Regulation of dctA and DctA by cAMP-CRP and EIIAGlc at the transcriptional and post-translational levels in E. coli: Consequences for aerobic uptake and metabolism of C4-dicarboxylates

Direct Stimulation ◽

E Coli ◽

Inducer Exclusion ◽

Specific Regulation

AbstractThe expression of dctA, encoding the aerobic C4-dicarboxylate (C4-DC) transporter DctA of Escherichia coli, and its use in the presence of alternative carbon sources was characterized. dctA is regulated by cAMP-CRP and substrates that control cAMP levels, either through the phosphotransferase system (PTS), or through their metabolic link to PEP synthesis. The data indicates that phosphorylation of the regulator EIIAGlc of the glucose-specific PTS represents the mediator for regulation. The dctA promotor region contains a class I CRP-binding site (position -81.5) and a DcuR-binding site (position -105.5). The response regulator DcuR of the C4-DC-activated DcuS-DcuR two-component system is known to stimulate expression of dctA, and cAMP-CRP is known to stimulate expression of dcuS-dcuR. Thus, activation of dctA expression by cAMP-CRP and DcuR is organized in a coherent feed-forward loop (FFL) where cAMP-CRP positively regulates the expression of dctA by direct stimulation and by stimulating the expression of dcuR. Stimulation by DcuR is presumed to require DNA bending by cAMP-CRP. In this way, CRP-FFL integrates carbon catabolite control and C4-DC-specific regulation. Moreover, EIIAGlc of the glucose-specific PTS strongly interacts with DctA, which could lead to substrate exclusion of C4-DCs when preferred carbon substrates such as sugars are present. Since C4-DCs are perceived in the periplasmic space by the sensor DcuS, the substrate exclusion is not linked to inducer exclusion, contrasting classical inducer exclusion known for the lactose permease LacY. Thus, aerobic C4-DC metabolism is tightly regulated at the transcriptional and post-translational levels, whereas uptake of L-aspartate by DcuA is essentially unaffected. Overall, transcriptional and post-translational regulation of dctA expression and DctA function efficiently fine-tunes C4-DC catabolism in response to other preferred carbon sources.

Oversized galactosides as a probe for conformational dynamics in LacY

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1800706115 ◽

2018 ◽

Vol 115 (16) ◽

pp. 4146-4151 ◽

Cited By ~ 3

Author(s):

Irina Smirnova ◽

Vladimir Kasho ◽

Xiaoxu Jiang ◽

Hong-Ming Chen ◽

Stephen G. Withers ◽

...

Keyword(s):

Binding Site ◽

Conformational Dynamics ◽

Dissociation Rate ◽

Binding Kinetics ◽

Lactose Permease ◽

E Coli ◽

Open Conformation ◽

Dissociation Rate Constants ◽

Kinetics Of ◽

Micromolar Range

Binding kinetics of α-galactopyranoside homologs with fluorescent aglycones of different sizes and shapes were determined with the lactose permease (LacY) of Escherichia coli by FRET from Trp151 in the binding site of LacY to the fluorophores. Fast binding was observed with LacY stabilized in an outward-open conformation (kon = 4–20 μM−1·s−1), indicating unobstructed access to the binding site even for ligands that are much larger than lactose. Dissociation rate constants (koff) increase with the size of the aglycone so that Kd values also increase but remain in the micromolar range for each homolog. Phe27 (helix I) forms an apparent constriction in the pathway for sugar by protruding into the periplasmic cavity. However, replacement of Phe27 with a bulkier Trp does not create an obstacle in the pathway even for large ligands, since binding kinetics remain unchanged. High accessibility of the binding site is also observed in a LacY/nanobody complex with partially blocked periplasmic opening. Remarkably, E. coli expressing WT LacY catalyzes transport of α- or β-galactopyranosides with oversized aglycones such as bodipy or Aldol518, which may require an extra space within the occluded intermediate. The results confirm that LacY specificity is strictly directed toward the galactopyranoside ring and also clearly indicate that the opening on the periplasmic side is sufficiently wide to accommodate the large galactoside derivatives tested here. We conclude that the actual pathway for the substrate entering from the periplasmic side is wider than the pore diameter calculated in the periplasmic-open X-ray structures.

Aer and Tsr guide Escherichia coli in spatial gradients of oxidizable substrates

Microbiology ◽

10.1099/mic.0.26304-0 ◽

2003 ◽

Vol 149 (9) ◽

pp. 2661-2667 ◽

Cited By ~ 34

Author(s):

Suzanne E. Greer-Phillips ◽

Gladys Alexandre ◽

Barry L. Taylor ◽

Igor B. Zhulin

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Carbon Sources ◽

Proton Motive Force ◽

Maximal Growth ◽

E Coli ◽

Spatial Gradients ◽

Energy Taxis ◽

Effective Growth

The Aer and Tsr chemoreceptors in Escherichia coli govern tactic responses to oxygen and redox potential that are parts of an overall behaviour known as energy taxis. They are also proposed to mediate responses to rapidly utilized carbon sources, glycerol and succinate, via the energy taxis mechanism. In this study, the Aer and Tsr proteins were individually expressed in an ‘all-transducer-knockout’ strain of E. coli and taxis was analysed in gradients of various oxidizable carbon sources. In addition to the known response to glycerol and succinate, it was found that Aer directed taxis towards ribose, galactose, maltose, malate, proline and alanine as well as the phosphotransferase system (PTS) carbohydrates glucose, mannitol, mannose, sorbitol and fructose, but not to aspartate, glutamate, glycine and arabinose. Tsr directed taxis towards sugars (including those transported by the PTS), but not to organic acids or amino acids. When a mutated Aer protein unable to bind the FAD cofactor was expressed in the receptor-less strain, chemotaxis was not restored to any substrate. Aer appears to mediate responses to rapidly oxidizable substrates, whether or not they are effective growth substrates, whereas Tsr appears to mediate taxis to substrates that support maximal growth, whether or not they are rapidly oxidizable. This correlates with the hypothesis that Aer and Tsr sense redox and proton motive force, respectively. Taken together, the results demonstrate that Aer and Tsr mediate responses to a broad range of chemicals and their attractant repertoires overlap with those of specialized chemoreceptors, namely Trg (ribose, galactose) and Tar (maltose).

Inactivation of the PTS as a Strategy to Engineer the Production of Aromatic Metabolites in Escherichia coli

Journal of Molecular Microbiology and Biotechnology ◽

10.1159/000380854 ◽

2015 ◽

Vol 25 (2-3) ◽

pp. 195-208 ◽

Cited By ~ 11

Author(s):

Susy Beatriz Carmona ◽

Fabián Moreno ◽

Francisco Bolívar ◽

Guillermo Gosset ◽

Adelfo Escalante

Keyword(s):

Escherichia Coli ◽

Carbon Source ◽

Transport Systems ◽

Adaptive Laboratory Evolution ◽

E Coli ◽

Inducer Exclusion ◽

Genetic Modifications ◽

Phosphate Donor ◽

Selection Of

Laboratory and industrial cultures of Escherichia coli employ media containing glucose which is mainly transported and phosphorylated by the phosphotransferase system (PTS). In these strains, 50% of the phosphoenolpyruvate (PEP), which results from the catabolism of transported glucose, is used as a phosphate donor for its phosphorylation and translocation by the PTS. This characteristic of the PTS limits the production of industrial biocommodities that have PEP as a precursor. Furthermore, when E. coli is exposed to carbohydrate mixtures, the PTS prevents expression of catabolic and non-PTS transport genes by carbon catabolite repression and inducer exclusion. In this contribution, we discuss the main strategies developed to overcome these potentially limiting effects in production strains. These strategies include adaptive laboratory evolution selection of PTS- Glc+ mutants, followed by the generation of strains that recover their ability to grow with glucose as a carbon source while allowing the simultaneous consumption of more than one carbon source. We discuss the benefits of using alternative glucose transport systems and describe the application of these strategies to E. coli strains with specific genetic modifications in target pathways. These efforts have resulted in significant improvements in the production of diverse biocommodities, including aromatic metabolites, biofuels and organic acids.

Thermodynamic mechanism for inhibition of lactose permease by the phosphotransferase protein IIAGlc

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1500891112 ◽

2015 ◽

Vol 112 (8) ◽

pp. 2407-2412 ◽

Cited By ~ 12

Author(s):

Parameswaran Hariharan ◽

Dhandayuthapani Balasubramaniam ◽

Alan Peterkofsky ◽

H. Ronald Kaback ◽

Lan Guan

Keyword(s):

Binding Sites ◽

Catabolite Repression ◽

Conformational Dynamics ◽

Titration Calorimetry ◽

Lactose Permease ◽

Inducer Exclusion ◽

Solvation Entropy ◽

Thermodynamic Mechanism ◽

Camp Levels

In a variety of bacteria, the phosphotransferase protein IIAGlcplays a key regulatory role in catabolite repression in addition to its role in the vectorial phosphorylation of glucose catalyzed by the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). The lactose permease (LacY) ofEscherichia colicatalyzes stoichiometric symport of a galactoside with an H+, using a mechanism in which sugar- and H+-binding sites become alternatively accessible to either side of the membrane. Both the expression (via regulation of cAMP levels) and the activity of LacY are subject to regulation by IIAGlc(inducer exclusion). Here we report the thermodynamic features of the IIAGlc–LacY interaction as measured by isothermal titration calorimetry (ITC). The studies show that IIAGlcbinds to LacY with aKdof about 5 μM and a stoichiometry of unity and that binding is driven by solvation entropy and opposed by enthalpy. Upon IIAGlcbinding, the conformational entropy of LacY is restrained, which leads to a significant decrease in sugar affinity. By suppressing conformational dynamics, IIAGlcblocks inducer entry into cells and favors constitutive glucose uptake and utilization. Furthermore, the studies support the notion that sugar binding involves an induced-fit mechanism that is inhibited by IIAGlcbinding. The precise mechanism of the inhibition of LacY by IIAGlcelucidated by ITC differs from the inhibition of melibiose permease (MelB), supporting the idea that permeases can differ in their thermodynamic response to binding IIAGlc.

A Novel Role for Enzyme I of the Vibrio cholerae Phosphoenolpyruvate Phosphotransferase System in Regulation of Growth in a Biofilm

10.1128/jb.01410-07 ◽

2007 ◽

Vol 190 (1) ◽

pp. 311-320 ◽

Cited By ~ 56

Author(s):

Laetitia Houot ◽

Paula I. Watnick

Keyword(s):

Vibrio Cholerae ◽

Sugar Transport ◽

Potent Inducer ◽

Inducer Exclusion ◽

Genes Encoding ◽

Cell Components ◽

Specific Regulation ◽

Enzyme I

ABSTRACT Glucose is a universal energy source and a potent inducer of surface colonization for many microbial species. Highly efficient sugar assimilation pathways ensure successful competition for this preferred carbon source. One such pathway is the phosphoenolpyruvate phosphotransferase system (PTS), a multicomponent sugar transport system that phosphorylates the sugar as it enters the cell. Components required for transport of glucose through the PTS include enzyme I, histidine protein, enzyme IIAGlc, and enzyme IIBCGlc. In Escherichia coli, components of the PTS fulfill many regulatory roles, including regulation of nutrient scavenging and catabolism, chemotaxis, glycogen utilization, catabolite repression, and inducer exclusion. We previously observed that genes encoding the components of the Vibrio cholerae PTS were coregulated with the vps genes, which are required for synthesis of the biofilm matrix exopolysaccharide. In this work, we identify the PTS components required for transport of glucose and investigate the role of each of these components in regulation of biofilm formation. Our results establish a novel role for the phosphorylated form of enzyme I in specific regulation of biofilm-associated growth. As the PTS is highly conserved among bacteria, the enzyme I regulatory pathway may be relevant to a number of biofilm-based infections.

The Escherichia coli BarA-UvrY Two-Component System Is Needed for Efficient Switching between Glycolytic and Gluconeogenic Carbon Sources

10.1128/jb.185.3.843-853.2003 ◽

2003 ◽

Vol 185 (3) ◽

pp. 843-853 ◽

Cited By ~ 74

Author(s):

Anna-Karin Pernestig ◽

Dimitris Georgellis ◽

Tony Romeo ◽

Kazushi Suzuki ◽

Henrik Tomenius ◽

...

Keyword(s):

Escherichia Coli ◽

Response Regulator ◽

Carbon Sources ◽

Regulation System ◽

Wild Type ◽

Two Component System ◽

Kinase Gene ◽

Component System ◽

E Coli ◽

Two Component

ABSTRACT The Escherichia coli BarA and UvrY proteins were recently demonstrated to constitute a novel two-component system, although its function has remained largely elusive. Here we show that mutations in the sensor kinase gene, barA, or the response regulator gene, uvrY, in uropathogenic E. coli drastically affect survival in long-term competition cultures. Using media with gluconeogenic carbon sources, the mutants have a clear growth advantage when competing with the wild type, but using media with carbon sources feeding into the glycolysis leads to a clear growth advantage for the wild type. Results from competitions with mutants in the carbon storage regulation system, CsrA/B, known to be a master switch between glycolysis and gluconeogenesis, led us to propose that the BarA-UvrY two-component system controls the Csr system. Taking these results together, we propose the BarA-UvrY two-component system is crucial for efficient adaptation between different metabolic pathways, an essential function for adaptation to a new environment.

Phosphorylation of HPr by the Bifunctional HPr Kinase/P-Ser-HPr Phosphatase from Lactobacillus casei Controls Catabolite Repression and Inducer Exclusion but Not Inducer Expulsion

10.1128/jb.182.9.2582-2590.2000 ◽

2000 ◽

Vol 182 (9) ◽

pp. 2582-2590 ◽

Cited By ~ 60

Author(s):

Valérie Dossonnet ◽

Vicente Monedero ◽

Monique Zagorec ◽

Anne Galinier ◽

Gaspar Pérez-Martínez ◽

...

Keyword(s):

Catabolite Repression ◽

Type Strain ◽

Lactobacillus Casei ◽

Wild Type Strain ◽

Carbon Sources ◽

Lag Phase ◽

Wild Type ◽

Inducer Exclusion ◽

In The Wild

ABSTRACT We have cloned and sequenced the Lactobacillus casei hprK gene encoding the bifunctional enzyme HPr kinase/P-Ser-HPr phosphatase (HprK/P). Purified recombinant L. casei HprK/P catalyzes the ATP-dependent phosphorylation of HPr, a phosphocarrier protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system at the regulatory Ser-46 as well as the dephosphorylation of seryl-phosphorylated HPr (P-Ser-HPr). The two opposing activities of HprK/P were regulated by fructose-1,6-bisphosphate, which stimulated HPr phosphorylation, and by inorganic phosphate, which stimulated the P-Ser-HPr phosphatase activity. A mutant producing truncated HprK/P was found to be devoid of both HPr kinase and P-Ser-HPr phosphatase activities. When hprK was inactivated, carbon catabolite repression of N-acetylglucosaminidase disappeared, and the lag phase observed during diauxic growth of the wild-type strain on media containing glucose plus either lactose or maltose was strongly diminished. In addition, inducer exclusion exerted by the presence of glucose on maltose transport in the wild-type strain was abolished in the hprK mutant. However, inducer expulsion ofmethyl β-d-thiogalactoside triggered by rapidly metabolizable carbon sources was still operative inptsH mutants altered at Ser-46 of HPr and thehprK mutant, suggesting that, in contrast to the model proposed for inducer expulsion in gram-positive bacteria, P-Ser-HPr might not be involved in this regulatory process.

Dual Role of Response Regulator StyR in Styrene Catabolism Regulation

Applied and Environmental Microbiology ◽

10.1128/aem.71.9.5411-5419.2005 ◽

2005 ◽

Vol 71 (9) ◽

pp. 5411-5419 ◽

Cited By ~ 22

Author(s):

Livia Leoni ◽

Giordano Rampioni ◽

Valeria Di Stefano ◽

Elisabetta Zennaro

Keyword(s):

Binding Site ◽

Glucose Repression ◽

Response Regulator ◽

Regulatory Element ◽

Carbon Sources ◽

Growth Conditions ◽

Integration Host Factor ◽

Two Component System ◽

Positive Role ◽

Proposed Model

ABSTRACT In Pseudomonas fluorescens ST, the promoter of the styrene catabolic operon, PstyA, is induced by styrene and repressed by the addition of preferred carbon sources. PstyA is regulated by the StyS/StyR two-component system. The integration host factor (IHF) also plays a positive role in PstyA regulation. Three distinct StyR binding sites, which have different affinities for this response regulator, have been characterized on PstyA. The high-affinity StyR binding site (STY2) is necessary for promoter activity. The DNA region upstream of STY2 contains a lower-affinity StyR binding site, STY1, that partially overlaps the IHF binding site. Deletion of this region, designated URE (upstream regulatory element), has a dual effect on the PstyA promoter, decreasing the styrene-dependent activity and partially relieving the glucose repression. The lowest-affinity StyR binding site (STY3) is located downstream of the transcription start point. Deletion of the URE region and inactivation of the STY3 site completely abolished glucose-mediated repression of PstyA. In the proposed model StyR can act either as an activator or as a repressor, depending on which sites it occupies in the different growth conditions. We suggest that the cellular levels of phosphorylated StyR, as determined by StyS sensor kinase activity, and the interplay of this molecule with IHF modulate the activity of the promoter in different growth conditions.

The Response Regulator SprE (RssB) Modulates Polyadenylation and mRNA Stability in Escherichia coli

10.1128/jb.00870-09 ◽

2009 ◽

Vol 191 (22) ◽

pp. 6812-6821 ◽

Cited By ~ 11

Author(s):

Valerie J. Carabetta ◽

Bijoy K. Mohanty ◽

Sidney R. Kushner ◽

Thomas J. Silhavy

Keyword(s):

Escherichia Coli ◽

Response Regulator ◽

Intracellular Localization ◽

Carbon Sources ◽

Adaptor Protein ◽

Deletion Mutants ◽

E Coli ◽

Specific Mrnas ◽

The Stability ◽

Polymerase I

ABSTRACT In Escherichia coli, the adaptor protein SprE (RssB) controls the stability of the alternate sigma factor RpoS (σ38 and σS). When nutrients are abundant, SprE binds RpoS and delivers it to ClpXP for degradation, but when carbon sources are depleted, this process is inhibited. It also has been noted that overproduction of SprE is toxic. Here we show that null mutations in pcnB, encoding poly(A) polymerase I (PAP I), and in hfq, encoding the RNA chaperone Hfq, suppress this toxicity. Since PAP I, in conjunction with Hfq, is responsible for targeting RNAs, including mRNAs, for degradation by adding poly(A) tails onto their 3′ ends, these data indicate that SprE helps modulate the polyadenylation pathway in E. coli. Indeed, in exponentially growing cells, sprE deletion mutants exhibit significantly reduced levels of polyadenylation and increased stability of specific mRNAs, similar to what is observed in a PAP I-deficient strain. In stationary phase, we show that SprE changes the intracellular localization of PAP I. Taken together, we propose that SprE plays a multifunctional role in controlling the transcriptome, regulating what is made via its effects on RpoS, and modulating what is degraded via its effects on polyadenylation and turnover of specific mRNAs.

The General Phosphotransferase System Proteins Localize to Sites of Strong Negative Curvature in Bacterial Cells

mBio ◽

10.1128/mbio.00443-13 ◽

2013 ◽

Vol 4 (5) ◽

Cited By ~ 24

Author(s):

Sutharsan Govindarajan ◽

Yair Elisha ◽

Keren Nevo-Dinur ◽

Orna Amster-Choder

Keyword(s):

Escherichia Coli ◽

Negative Curvature ◽

Carbon Sources ◽

Bacterial Cells ◽

Content Type ◽

E Coli ◽

Negatively Curved ◽

Subcellular Domains ◽

Geometric Cues

ABSTRACTThe bacterial cell poles are emerging as subdomains where many cellular activities take place, but the mechanisms for polar localization are just beginning to unravel. The general phosphotransferase system (PTS) proteins, enzyme I (EI) and HPr, which control preferential use of carbon sources in bacteria, were recently shown to localize near theEscherichia colicell poles. Here, we show that EI localization does not depend on known polar constituents, such as anionic lipids or the chemotaxis receptors, and on the cell division machinery, nor can it be explained by nucleoid occlusion or localized translation. Detection of the general PTS proteins at the budding sites of endocytotic-like membrane invaginations in spherical cells and their colocalization with the negative curvature sensor protein DivIVA suggest that geometric cues underlie localization of the PTS system. Notably, the kinetics of glucose uptake by spherical and rod-shapedE. colicells are comparable, implying that negatively curved “pole-like” sites support not only the localization but also the proper functioning of the PTS system in cells with different shapes. Consistent with the curvature-mediated localization model, we observed the EI protein fromBacillus subtilisat strongly curved sites in bothB. subtilisandE. coli. Taken together, we propose that changes in cell architecture correlate with dynamic survival strategies that localize central metabolic systems like the PTS to subcellular domains where they remain active, thus maintaining cell viability and metabolic alertness.IMPORTANCEDespite their tiny size and the scarcity of membrane-bounded organelles, bacteria are capable of sorting macromolecules to distinct subcellular domains, thus optimizing functionality of vital processes. Understanding the cues that organize bacterial cells should provide novel insights into the complex organization of higher organisms. Previously, we have shown that the general proteins of the phosphotransferase system (PTS) signaling system, which governs utilization of carbon sources in bacteria, localize to the poles ofEscherichia colicells. Here, we show that geometric cues, i.e., strong negative membrane curvature, mediate positioning of the PTS proteins. Furthermore, localization to negatively curved regions seems to support the PTS functionality.