibited by the protonophore carbonyl cyanide m-chlorophenylhydrazone under neutral as well as alkaline conditions but not by the sodium motor-specific inhibitor phenamil. We conclude that the E. coli proton motor components MotA and MotB can function in place of the motor proteins ofV. cholerae and that the hybrid motors are driven by the proton motive force.

scholarly journals A T7 phage factor required for managing RpoS inEscherichia coli

2018 ◽  
Author(s):  
Aline Tabib-Salazar ◽  
Bing Liu ◽  
Declan Barker ◽  
Lynn Burchell ◽  
Udi Qimron ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Stationary Phase ◽  
Stationary Phases ◽  
Specific Inhibitor ◽  
E Coli ◽  
Bacterial Transcription ◽  
Bacterial Rna ◽  
Phage T7 ◽  
T7 Phage

T7 development inEscherichia colirequires the inhibition of the housekeeping form of the bacterial RNA polymerase (RNAP), Eσ70, by two T7 proteins: Gp2 and Gp5.7. While the biological role of Gp2 is well understood, that of Gp5.7 remains to be fully deciphered. Here, we present results from functional and structural analyses to reveal that Gp5.7 primarily serves to inhibit EσS, the predominant form of the RNAP in the stationary phase of growth, which accumulates in exponentially growingE. colias a consequence of buildup of guanosine pentaphosphate ((p)ppGpp) during T7 development. We further demonstrate a requirement of Gp5.7 for T7 development inE. colicells in the stationary phase of growth. Our finding represents a paradigm for how some lytic phages have evolved distinct mechanisms to inhibit the bacterial transcription machinery to facilitate phage development in bacteria in the exponential and stationary phases of growth.Significance statementVirus that infect bacteria (phages) represent the most abundant living entities on the planet and many aspects of our fundamental knowledge of phage-bacteria relationships have been derived in the context of exponentially growing bacteria. In the case of the prototypicalEscherichia coliphage T7, specific inhibition of the housekeeping form of the RNA polymerase (Eσ70) by a T7 protein, called Gp2, is essential for the development of viral progeny. We now reveal that T7 uses a second specific inhibitor that selectively inhibits the stationary phase RNAP (EσS), which enables T7 to develop well in exponentially growing and stationary phase bacteria. The results have broad implications for our understanding of phage-bacteria relationships and therapeutic application of phages.

scholarly journals Effect of pH-Regulation on the Capture of Lipopolysaccharides from E. coli EH100 by Four-Antennary Oligoglycines in Aqueous Medium

Materials ◽  
2021 ◽  
Vol 14 (24) ◽  
pp. 7659
Author(s):  
Anna Y. Gyurova ◽  
Kaloyan Berberov ◽  
Alexander Chinarev ◽  
Ljubomir Nikolov ◽  
Daniela Karashanova ◽  
...  
Keyword(s):  
Profile Analysis ◽  
Ph Regulation ◽  
Thin Liquid Film ◽  
Fine Tuning ◽  
Aqueous Systems ◽  
E Coli ◽  
Ph Values ◽  
Transmission Electron ◽  
Wide Range ◽  
Bacterial Lipopolysaccharides

Bacterial lipopolysaccharides (LPS) are designated as endotoxins, because they cause fever and a wide range of pathologies in humans. It is important to develop effective methodologies to detect trace quantities of LPS in aqueous systems. The present study develops a fine-tuning procedure for the entrapment of trace quantities of LPS from E. coli EH100. The capture agents are self-assemblies (tectomers) formed by synthetic four-antennary oligoglycine (C-(CH2-NH-Gly7)4, T4). Based on previously performed investigations of bulk and adsorption-layer properties of aqueous solutions containing T4 and LPS, the optimal conditions for the entrapment interactions are further fine-tuned by the pH regulation of aqueous systems. A combined investigation protocol is developed, including dynamic light scattering, profile analysis tensiometry, microscopic thin-liquid-film techniques, and transmission electron microscopy. The key results are: (1) two types of complexes between T4 and LPS are generated—amphiphilic species and “sandwich-like” hydrophilic entities; the complexes are smaller at lower pH, and larger at higher pH; (2) an optimum range of pH values is established within which the whole quantity of the LPS is entrapped by the tectomers, namely pH = 5.04–6.30. The obtained data substantiate the notion that T4 may be used for an effective capture and the removal of traces of endotoxins in aqueous systems.

scholarly journals Genome-wide mutational diversity inEscherichia colipopulation evolving in prolonged stationary-phase

2017 ◽  
Author(s):  
Savita Chib ◽  
Farhan Ali ◽  
Aswin Sai Narain Seshasayee
Keyword(s):  
Stationary Phase ◽  
Metabolic Pathways ◽  
Natural Environments ◽  
Lysogeny Broth ◽  
Neutral Drift ◽  
E Coli ◽  
Genome Wide ◽  
Wide Range ◽  
Regulatory Mutations ◽  
Over Time

AbstractProlonged stationary-phase is an approximation of natural environments presenting a range of stresses. Survival in prolonged stationary-phase requires alternative metabolic pathways for survival. This study describes the repertoire of mutations accumulating in starvingE. colipopulations in lysogeny broth. A wide range of mutations accumulate over the course of one month in stationary-phase. SNPs constitute 64% of all mutations. A majority of these mutations are non-synonymous and are located at conserved loci. There is an increase in genetic diversity in the evolving populations over time. Computer simulations of evolution in stationary phase suggest that the maximum frequency obtained by mutations in our experimental populations can not be explained by neutral drift. Moreover there is frequent genetic parallelism across populations suggesting that these mutations are under positive selection. Finally functional analysis of mutations suggests that regulatory mutations are frequent targets of selection.

scholarly journals Indole Pulse Signalling Regulates the Cytoplasmic pH ofE. coliin a Memory-Like Manner

2018 ◽  
Author(s):  
Ashraf Zarkan ◽  
Santiago Caño Muñiz ◽  
Jinbo Zhu ◽  
Kareem Al Nahas ◽  
Jehangir Cama ◽  
...  
Keyword(s):  
Stationary Phase ◽  
Ph Regulation ◽  
Critical Role ◽  
Bacterial Cells ◽  
Cytoplasmic Ph ◽  
E Coli ◽  
Degree Of Protection ◽  
Bacterial Cell Membrane ◽  
Ph Range ◽  
Phase Entry

SUMMARYBacterial cells are critically dependent upon pH regulation. Most proteins function over a limited pH range and the pH gradient across the bacterial cell membrane is central to energy production and transduction1. Here we demonstrate that indole plays a critical role in the regulation of the cytoplasmic pH ofE. coli. Indole is an aromatic molecule with diverse signalling roles that in bacteria is produced from tryptophan by the enzyme tryptophanase (TnaA)2. Two modes of indole signalling have been described: persistent and pulse signalling. The latter is illustrated by the brief but intense elevation of intracellular indole during stationary phase entry3,4. We show thatE. colicells growing under conditions where no indole is produced maintain their cytoplasmic pH at 7.8 ± 0.2. In contrast, under conditions permitting indole production, pH is maintained at 7.2 ± 0.2. Experiments where indole was added experimentally to non-producing cultures showed that pH regulation results from pulse, rather than persistent, indole signalling. Furthermore, the application of an artificial pulse of either of two non-biological proton ionophores (DNP or CCCP) caused a similar effect, suggesting that the relevant property of indole in this context is its ability to conduct protons across the cytoplasmic membrane5. Additionally, we show that the effect of the indole pulse that occurs normally during stationary phase entry in rich medium remains as a “memory” to maintain the correct cytoplasmic pH until entry into the next stationary phase. The indole-mediated reduction in cytoplasmic pH may explain why indole providesE. coliwith a degree of protection against stresses, including some bactericidal antibiotics.

scholarly journals The E. coli molecular phenotype under different growth conditions

2016 ◽  
Author(s):  
Mehmet U. Caglar ◽  
John R. Houser ◽  
Craig S. Barnhart ◽  
Daniel R. Boutz ◽  
Sean M. Carroll ◽  
...  
Keyword(s):  
Stationary Phase ◽  
Carbon Sources ◽  
Growth Conditions ◽  
Central Carbon Metabolism ◽  
Growth Media ◽  
Data Set ◽  
E Coli ◽  
Wide Range ◽  
Time Courses

AbstractModern systems biology requires extensive, carefully curated measurements of cellular components in response to different environmental conditions. While high-throughput methods have made transcriptomics and proteomics datasets widely accessible and relatively economical to generate, systematic measurements of both mRNA and protein abundances under a wide range of different conditions are still relatively rare. Here we present a detailed, genome-wide transcriptomics and proteomics dataset of E. coli grown under 34 different conditions. Additionally, we provide measurements of doubling times and in-vivo metabolic fluxes through the central carbon metabolism. We manipulate concentrations of sodium and magnesium in the growth media, and we consider four different carbon sources glucose, gluconate, lactate, and glycerol. Moreover, samples are taken both in exponential and stationary phase, and we include two extensive time-courses, with multiple samples taken between 3 hours and 2 weeks. We find that exponential-phase samples systematically differ from stationary-phase samples, in particular at the level of mRNA. Regulatory responses to different carbon sources or salt stresses are more moderate, but we find numerous differentially expressed genes for growth on gluconate and under salt and magnesium stress. Our data set provides a rich resource for future computational modeling of E. coli gene regulation, transcription, and translation.

Small Molecule Permeation Across Membrane Channels: Chemical Modification to Quantify Transport Across OmpF

2018 ◽  
Author(s):  
Jiajun Wang ◽  
Jayesh Arun Bafna ◽  
Satya Prathyusha Bhamidimarri ◽  
Mathias Winterhalter
Keyword(s):  
Dwell Time ◽  
Covalent Binding ◽  
Channel Structure ◽  
Ion Current ◽  
E Coli ◽  
Current Fluctuation ◽  
Wide Range ◽  
Outer Membrane Channel ◽  
Biological Channels ◽  
Constriction Region

Biological channels facilitate the exchange of small molecules across membranes, but surprisingly there is a lack of general tools for the identification and quantification of transport (i.e., translocation and binding). Analyzing the ion current fluctuation of a typical channel with its constriction region in the middle does not allow a direct conclusion on successful transport. For this, we created an additional barrier acting as a molecular counter at the exit of the channel. To identify permeation, we mainly read the molecule residence time in the channel lumen as the indicator whether the molecule reached the exit of the channel. As an example, here we use the well-studied porin, OmpF, an outer membrane channel from <i>E. coli</i>. Inspection of the channel structure suggests that aspartic acid at position 181 is located below the constriction region (CR) and we subsequently mutated this residue to cysteine, where else cysteine free and functionalized it by covalent binding with 2-sulfonatoethyl methanethiosulfonate (MTSES) or the larger glutathione (GLT) blockers. Using the dwell time as the signal for transport, we found that both mono-arginine and tri-arginine permeation process is prolonged by 20% and 50% respectively through OmpF<sub>E181C</sub>MTSES, while the larger sized blocker modification OmpF<sub>E181C</sub>GLT drastically decreased the permeation of mono-arginine by 9-fold and even blocked the pathway of the tri-arginine. In case of the hepta-arginine as substrate, both chemical modifications led to an identical ‘blocked’ pattern observed by the dwell time of ion current fluctuation of the OmpF<sub>wt</sub>. As an instance for antibiotic permeation, we analyzed norfloxacin, a fluoroquinolone antimicrobial agent. The modulation of the interaction dwell time suggests possible successful permeation of norfloxacin across OmpF<sub>wt</sub>. This approach may discriminate blockages from translocation events for a wide range of substrates. A potential application could be screening for scaffolds to improve the permeability of antibiotics.

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