Cross-Talk between the Canonical and the Nitrogen-Related Phosphotransferase Systems Modulates Synthesis of the KdpFABC Potassium Transporter in Escherichia coli

2015 ◽  
Vol 25 (2-3) ◽  
pp. 168-177 ◽  
Author(s):  
Denise Lüttmann ◽  
Yvonne Göpel ◽  
Boris Görke
Keyword(s):  
Escherichia Coli ◽  
Carbon Source ◽  
Cross Talk ◽  
Growth Stage ◽  
Inhibit Activity ◽  
Wild Type ◽  
Phosphotransferase System ◽  
Potassium Transporter ◽  
Phosphoryl Groups ◽  
Major Target

Many Proteobacteria possess the regulatory nitrogen-related phosphotransferase system (PTS<sup>Ntr</sup>), which operates in parallel to the transport PTS. PTS<sup>Ntr</sup> is composed of the proteins EI<sup>Ntr</sup> and NPr and the final phosphate acceptor EIIA<sup>Ntr</sup>. Both PTSs can exchange phosphoryl groups among each other. Proteins governing K<sup>+</sup> uptake represent a major target of PTS<sup>Ntr</sup> in <i>Escherichia coli</i>. Nonphosphorylated EIIA<sup>Ntr</sup> binds and stimulates the K<sup>+</sup> sensor KdpD, which activates expression of the <i>kdpFABC</i> operon encoding a K<sup>+</sup> transporter. Here we show that this regulation also operates in an <i>ilvG</i><sup><i>+</i></sup> strain ruling out previous concern about interference with a nonfunctional <i>ilvG</i> allele present in many strains. Furthermore, we analyzed phosphorylation of EIIA<sup>Ntr</sup>. In wild-type cells EIIA<sup>Ntr</sup> is predominantly phosphorylated, regardless of the growth stage and the utilized carbon source. However, cross-phosphorylation of EIIA<sup>Ntr</sup> by the transport PTS becomes apparent in the absence of EI<sup>Ntr</sup>: EIIA<sup>Ntr</sup> is predominantly nonphosphorylated when cells grow on a PTS sugar and phosphorylated when a non-PTS carbohydrate is utilized. These differences in phosphorylation are transduced into corresponding <i>kdpFABC</i> transcription levels. Thus, the transport PTS may affect phosphorylation of EIIA<sup>Ntr</sup> and accordingly modulate processes controlled by EIIA<sup>Ntr</sup>. Our data suggest that this cross-talk becomes most relevant under conditions that would inhibit activity of EI<sup>Ntr</sup>.

scholarly journals The Phosphohistidine Phosphatase SixA Targets a Phosphotransferase System

mBio ◽  
2018 ◽  
Vol 9 (6) ◽  
Author(s):  
Jane E. Schulte ◽  
Mark Goulian
Keyword(s):  
Escherichia Coli ◽  
Protein Phosphatases ◽  
Growth Defect ◽  
Phosphotransferase System ◽  
Protein Activity ◽  
Content Type ◽  
Bacterial Physiology ◽  
Phosphoryl Groups ◽  
Regulate Protein ◽  
Protein Components

ABSTRACTSixA, a well-conserved protein found in proteobacteria, actinobacteria, and cyanobacteria, is the only reported example of a bacterial phosphohistidine phosphatase. A single protein target of SixA has been reported to date: theEscherichia colihistidine kinase ArcB. The present work analyzes an ArcB-independent growth defect of asixAdeletion inE. coli. A screen for suppressors, analysis of various mutants, and phosphorylation assays indicate that SixA modulates phosphorylation of the nitrogen-related phosphotransferase system (PTSNtr). The PTSNtris a widely conserved bacterial pathway that regulates diverse metabolic processes through the phosphorylation states of its protein components, EINtr, NPr, and EIIANtr, which receive phosphoryl groups on histidine residues. However, a mechanism for dephosphorylating this system has not been reported. The results presented here suggest a model in which SixA removes phosphoryl groups from the PTSNtrby acting on NPr. This work uncovers a new role for the phosphohistidine phosphatase SixA and, through factors that affect SixA expression or activity, may point to additional inputs that regulate the PTSNtr.IMPORTANCEOne common means to regulate protein activity is through phosphorylation. Protein phosphatases exist to reverse this process, returning the protein to the unphosphorylated form. The vast majority of protein phosphatases that have been identified target phosphoserine, phosphotheronine, and phosphotyrosine. A widely conserved phosphohistidine phosphatase was identified inEscherichia coli20 years ago but remains relatively understudied. The present work shows that this phosphatase modulates the nitrogen-related phosphotransferase system, a pathway that is regulated by nitrogen and carbon metabolism and affects diverse aspects of bacterial physiology. Until now, there was no known mechanism for removing phosphoryl groups from this pathway.

Mannose utilization in Escherichia coli requires cyclic AMP but not an exogenous inducer

1980 ◽  
Vol 26 (12) ◽  
pp. 1508-1511 ◽  
Author(s):  
Ann D. E. Fraser ◽  
Hiroshi Yamazaki
Keyword(s):  
Escherichia Coli ◽  
Carbon Source ◽  
Cyclic Amp ◽  
Adenylyl Cyclase ◽  
Sole Carbon Source ◽  
Receptor Protein ◽  
Deficient Mutant ◽  
Phosphotransferase System ◽  
Cyclic Amp Receptor Protein ◽  
Cyclic Monophosphate

It has not been clarified whether the utilization of mannose by Escherichia coli requires adenosine 3′,5′-cyclic monophosphate (cyclic AMP). Using an adenylyl cyclase deficient mutant (CA8306B) and a cyclic AMP receptor protein (CRP) deficient mutant (5333B) we have shown that the utilization of mannose is dependent on the cyclic AMP–CRP complex. 2-Deoxyglucose (DG) is a nonmetabolizable glucose analog specific for the phosphotransferase system (PTS) which transports mannose (termed here PTSM). Growth of CA8306B on glycerol is unaffected by addition of the analog, whereas growth of the strain on glycerol plus cyclic AMP ceases im mediately upon addition of DG. These results suggest that the formation of PTSM is dependent on cyclic AMP. In addition, CA8306B grown on glycerol plus cyclic AMP can immediately utilize mannose when transferred to a medium containing mannose as a sole carbon source, whereas the same strain grown on glycerol without cyclic AMP cannot utilize mannose when so transferred. These results suggest that the formation of PTSM does not require an exogenous inducer.

scholarly journals Glucose transport of Escherichia coli growing in glucose-limited continuous culture

1979 ◽  
Vol 178 (1) ◽  
pp. 97-101 ◽  
Author(s):  
I S Hunter ◽  
H L Kornberg
Keyword(s):  
Escherichia Coli ◽  
Continuous Culture ◽  
Glucose Concentration ◽  
Wild Type ◽  
Phosphotransferase System ◽  
Glucose Limitation ◽  
Low Glucose ◽  
Rate Limiting ◽  
Excess Glucose ◽  
Stimulation Of

Dilute cultures of wild-type Escherichia coli K12 and of derivatives impaired in one or other Enzyme-II component of the glucose phosphotransferase system were grown in continuous culture under glucose limitation. Cells harvested from the chemostat took up [U-14C]glucose from 0.1 mM solutions at rates directly related to the rates at which those cells had grown; the activity of the phosphotransferase system in those cells, rendered permeable with optimal accounts of toluene, parallels the ability of the cells to take up glucose. The capacity of these systems was rate-limiting for growth under the negligibly low glucose concentration in the chemostat, but was adequate to account for the stimulation of respiration observed when the cells were presented suddenly with excess glucose.

scholarly journals Alterations in Protein Expression Caused by thehha Mutation in Escherichia coli: Influence of Growth Medium Osmolarity

1999 ◽  
Vol 181 (10) ◽  
pp. 3018-3024 ◽  
Author(s):  
Carlos Balsalobre ◽  
Jörgen Johansson ◽  
Bernt Eric Uhlin ◽  
Antonio Juárez ◽  
Francisco J. Muñoa
Keyword(s):  
Escherichia Coli ◽  
Growth Medium ◽  
Gel Electrophoresis ◽  
Catabolite Repression ◽  
Protein Pattern ◽  
Polyacrylamide Gel Electrophoresis ◽  
Wild Type ◽  
Phosphotransferase System ◽  
New Family ◽  
Medium Osmolarity

ABSTRACT The Hha protein belongs to a new family of regulators involved in the environmental regulation of virulence factors. The aim of this work was to study the effect of the hha mutation on the overall protein pattern of Escherichia coli cells by two-dimensional polyacrylamide gel electrophoresis. The growth medium osmolarity clearly influenced the effect of the hhamutation. The number of proteins whose expression was altered inhha cells, compared with wild-type cells, was three times larger at a high osmolarity than at a low osmolarity. Among the proteins whose expression was modified by the hha allele, both OmpA and protein IIAGlc of the phosphotransferase system could be identified. As this latter enzyme participates in the regulation of the synthesis of cyclic AMP and hence influences the catabolite repression system, we tested whether the expression of thelacZ gene was also modified in hha mutants. This was the case, suggesting that at least some of the pleiotropic effects of the hha mutation could be caused by its effect on the catabolite repression system.

scholarly journals A mutant phosphofructokinase produces a futile cycle during gluconeogenesis in Escherichia coli

1997 ◽  
Vol 327 (3) ◽  
pp. 675-684 ◽  
Author(s):  
C. Juan TORRES ◽  
Victoria GUIXÉ ◽  
Jorge BABUL
Keyword(s):  
Escherichia Coli ◽  
Carbon Source ◽  
Kinetic Equations ◽  
British Library ◽  
Wild Type ◽  
Futile Cycle ◽  
Co2 Output ◽  
Futile Cycling ◽  
Fructose 1,6 Bisphosphate

Strains of Escherichia coli bearing different forms of phosphofructokinase were used to assess the occurrence of futile cycling in cell resuspensions supplied with glycerol as gluconeogenic carbon source. A model was used to simulate results of different kinds of experiments for different levels of futile cycle. The main predictions of the model were experimentally confirmed in a strain with a mutant phosphofructokinase-2 (phosphofructokinase-2*) which is not inhibited by MgATP. The intracellular fructose 1,6-bisphosphate concentration reaches significantly higher levels in the mutant-bearing strain than in strains with either phosphofructokinase-1 or -2. Also, this strain showed a higher rate and level of in vivo radioactive labelling of fructose 1,6-bisphosphate, from a trace of [U-14C]glucose supplied during gluconeogenesis, indicating higher kinase activity in these conditions. Cell resuspensions of the mutant-bearing strain produced higher levels of radioactively labelled CO2 when supplied with [U-14C]glycerol as the only carbon source. Simultaneously, fewer glycerol carbons were incorporated into HClO4-insoluble macromolecules. Finally, radioactive CO2 output was measured in resuspensions supplied with glycerol as the major carbon source with traces of either [1-14C]glucose or [6-14C]glucose. It was found that, whereas in the strains with either of the wild-type phosphofructokinase isoenzymes, radioactive CO2 output from [1-14C]glucose was higher than with [6-14C]glucose, the reverse is found for the strain with phosphofructokinase-2*. This result also agrees with the corresponding prediction of the model. Using the radioactivity flux rates predicted by the model, an explanation linking the futile cycle to the differential labelling of CO2 is advanced. Finally, on the basis of these results it is proposed that strains bearing phosphofructokinase-2* sustain higher rates of futile cycling during gluconeogenesis than strains bearing either of the wild-type isoforms of phosphofructokinase. The kinetic equations and parameter values used for the model simulations are given in Supplementary Publication SUP 50183 (8 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1997) 321, 8.

scholarly journals Identification of a Phosphotransferase System of Escherichia coli Required for Growth on N-Acetylmuramic Acid

2004 ◽  
Vol 186 (8) ◽  
pp. 2385-2392 ◽  
Author(s):  
Ulrike Dahl ◽  
Tina Jaeger ◽  
Bao Trâm Nguyen ◽  
Julia M. Sattler ◽  
Christoph Mayer
Keyword(s):  
Escherichia Coli ◽  
Energy Analysis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sole Source ◽  
Wild Type ◽  
Phosphotransferase System ◽  
E Coli ◽  
Glucose Transport System ◽  
Single Polypeptide

ABSTRACT We report here that wild-type Escherichia coli grows on N-acetylmuramic acid (MurNAc) as the sole source of carbon and energy. Analysis of mutants defective in N-acetylglucosamine (GlcNAc) catabolism revealed that the catabolic pathway for MurNAc merges into the GlcNAc pathway on the level of GlcNAc 6-phosphate. Furthermore, analysis of mutants defective in components of the phosphotransferase system (PTS) revealed that a PTS is essential for growth on MurNAc. However, neither the glucose-, mannose/glucosamine-, nor GlcNAc-specific PTS (PtsG, ManXYZ, and NagE, respectively) was found to be necessary. Instead, we identified a gene at 55 min on the E. coli chromosome that is responsible for MurNAc uptake and growth. It encodes a single polypeptide consisting of the EIIB and C domains of a so-far-uncharacterized PTS that was named murP. MurP lacks an EIIA domain and was found to require the activity of the crr-encoded enzyme IIA-glucose (EIIAGlc), a component of the major glucose transport system for growth on MurNAc. murP deletion mutants were unable to grow on MurNAc as the sole source of carbon; however, growth was rescued by providing murP in trans expressed from an isopropylthiogalactopyranoside-inducible plasmid. A functional His6 fusion of MurP was constructed, isolated from membranes, and identified as a polypeptide with an apparent molecular mass of 37 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Close homologs of MurP were identified in the genome of several bacteria, and we believe that these organisms might also be able to utilize MurNAc.

scholarly journals Why Does Escherichia coli Grow More Slowly on Glucosamine than on N-Acetylglucosamine? Effects of Enzyme Levels and Allosteric Activation of GlcN6P Deaminase (NagB) on Growth Rates

2005 ◽  
Vol 187 (9) ◽  
pp. 2974-2982 ◽  
Author(s):  
Laura I. Álvarez-Añorve ◽  
Mario L. Calcagno ◽  
Jacqueline Plumbridge
Keyword(s):  
Escherichia Coli ◽  
Growth Rates ◽  
Sugar Transport ◽  
Point Mutations ◽  
Sole Source ◽  
Wild Type ◽  
Phosphotransferase System ◽  
Allosteric Activation

ABSTRACT Wild-type Escherichia coli grows more slowly on glucosamine (GlcN) than on N-acetylglucosamine (GlcNAc) as a sole source of carbon. Both sugars are transported by the phosphotransferase system, and their 6-phospho derivatives are produced. The subsequent catabolism of the sugars requires the allosteric enzyme glucosamine-6-phosphate (GlcN6P) deaminase, which is encoded by nagB, and degradation of GlcNAc also requires the nagA-encoded enzyme, N-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase. We investigated various factors which could affect growth on GlcN and GlcNAc, including the rate of GlcN uptake, the level of induction of the nag operon, and differential allosteric activation of GlcN6P deaminase. We found that for strains carrying a wild-type deaminase (nagB) gene, increasing the level of the NagB protein or the rate of GlcN uptake increased the growth rate, which showed that both enzyme induction and sugar transport were limiting. A set of point mutations in nagB that are known to affect the allosteric behavior of GlcN6P deaminase in vitro were transferred to the nagB gene on the Escherichia coli chromosome, and their effects on the growth rates were measured. Mutants in which the substrate-induced positive cooperativity of NagB was reduced or abolished grew even more slowly on GlcN than on GlcNAc or did not grow at all on GlcN. Increasing the amount of the deaminase by using a nagC or nagA mutation to derepress the nag operon improved growth. For some mutants, a nagA mutation, which caused the accumulation of the allosteric activator GlcNAc6P and permitted allosteric activation, had a stronger effect than nagC. The effects of the mutations on growth in vivo are discussed in light of their in vitro kinetics.

scholarly journals Substrate Specificity and Signal Transduction Pathways in the Glucose-Specific Enzyme II (EIIGlc) Component of the Escherichia coli Phosphotransferase System

2000 ◽  
Vol 182 (16) ◽  
pp. 4437-4442 ◽  
Author(s):  
Lucinda Notley-McRobb ◽  
Thomas Ferenci
Keyword(s):  
Escherichia Coli ◽  
Signal Transduction ◽  
Substrate Specificity ◽  
Dependent Manner ◽  
Wild Type ◽  
Specific Enzyme ◽  
Phosphotransferase System ◽  
Transmembrane Segments ◽  
In The Wild ◽  
Second Category

ABSTRACT Escherichia coli adapted to glucose-limited chemostats contained mutations in ptsG resulting in V12G, V12F, and G13C substitutions in glucose-specific enzyme II (EIIGlc) and resulting in increased transport of glucose and methyl-α-glucoside. The mutations also resulted in faster growth on mannose and glucosamine in a PtsG-dependent manner. By use of enhanced growth on glucosamine for selection, four further sites were identified where substitutions caused broadened substrate specificity (G176D, A288V, G320S, and P384R). The altered amino acids include residues previously identified as changing the uptake of ribose, fructose, and mannitol. The mutations belonged to two classes. First, at two sites, changes affected transmembrane residues (A288V and G320S), probably altering sugar selectivity directly. More remarkably, the five other specificity mutations affected residues unlikely to be in transmembrane segments and were additionally associated with increasedptsG transcription in the absence of glucose. Increased expression of wild-type EIIGlc was not by itself sufficient for growth with other sugars. A model is proposed in which the protein conformation determining sugar accessibility is linked to transcriptional signal transduction in EIIGlc. The conformation of EIIGlc elicited by either glucose transport in the wild-type protein or permanently altered conformation in the second category of mutants results in altered signal transduction and interaction with a regulator, probably Mlc, controlling the transcription of pts genes.

scholarly journals Metabolic Flux Analysis of Escherichia coli creB and arcA Mutants Reveals Shared Control of Carbon Catabolism under Microaerobic Growth Conditions

2009 ◽  
Vol 191 (17) ◽  
pp. 5538-5548 ◽  
Author(s):  
Pablo I. Nikel ◽  
Jiangfeng Zhu ◽  
Ka-Yiu San ◽  
Beatriz S. Méndez ◽  
George N. Bennett
Keyword(s):  
Escherichia Coli ◽  
Carbon Source ◽  
Type Strain ◽  
Wild Type Strain ◽  
Tricarboxylic Acid Cycle ◽  
Pyruvate Dehydrogenase Complex ◽  
Building Blocks ◽  
Flux Analysis ◽  
Wild Type ◽  
Mutant Strains

ABSTRACT Escherichia coli has several elaborate sensing mechanisms for response to availability of oxygen and other electron acceptors, as well as the carbon source in the surrounding environment. Among them, the CreBC and ArcAB two-component signal transduction systems are responsible for regulation of carbon source utilization and redox control in response to oxygen availability, respectively. We assessed the role of CreBC and ArcAB in regulating the central carbon metabolism of E. coli under microaerobic conditions by means of 13C-labeling experiments in chemostat cultures of a wild-type strain, ΔcreB and ΔarcA single mutants, and a ΔcreB ΔarcA double mutant. Continuous cultures were conducted at D = 0.1 h−1 under carbon-limited conditions with restricted oxygen supply. Although all experimental strains metabolized glucose mainly through the Embden-Meyerhof-Parnas pathway, mutant strains had significantly lower fluxes in both the oxidative and the nonoxidative pentose phosphate pathways. Significant differences were also found at the pyruvate branching point. Both pyruvate-formate lyase and the pyruvate dehydrogenase complex contributed to acetyl-coenzyme A synthesis from pyruvate, and their activity seemed to be modulated by both ArcAB and CreBC. Strains carrying the creB deletion showed a higher biomass yield on glucose compared to the wild-type strain and its ΔarcA derivative, which also correlated with higher fluxes from building blocks to biomass. Glyoxylate shunt and lactate dehydrogenase were active mainly in the ΔarcA strain. Finally, it was observed that the tricarboxylic acid cycle reactions operated in a rather cyclic fashion under our experimental conditions, with reduced activity in the mutant strains.

Biofilm Growth of Escherichia coli Is Subject to cAMP-Dependent and cAMP-Independent Inhibition

2015 ◽  
Vol 25 (2-3) ◽  
pp. 209-225 ◽  
Author(s):  
Sarah L. Sutrina ◽  
Kia Daniel ◽  
Michael Lewis ◽  
Naomi T. Charles ◽  
Cherysa K.E. Anselm ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Catabolite Repression ◽  
Ph Effects ◽  
Wild Type ◽  
Inhibitory Effects ◽  
Phosphotransferase System ◽  
Biofilm Growth ◽  
E Coli ◽  
Low Concentrations ◽  
High Concentrations

We established that <i>Escherichia coli </i>strain 15 (ATCC 9723) produces both curli and cellulose, and forms robust biofilms. Since this strain is wild type with respect to the phosphoenolpyruvate:sugar phosphotransferase system (PTS), it is an ideal strain in which to investigate the effects of the PTS on the biofilm growth of <i>E. coli</i>. We began by looking into the effects of PTS and non-PTS sugars on the biofilm growth of this strain. All the sugars tested tended to activate biofilm growth at low concentrations but to inhibit biofilm growth at high concentrations. Acidification of the medium was an inhibitory factor in the absence of buffer, but buffering to prevent a pH drop did not prevent the inhibitory effects of the sugars. The concentration at which inhibition set in varied from sugar to sugar. For most sugars, cyclic (c)AMP counteracted the inhibition at the lowest inhibitory concentrations but became ineffective at higher concentrations. Our results suggest that cAMP-dependent catabolite repression, which is mediated by the PTS in <i>E. coli</i>, plays a role in the regulation of biofilm growth in response to sugars. cAMP-independent processes, possibly including Cra, also appear to be involved, in addition to pH effects.

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