Control of Transposon-Mediated Directed Mutation by the Escherichia coli Phosphoenolpyruvate:Sugar Phosphotransferase System

The phosphoenolpyruvate:sugar phosphotransferase system (PTS) has been shown to control transport, cell metabolism and gene expression. We here present results supporting the novel suggestion that in certain instances it also regulates the mutation rate. Directed mutations are defined as mutations that occur at higher frequencies when beneficial than when neutral or detrimental. To date, the occurrence of directed point mutations has not been documented and confirmed, but a few examples of transposon-mediated directed mutations have been reported. Here we focus on the first and best-studied example of directed mutation, which involves the regulation of insertion sequence-5 hopping into a specific site upstream of the glpFK glycerol utilization operon in Escherichia coli. This insertional event specifically activates expression of the glpFK operon, allowing the growth of wild-type cells with glycerol as a carbon source in the presence of nonmetabolizable glucose analogues which normally block glycerol utilization. The sugar-transporting PTS controls this process by regulating levels of cytoplasmic glycerol-3-phosphate and cyclic (c)AMP as established in previous publications. Direct involvement of the glycerol repressor, GlpR, and the cAMP receptor protein, Crp, in the regulation of transposon-mediated directed mutation has been demonstrated.

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A Global Regulatory Role of Gluconeogenic Genes in Escherichia coli Revealed by Transcriptome Network Analysis

Journal of Biological Chemistry ◽

10.1074/jbc.m508202200 ◽

2005 ◽

Vol 280 (43) ◽

pp. 36079-36087 ◽

Cited By ~ 57

Author(s):

Katy C. Kao ◽

Linh M. Tran ◽

James C. Liao

Keyword(s):

Escherichia Coli ◽

Dynamic Environment ◽

Regulatory System ◽

Underlying Mechanism ◽

Receptor Protein ◽

Phosphotransferase System ◽

Global Regulators ◽

Transcription Regulators ◽

Camp Receptor ◽

Global Regulatory System

In bacterial adaptation to the dynamic environment, metabolic genes are typically thought to be the executors, whereas global transcription regulators are regarded as the decision makers. Although the feedback from metabolic consequence is believed to be important, much less is understood. This work demonstrates that the gluconeogenic genes in Escherichia coli, ppsA, sfcA, and maeB, provide a feedback loop to the global regulator, cAMP receptor protein (CRP), in carbon source transition. Disruption of one of the gluconeogenic pathways has no phenotype in balanced growth, but causes a significant delay in the diauxic transition from glucose to acetate. To investigate the underlying mechanism, we measured the transcriptome profiles during the transition using DNA microarray, and network component analysis was employed to obtain the transcription factor activities. Results showed that one of the global regulators, CRP, was insufficiently activated during the transition in the ppsA deletion mutant. Indeed, addition of cAMP partially rescued the delay in transition. These results suggest that the gluconeogenic flux to phosphoenolpyruvate is important for full activation of adenylate cyclase through the phosphorylated enzyme IIAglu of the phosphotransferase system. Reduction of this flux causes insufficient activation of CRP and a global metabolic deficiency, which exemplifies a significant feedback interaction from metabolism to the a global regulatory system.

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Mutations That StimulateflhDCExpression in Escherichia coli K-12

Journal of Bacteriology ◽

10.1128/jb.00455-15 ◽

2015 ◽

Vol 197 (19) ◽

pp. 3087-3096 ◽

Cited By ~ 20

Author(s):

Karen A. Fahrner ◽

Howard C. Berg

Keyword(s):

Escherichia Coli ◽

Transcription Factors ◽

Insertion Sequence ◽

Receptor Protein ◽

Upstream Region ◽

Single Nucleotide ◽

Content Type ◽

Sequence Elements ◽

Camp Receptor ◽

K 12

ABSTRACTMotility is a beneficial attribute that enables cells to access and explore new environments and to escape detrimental ones. The organelle of motility inEscherichia coliis the flagellum, and its production is initiated by the activating transcription factors FlhD and FlhC. The expression of these factors by theflhDCoperon is highly regulated and influenced by environmental conditions. TheflhDCpromoter is recognized by σ70and is dependent on the transcriptional activator cyclic AMP (cAMP)-cAMP receptor protein complex (cAMP-CRP). A number of K-12 strains exhibit limited motility due to low expression levels offlhDC. We report here a large number of mutations that stimulateflhDCexpression in such strains. They include single nucleotide changes in the −10 element of the promoter, in the promoter spacer, and in the cAMP-CRP binding region. In addition, we show that insertion sequence (IS) elements or a kanamycin gene located hundreds of base pairs upstream of the promoter can effectively enhance transcription, suggesting that the topology of a large upstream region plays a significant role in the regulation offlhDCexpression. None of the mutations eliminated the requirement for cAMP-CRP for activation. However, several mutations allowed expression in the absence of the nucleoid organizing protein, H-NS, which is normally required forflhDCexpression.IMPORTANCETheflhDCoperon ofEscherichia coliencodes transcription factors that initiate flagellar synthesis, an energetically costly process that is highly regulated. Few deregulating mutations have been reported thus far. This paper describes new single nucleotide mutations that stimulateflhDCexpression, including a number that map to the promoter spacer region. In addition, this work shows that insertion sequence elements or a kanamycin gene located far upstream from the promoter or repressor binding sites also stimulate transcription, indicating a role of regional topology in the regulation offlhDCexpression.

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Novel mechanisms of TolC-independent decreased bile-salt susceptibility in Escherichia coli

FEMS Microbiology Letters ◽

10.1093/femsle/fnaa083 ◽

2020 ◽

Vol 367 (10) ◽

Author(s):

Vuong Van Hung Le ◽

Patrick J Biggs ◽

David Wheeler ◽

Ieuan G Davies ◽

Jasna Rakonjac

Keyword(s):

Escherichia Coli ◽

Efflux Pump ◽

Sodium Deoxycholate ◽

Coli Strain ◽

Receptor Protein ◽

Phosphotransferase System ◽

Multidrug Efflux Pump ◽

Knock Out ◽

E Coli ◽

Camp Receptor

ABSTRACT Bile salts, including sodium deoxycholate (DOC), are secreted into the intestine to aid fat digestion and contribute to antimicrobial protection. Gram-negative pathogens such as Escherichia coli, however, are highly resistant to DOC, using multiple mechanisms of which the multidrug efflux pump AcrAB-TolC is the dominant one. Given that TolC-mediated efflux masks the interaction of DOC with potential targets, we sought to identify those targets by identifying genes whose mutations cause an increase in the MIC to DOC relative to the ∆tolC parental strain, that lacks TolC-associated functional efflux pumps. Using a mutant screen, we isolated twenty independent spontaneous mutants that had a higher MICDOC than the E. coli parental ∆tolC strain. Whole genome sequencing of these mutants mapped most mutations to the ptsI or cyaA gene. Analysis of knock-out mutants and complementation showed that elimination of PtsI, a component of the carbohydrate phosphotransferase system, or one of the two key proteins involved in cAMP synthesis and signaling, adenylate cyclase (CyaA) or cAMP receptor protein (Crp) causes low-level increased resistance of a ∆tolC E. coli strain to DOC.

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Control of Transposon-mediated Activation of theglpFKOperon ofEscherichia coliby two DNA binding Proteins

10.1101/046649 ◽

2016 ◽

Author(s):

Zhongge Zhang ◽

Milton H. Saier

Keyword(s):

Insertion Sequence ◽

Gene Activation ◽

Regulatory Region ◽

Receptor Protein ◽

Upstream Region ◽

Upstream Regulatory Region ◽

Wild Type ◽

Transposon Insertion ◽

Glycerol Utilization ◽

Camp Receptor

AbstractEscherichia colicells deleted for the cyclic AMP (cAMP) receptor protein (Crp) gene (Δcrp) cannot utilize glycerol because cAMP-Crp is a required positive activator of glycerol utilization operonglpFK. We have previously shown that a transposon, Insertion Sequence 5 (IS5), can reversibly insert into the upstream regulatory region of the operon so as to activateglpFKand enable glycerol utilization. GlpR, which repressesglpFKtranscription, binds to theglpFKupstream region near the site of IS5insertion, and prevents insertion. We here show that the cAMP-Crp complex, which also binds to theglpFKupstream regulatory region, also inhibits IS5hopping into the activating site. This finding allowed us to identify conditions under which wild type cells can acquireglpFK-activating IS5insertions. Maximal rates of IS5insertion into the activating site require the presence of glycerol as well as a non-metabolizable sugar analogue that lowers cytoplasmic cAMP concentrations. Under these conditions, IS5insertional mutants accumulate and outcompete the wild type cells. Because of the widespread distribution of glucose analogues in nature, this mechanism of gene activation could have evolved by natural selection.

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Fluorescence quenching and kinetic studies of conformational changes induced by DNA and cAMP binding to cAMP receptor protein from Escherichia coli

FEBS Journal ◽

10.1111/j.1742-4658.2005.04540.x ◽

2005 ◽

Vol 272 (5) ◽

pp. 1103-1116 ◽

Cited By ~ 8

Author(s):

Magdalena Tworzydło ◽

Agnieszka Polit ◽

Jan Mikołajczak ◽

Zygmunt Wasylewski

Keyword(s):

Escherichia Coli ◽

Fluorescence Quenching ◽

Conformational Changes ◽

Kinetic Studies ◽

Receptor Protein ◽

Camp Receptor Protein ◽

Camp Receptor

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Heterologous cooperativity in Escherichia coli. The CytR repressor both contacts DNA and the cAMP receptor protein when binding to the deoP2 promoter.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)55198-x ◽

1991 ◽

Vol 266 (27) ◽

pp. 17804-17808

Author(s):

H. Pedersen ◽

L. Søgaard-Andersen ◽

B. Holst ◽

P. Valentin-Hansen

Keyword(s):

Escherichia Coli ◽

Receptor Protein ◽

Camp Receptor Protein ◽

Camp Receptor

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Interactions between the Escherichia coli cAMP receptor protein and the C-terminal domain of the α subunit of RNA polymerase at Class I promoters

Biochemical Journal ◽

10.1042/bj3370415 ◽

1999 ◽

Vol 337 (3) ◽

pp. 415-423 ◽

Cited By ~ 8

Author(s):

Emma C. LAW ◽

Nigel J. SAVERY ◽

Stephen J. W. BUSBY

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Transcription Initiation ◽

Class I ◽

Receptor Protein ◽

Camp Receptor Protein ◽

Α Subunit ◽

Terminal Domain ◽

Up Elements ◽

Camp Receptor

The Escherichia coli cAMP receptor protein (CRP) is a factor that activates transcription at over 100 target promoters. At Class I CRP-dependent promoters, CRP binds immediately upstream of RNA polymerase and activates transcription by making direct contacts with the C-terminal domain of the RNA polymerase α subunit (αCTD). Since αCTD is also known to interact with DNA sequence elements (known as UP elements), we have constructed a series of semi-synthetic Class I CRP-dependent promoters, carrying both a consensus DNA-binding site for CRP and a UP element at different positions. We previously showed that, at these promoters, the CRP–αCTD interaction and the CRP–UP element interaction contribute independently and additively to transcription initiation. In this study, we show that the two halves of the UP element can function independently, and that, in the presence of the UP element, the best location for the DNA site for CRP is position -69.5. This suggests that, at Class I CRP-dependent promoters where the DNA site for CRP is located at position -61.5, the two αCTDs of RNA polymerase are not optimally positioned. Two experiments to test this hypothesis are presented.

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

Canadian Journal of Microbiology ◽

10.1139/m80-251 ◽

1980 ◽

Vol 26 (12) ◽

pp. 1508-1511 ◽

Cited By ~ 8

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.

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