The CreC Regulator of Escherichia coli, a New Target for Metabolic Manipulations

ABSTRACTThe CreBC (carbon source-responsive) two-component regulation system ofEscherichia coliaffects a number of functions, including intermediary carbon catabolism. The impacts of differentcreCmutations (a ΔcreCmutant and a mutant carrying the constitutivecreC510allele) on bacterial physiology were analyzed in glucose cultures under three oxygen availability conditions. Differences in the amounts of extracellular metabolites produced were observed in the null mutant compared to the wild-type strain and the mutant carryingcreC510and shown to be affected by oxygen availability. The ΔcreCstrain secreted more formate, succinate, and acetate but less lactate under low aeration. These metabolic changes were associated with differences in AckA and LdhA activities, both of which were affected by CreC. Measurement of the NAD(P)H/NAD(P)+ratios showed that thecreC510strain had a more reduced intracellular redox state, while the opposite was observed for the ΔcreCmutant, particularly under intermediate oxygen availability conditions, indicating that CreC affects redox balance. The null mutant formed more succinate than the wild-type strain under both low aeration and no aeration. Overexpression of the genes encoding phosphoenolpyruvate carboxylase fromE. coliand a NADH-forming formate dehydrogenase fromCandida boidiniiin the ΔcreCmutant further increased the yield of succinate on glucose. Interestingly, the elimination ofackAandadhEdid not significantly improve the production of succinate. The diverse metabolic effects of this regulator on the central biochemical network ofE. colimake it a good candidate for metabolic-engineering manipulations to enhance the formation of bioproducts, such as succinate.

Download Full-text

Genetic Analysis of the Role ofyfiRin the Ability of Escherichia coli CFT073 To Control Cellular Cyclic Dimeric GMP Levels and To Persist in the Urinary Tract

Infection and Immunity ◽

10.1128/iai.01396-12 ◽

2013 ◽

Vol 81 (9) ◽

pp. 3089-3098 ◽

Cited By ~ 20

Author(s):

Erica L. Raterman ◽

Daniel D. Shapiro ◽

Daniel J. Stevens ◽

Kevin J. Schwartz ◽

Rodney A. Welch

Keyword(s):

Escherichia Coli ◽

Urinary Tract ◽

Type Strain ◽

Wild Type Strain ◽

Wild Type ◽

Cellulose Production ◽

Content Type ◽

E Coli ◽

Successful Infection ◽

Tract Infections

ABSTRACTDuring urinary tract infections (UTIs), uropathogenicEscherichia colimust maintain a delicate balance between sessility and motility to achieve successful infection of both the bladder and kidneys. Previous studies showed that cyclic dimeric GMP (c-di-GMP) levels aid in the control of the transition between motile and nonmotile states inE. coli. TheyfiRNBlocus inE. coliCFT073 contains genes for YfiN, a diguanylate cyclase, and its activity regulators, YfiR and YfiB. Deletion ofyfiRyielded a mutant that was attenuated in both the bladder and the kidneys when tested in competition with the wild-type strain in the murine model of UTI. A doubleyfiRNmutant was not attenuated in the mouse model, suggesting that unregulated YfiN activity and likely increased cytoplasmic c-di-GMP levels cause a survival defect. Curli fimbriae and cellulose production were increased in theyfiRmutant. Expression ofyhjH, a gene encoding a proven phosphodiesterase, in CFT073 ΔyfiRsuppressed the overproduction of curli fimbriae and cellulose and further verified that deletion ofyfiRresults in c-di-GMP accumulation. Additional deletion ofcsgDandbcsA, genes necessary for curli fimbriae and cellulose production, respectively, returned colonization levels of theyfiRdeletion mutant to wild-type levels. Peroxide sensitivity assays and iron acquisition assays displayed no significant differences between theyfiRmutant and the wild-type strain. These results indicate that dysregulation of c-di-GMP production results in pleiotropic effects that disableE. coliin the urinary tract and implicate the c-di-GMP regulatory system as an important factor in the persistence of uropathogenicE. coli in vivo.

Download Full-text

Fumarate-Mediated Persistence of Escherichia coli against Antibiotics

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01794-15 ◽

2016 ◽

Vol 60 (4) ◽

pp. 2232-2240 ◽

Cited By ~ 19

Author(s):

Jun-Seob Kim ◽

Da-Hyeong Cho ◽

Paul Heo ◽

Suk-Chae Jung ◽

Myungseo Park ◽

...

Keyword(s):

Escherichia Coli ◽

Type Strain ◽

Molecular Mechanisms ◽

Wild Type Strain ◽

Wild Type ◽

Content Type ◽

E Coli ◽

Quiescent Cells ◽

Transport Chain ◽

Genetic Perturbations

ABSTRACTBacterial persisters are a small fraction of quiescent cells that survive in the presence of lethal concentrations of antibiotics. They can regrow to give rise to a new population that has the same vulnerability to the antibiotics as did the parental population. Although formation of bacterial persisters in the presence of various antibiotics has been documented, the molecular mechanisms by which these persisters tolerate the antibiotics are still controversial. We found that amplification of the fumarate reductase operon (FRD) inEscherichia coliled to a higher frequency of persister formation. The persister frequency ofE. coliwas increased when the cells contained elevated levels of intracellular fumarate. Genetic perturbations of the electron transport chain (ETC), a metabolite supplementation assay, and even the toxin-antitoxin-relatedhipA7mutation indicated that surplus fumarate markedly elevated theE. colipersister frequency. AnE. colistrain lacking succinate dehydrogenase (SDH), thereby showing a lower intracellular fumarate concentration, was killed ∼1,000-fold more effectively than the wild-type strain in the stationary phase. It appears thatSDHandFRDrepresent a paired system that gives rise to and maintainsE. colipersisters by producing and utilizing fumarate, respectively.

Download Full-text

Finding Regulators Associated with the Expression of the Long Polar Fimbriae in Enteropathogenic Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.00509-15 ◽

2015 ◽

Vol 197 (23) ◽

pp. 3658-3665 ◽

Cited By ~ 2

Author(s):

Jia Hu ◽

Brittany N. Ross ◽

Roberto J. Cieza ◽

Alfredo G. Torres

Keyword(s):

Escherichia Coli ◽

Type Strain ◽

Wild Type Strain ◽

Wild Type ◽

Content Type ◽

E Coli ◽

Colonization Factor ◽

Initial Adhesion ◽

Intestinal Adhesion

ABSTRACTEnteropathogenicEscherichia coli(EPEC) is a human pathogen that requires initial adhesion to the intestine in order to cause disease. Multiple adhesion factors have been identified inE. colistrains, among them the long polar fimbriae (Lpf), a colonization factor associated with intestinal adhesion. The conditions of Lpf expression are well understood in enterohemorrhagicE. coli(EHEC); however, the expression of EPEClpfhas been found to be repressed under anyin vitrocondition tested. Therefore, we decided to identify those factors silencing expression of EPEClpf. Because histone-like nucleoid structuring protein (H-NS) is a known repressor of EHEClpf, we tested it and found that H-NS is a repressor of EPEClpf. We also found that the adhesion of the EPEC Δhnsstrain was significantly enhanced compared to the wild-type strain. Becauselpfexpression was modestly increased in thehnsmutant, transposon mutagenesis was performed to find a strain displaying higherlpfexpression than EPEC Δhns. One Tn5insertion was identified within theyhjXgene, and furtherin vitrocharacterization revealed increasedlpfexpression and adhesion to Caco-2 cells compared with EPEC Δhns. However, in a murine model of intestinal infection, the EPEC Δhnsand EPEC ΔhnsTn5mutants had only a slight change in colonization pattern compared to the wild-type strain. Our data showed that EPEC Lpf is transcribed, but its role in EPEC intestinal colonization requires further analysis.IMPORTANCEData are presented demonstrating that the long polar fimbriae (lpf) operon in enteropathogenicE. coli(EPEC) is highly regulated; however, derepression occurs by mutagenizing two proteins associated with its control. The study demonstrates that the EPEClpfoperon can be expressed and, therefore, participates in the EPEC adherence phenotype.

Download Full-text

The Small RNA RyhB Contributes to Siderophore Production and Virulence of Uropathogenic Escherichia coli

Infection and Immunity ◽

10.1128/iai.02287-14 ◽

2014 ◽

Vol 82 (12) ◽

pp. 5056-5068 ◽

Cited By ~ 34

Author(s):

Gaëlle Porcheron ◽

Rima Habib ◽

Sébastien Houle ◽

Mélissa Caza ◽

François Lépine ◽

...

Keyword(s):

Oxidative Stress ◽

Escherichia Coli ◽

Urinary Tract ◽

Type Strain ◽

Iron Homeostasis ◽

Wild Type Strain ◽

Iron Acquisition ◽

Wild Type ◽

Content Type ◽

E Coli

ABSTRACTInEscherichia coli, the small regulatory noncoding RNA (sRNA) RyhB and the global ferric uptake regulator (Fur) mediate iron acquisition and storage control. Iron is both essential and potentially toxic for most living organisms, making the precise maintenance of iron homeostasis necessary for survival. While the roles of these regulators in iron homeostasis have been well studied in a nonpathogenicE. colistrain, their impact on the production of virulence-associated factors is still unknown for a pathogenicE. colistrain. We thus investigated the roles of RyhB and Fur in iron homeostasis and virulence of the uropathogenicE. coli(UPEC) strain CFT073. In a murine model of urinary tract infection (UTI), deletion offuralone did not attenuate virulence, whereas a ΔryhBmutant and a ΔfurΔryhBdouble mutant showed significantly reduced bladder colonization. The Δfurmutant was more sensitive to oxidative stress and produced more of the siderophores enterobactin, salmochelins, and aerobactin than the wild-type strain. In contrast, while RyhB was not implicated in oxidative stress resistance, the ΔryhBmutant produced lower levels of siderophores. This decrease was correlated with the downregulation ofshiA(encoding a transporter of shikimate, a precursor of enterobactin and salmochelin biosynthesis) andiucD(involved in aerobactin biosynthesis) in this mutant grown in minimal medium or in human urine.iucDwas also downregulated in bladders infected with the ΔryhBmutant compared to those infected with the wild-type strain. Our results thus demonstrate that the sRNA RyhB is involved in production of iron acquisition systems and colonization of the urinary tract by pathogenicE. coli.

Download Full-text

Quorum Sensing Is a Global Regulatory Mechanism in Enterohemorrhagic Escherichia coli O157:H7

Journal of Bacteriology ◽

10.1128/jb.183.17.5187-5197.2001 ◽

2001 ◽

Vol 183 (17) ◽

pp. 5187-5197 ◽

Cited By ~ 302

Author(s):

Vanessa Sperandio ◽

Alfredo G. Torres ◽

Jorge A. Girón ◽

James B. Kaper

Keyword(s):

Escherichia Coli ◽

Quorum Sensing ◽

Type Strain ◽

Wild Type Strain ◽

Regulatory Mechanism ◽

Sos Response ◽

Enterohemorrhagic Escherichia Coli ◽

Trna Genes ◽

Wild Type ◽

E Coli

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is responsible for outbreaks of bloody diarrhea and hemolytic-uremic syndrome in many countries. EHEC virulence mechanisms include the production of Shiga toxins (Stx) and formation of attaching and effacing (AE) lesions on intestinal epithelial cells. We recently reported that genes involved in the formation of the AE lesion were regulated by quorum sensing through autoinducer-2, which is synthesized by the product of the luxS gene. In this study we hybridized an E. coli gene array with cDNA synthesized from RNA that was extracted from EHEC strain 86-24 and its isogenicluxS mutant. We observed that 404 genes were regulated by luxS at least fivefold, which comprises approximately 10% of the array genes; 235 of these genes were up-regulated and 169 were down-regulated in the wild-type strain compared to in theluxS mutant. Down-regulated genes included several involved in cell division, as well as ribosomal and tRNA genes. Consistent with this pattern of gene expression, theluxS mutant grows faster than the wild-type strain (generation times of 37.5 and 60 min, respectively, in Dulbecco modified Eagle medium). Up-regulated genes included several involved in the expression and assembly of flagella, motility, and chemotaxis. Using operon::lacZ fusions to class I, II, and III flagellar genes, we were able to confirm this transcriptional regulation. We also observed fewer flagella by Western blotting and electron microscopy and decreased motility halos in semisolid agar in the luxS mutant. The average swimming speeds for the wild-type strain and the luxS mutant are 12.5 and 6.6 μm/s, respectively. We also observed an increase in the production of Stx due to quorum sensing. Genes encoding Stx, which are transcribed along with λ-like phage genes, are induced by an SOS response, and genes involved in the SOS response were also regulated by quorum sensing. These results indicate that quorum sensing is a global regulatory mechanism for basic physiological functions of E. coli as well as for virulence factors.

Download Full-text

The RcsCB His-Asp Phosphorelay System Is Essential To Overcome Chlorpromazine-Induced Stress in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.184.10.2850-2853.2002 ◽

2002 ◽

Vol 184 (10) ◽

pp. 2850-2853 ◽

Cited By ~ 39

Author(s):

Annie Conter ◽

Rachel Sturny ◽

Claude Gutierrez ◽

Kaymeuang Cam

Keyword(s):

Escherichia Coli ◽

Type Strain ◽

Wild Type Strain ◽

Cell Envelope ◽

Wild Type ◽

E Coli ◽

Induced Stress ◽

Mutant Strains ◽

Molecular Nature

ABSTRACT The RcsCB His-Asp phosphorelay system regulates the expression of several genes of Escherichia coli, but the molecular nature of the inducing signal is still unknown. We show here that treatment of an exponentially growing culture of E. coli with the cationic amphipathic compound chlorpromazine (CPZ) stimulates expression of a set of genes positively regulated by the RcsCB system. This induction is abolished in rcsB or rcsC mutant strains. In addition, treatment with CPZ inhibits growth. The wild-type strain is able to recover from this inhibition and resume growth after a period of adaptation. In contrast, strains deficient in the RcsCB His-Asp phosphorelay system are hypersensitive to CPZ. These results suggest that cells must express specific RcsCB-regulated genes in order to cope with the CPZ-induced stress. This is the first report of the essential role of the RcsCB system in a stress situation. These results also strengthen the notion that alterations of the cell envelope induce a signal recognized by the RcsC sensor.

Download Full-text

Expression of Different-Size Transcripts from theclpP-clpX Operon of Escherichia coli during Carbon Deprivation

Journal of Bacteriology ◽

10.1128/jb.182.23.6630-6637.2000 ◽

2000 ◽

Vol 182 (23) ◽

pp. 6630-6637 ◽

Cited By ~ 13

Author(s):

Chin Li ◽

Yi Ping Tao ◽

Lee D. Simon

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Type Strain ◽

Wild Type Strain ◽

Transcription Termination ◽

Carbon Starvation ◽

Lower Percentage ◽

Wild Type ◽

Lower Efficiency ◽

E Coli

ABSTRACT Transcription of the clpP-clpX operon ofEscherichia coli leads to the production of two different sizes of transcripts. In log phase, the level of the longer transcript is higher than the level of the shorter transcript. Soon after the onset of carbon starvation, the level of the shorter transcript increases significantly, and the level of the longer transcript decreases. The longer transcript consists of the entireclpP-clpX operon, whereas the shorter transcript contains the entire clpP gene but none of the clpXcoding sequence. The RpoH protein is required for the increase in the level of the shorter transcript during carbon starvation. Primer extension experiments suggest that there is increased usage of the ς32-dependent promoter of the clpP-clpXoperon within 15 min after the start of carbon starvation. Expression of the clpP-clpX operon from the promoters upstream of theclpP gene decreases to a very low level by 20 min after the onset of carbon starvation. Various pieces of evidence suggest, though they do not conclusively prove, that production of the shorter transcript may involve premature termination of the longer transcript. The half-life of the shorter transcript is much less than that of the longer transcript during carbon starvation. E. coli rpoBmutations that affect transcription termination efficiency alter the ratio of the shorter clpP-clpX transcript to the longer transcript. The E. coli rpoB3595 mutant, with an RNA polymerase that terminates transcription with lower efficiency than the wild type, accumulates a lower percentage of the shorter transcript during carbon starvation than does the isogenic wild-type strain. In contrast, the rpoB8 mutant, with an RNA polymerase that terminates transcription with higher efficiency than the wild type, produces a higher percentage of the shorter clpP-clpXtranscript when E. coli is in log phase. These and other data are consistent with the hypothesis that the shorter transcript results from premature transcription termination during production of the longer transcript.

Download Full-text

DOSEc, a Heme-Regulated Phosphodiesterase, Plays an Important Role in the Regulation of the Cyclic AMP Level in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.187.19.6678-6682.2005 ◽

2005 ◽

Vol 187 (19) ◽

pp. 6678-6682 ◽

Cited By ~ 16

Author(s):

Tokiko Yoshimura-Suzuki ◽

Ikuko Sagami ◽

Nao Yokota ◽

Hirofumi Kurokawa ◽

Toru Shimizu

Keyword(s):

Escherichia Coli ◽

Cyclic Amp ◽

Type Strain ◽

Redox State ◽

Wild Type Strain ◽

Functional Domain ◽

Wild Type ◽

Aerobic Conditions ◽

E Coli ◽

Knockout Strain

ABSTRACT Heme-regulated phosphodiesterase from Escherichia coli (DOSEc) catalyzes the hydrolysis of cyclic AMP (cAMP) in vitro and is regulated by the redox state of the bound heme. Changes in the redox state result in alterations in the three-dimensional structure of the enzyme, which is then transmitted to the functional domain to switch catalysis on or off. Because DOSEc was originally cloned from E. coli genomic DNA, it has not been known whether it is actually expressed in wild-type E. coli. In addition, the turnover number of DOSEc using cAMP as a substrate is only 0.15 min−1, which is relatively low for a physiologically relevant enzyme. In the present study, we demonstrated for the first time that the DOSEc gene and protein are expressed in wild-type E. coli, especially under aerobic conditions. We also developed a DOSEc gene knockout strain (Δdos). Interestingly, the knockout of dos caused excess accumulation of intracellular cAMP (26-fold higher than in the wild-type strain) under aerobic conditions, whereas accumulation of cAMP was not observed under anaerobic conditions. We also found differences in cell morphology and growth rate between the mutant cells and the wild-type strain. The changes in the knockout strain were partially complemented by introducing an expression plasmid for dos. Thus, the present study revealed that expression of DOSEc is regulated according to environmental O2 availability at the transcriptional level and that the concentration of cAMP in cells is regulated by DOSEc expression.

Download Full-text

Escherichia coliO157:H7 responds to phosphate starvation by modifying LPS involved in biofilm formation

10.1101/536201 ◽

2019 ◽

Cited By ~ 3

Author(s):

Philippe Vogeleer ◽

Antony T. Vincent ◽

Samuel M. Chekabab ◽

Steve J. Charette ◽

Alexey Novikov ◽

...

Keyword(s):

Escherichia Coli ◽

Biofilm Formation ◽

Inorganic Phosphate ◽

Type Strain ◽

Wild Type Strain ◽

Pho Regulon ◽

Wild Type ◽

E Coli ◽

Pi Starvation ◽

Pst System

ABSTRACTIn open environments such as water, enterohemorrhagicEscherichia coliO157:H7 responds to inorganic phosphate (Pi) starvation by inducing the Pho regulon controlled by PhoB. The phosphate-specific transport (Pst) system is the high-affinity Pi transporter. In the Δpstmutant, PhoB is constitutively activated and regulates the expression of genes from the Pho regulon. InE. coliO157:H7, the Δpstmutant, biofilm, and autoagglutination were increased. In the double-deletion mutant ΔpstΔphoB, biofilm and autoagglutination were similar to the wild-type strain, suggesting that PhoB is involved. We investigated the relationship between PhoB activation and enhanced biofilm formation by screening a transposon mutant library derived from Δpstmutant for decreased autoagglutination and biofilms mutants. Lipopolysaccharide (LPS) genes involved in the synthesis of the LPS core were identified. Transcriptomic studies indicate the influence of Pi-starvation andpstmutation on LPS biosynthetic gene expression. LPS analysis indicated that the O-antigen was deficient in the Δpstmutant. Interestingly,waaH, encoding a glycosyltransferase associated with LPS modifications inE. coliK-12, was highly expressed in the Δpstmutant ofE. coliO157:H7. Deletion ofwaaHfrom the Δpstmutant and from the wild-type strain grown in Pi-starvation conditions decreased the biofilm formation but without affecting LPS. Our findings suggest that LPS core is involved in the autoagglutination and biofilm phenotypes of the Δpstmutant and that WaaH plays a role in biofilm in response to Pi-starvation. This study highlights the importance of Pi-starvation in biofilm formation of E. coli O157:H7, which may affect its transmission and persistence.IMPORTANCEEnterohemorrhagicEscherichia coliO157:H7 is a human pathogen responsible for bloody diarrhea and renal failures. In the environment, O157:H7 can survive for prolonged periods of time under nutrient-deprived conditions. Biofilms are thought to participate in this environmental lifestyle. Previous reports have shown that the availability of extracellular inorganic phosphate (Pi) affected bacterial biofilm formation; however, nothing was known about O157:H7 biofilm formation. Our results show that O157:H7 membrane undergoes modifications upon PhoB activation leading to increased biofilm formation. A mutation in the Pst system results in reduced amount of the smooth type LPS and that this could influence the biofilm composition. This demonstrates how theE. coliO157:H7 adapts to Pi starvation increasing its ability to occupy different ecological niches.

Download Full-text

The dependence of Escherichia coli asparaginase II formation on cyclic AMP and cyclic AMP receptor protein

Canadian Journal of Microbiology ◽

10.1139/m78-104 ◽

1978 ◽

Vol 24 (5) ◽

pp. 629-631 ◽

Cited By ~ 3

Author(s):

La Verne Russell ◽

Hiroshi Yamazaki

Keyword(s):

Escherichia Coli ◽

Cyclic Amp ◽

Type Strain ◽

Wild Type Strain ◽

Anaerobic Growth ◽

Receptor Protein ◽

Wild Type ◽

Cyclic Amp Receptor Protein ◽

E Coli ◽

Cyclic Amp Synthesis

The amount of asparaginase II in an Escherichia coli wild-type strain (cya+, crp+) markedly increased upon a shift from aerobic to anaerobic growth. However, no such increase occurred in a mutant (cya) lacking cyclic AMP synthesis unless supplemented with exogenous cyclic AMP. Since a mutant (crp) deficient in cyclic AMP receptor protein also did not support the anaerobic formation of this enzyme, it is concluded that the formation of E. coli asparaginase II depends on both cyclic AMP and cyclic AMP receptor protein.

Download Full-text