scholarly journals Inducible l-Alanine Exporter Encoded by the Novel GeneygaW(alaE) in Escherichia coli

2011 ◽  
Vol 77 (12) ◽  
pp. 4027-4034 ◽  
Author(s):  
Hatsuhiro Hori ◽  
Hiroshi Yoneyama ◽  
Ryuta Tobe ◽  
Tasuke Ando ◽  
Emiko Isogai ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Wild Type Strain ◽  
Chemical Mutagenesis ◽  
The Novel ◽  
Wild Type ◽  
Content Type ◽  
E Coli ◽  
Export Rate ◽  
In The Wild ◽  
Export System

ABSTRACTWe previously isolated a mutant hypersensitive tol-alanyl-l-alanine from a non-l-alanine-metabolizingEscherichia colistrain and found that it lacked an induciblel-alanine export system. Consequently, this mutant showed a significant accumulation of intracellularl-alanine and a reduction in thel-alanine export rate compared to the parent strain. When the mutant was used as a host to clone a gene(s) that complements the dipeptide-hypersensitive phenotype, two uncharacterized genes,ygaWandytfF, and two characterized genes,yddGandyeaS, were identified. Overexpression of each gene in the mutant resulted in a decrease in the intracellularl-alanine level and enhancement of thel-alanine export rate in the presence of the dipeptide, suggesting that their products function as exporters ofl-alanine. SinceygaWexhibited the most striking impact on both the intra- and the extracellularl-alanine levels among the four genes identified, we disrupted theygaWgene in the non-l-alanine-metabolizing strain. The resulting isogenic mutant showed the same intra- and extracellularl-alanine levels as observed in the dipeptide-hypersensitive mutant obtained by chemical mutagenesis. When each gene was overexpressed in the wild-type strain, which does not intrinsically excrete alanine, only theygaWgene conferred on the cells the ability to excrete alanine. In addition, expression of theygaWgene was induced in the presence of the dipeptide. On the basis of these results, we concluded that YgaW is likely to be the physiologically most relevant exporter forl-alanine inE. coliand proposed that the gene be redesignatedalaEforalanineexport.

scholarly journals Plasmid DNA Production in Proteome-Reduced Escherichia coli

2020 ◽  
Vol 8 (9) ◽  
pp. 1444
Author(s):  
Mitzi de la Cruz ◽  
Elisa A. Ramírez ◽  
Juan-Carlos Sigala ◽  
José Utrilla ◽  
Alvaro R. Lara
Keyword(s):  
Escherichia Coli ◽  
Plasmid Dna ◽  
Type Strain ◽  
Wild Type Strain ◽  
The Novel ◽  
Wild Type ◽  
Batch Mode ◽  
Cell Factories ◽  
Starting Point ◽  
In The Wild

The design of optimal cell factories requires engineering resource allocation for maximizing product synthesis. A recently developed method to maximize the saving in cell resources released 0.5% of the proteome of Escherichia coli by deleting only three transcription factors. We assessed the capacity for plasmid DNA (pDNA) production in the proteome-reduced strain in a mineral medium, lysogeny, and terrific broths. In all three cases, the pDNA yield from biomass was between 33 and 53% higher in the proteome-reduced than in its wild type strain. When cultured in fed-batch mode in shake-flask, the proteome-reduced strain produced 74.8 mg L−1 pDNA, which was four times greater than its wild-type strain. Nevertheless, the pDNA supercoiled fraction was less than 60% in all cases. Deletion of recA increased the pDNA yields in the wild type, but not in the proteome-reduced strain. Furthermore, recA mutants produced a higher fraction of supercoiled pDNA, compared to their parents. These results show that the novel proteome reduction approach is a promising starting point for the design of improved pDNA production hosts.

scholarly journals 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

2013 ◽  
Vol 81 (9) ◽  
pp. 3089-3098 ◽  
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.

scholarly journals Fumarate-Mediated Persistence of Escherichia coli against Antibiotics

2016 ◽  
Vol 60 (4) ◽  
pp. 2232-2240 ◽  
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.

scholarly journals mhpTEncodes an Active Transporter Involved in 3-(3-Hydroxyphenyl)Propionate Catabolism by Escherichia coli K-12

2013 ◽  
Vol 79 (20) ◽  
pp. 6362-6368 ◽  
Author(s):  
Ying Xu ◽  
Bing Chen ◽  
Hongjun Chao ◽  
Ning-Yi Zhou
Keyword(s):  
Escherichia Coli ◽  
Wild Type Strain ◽  
Fluorescent Protein ◽  
Gene Knockout ◽  
Transport Activity ◽  
Wild Type ◽  
Content Type ◽  
E Coli ◽  
Green Fluorescent ◽  
K 12

ABSTRACTEscherichia coliK-12 utilizes 3-(3-hydroxyphenyl)propionate (3HPP) as a sole carbon and energy source. Among the genes in its catabolic cluster in the genome,mhpTwas proposed to encode a hypothetical transporter. Since no transporter for 3HPP uptake has been identified, we investigated whether MhpT is responsible for 3HPP uptake. MhpT fused with green fluorescent protein was found to be located at the periphery of cells by confocal microscopy, consistent with localization to the cytoplasmic membrane. Gene knockout and complementation studies clearly indicated thatmhpTis essential for 3HPP catabolism inE. coliK-12 W3110 at pH 8.2. Uptake assays with14C-labeled substrates demonstrated that strain W3110 and strain W3110ΔmhpTcontaining recombinant MhpT specifically transported 3HPP but not benzoate, 3-hydroxybenzoate, or gentisate into cells. Energy dependence assays suggested that MhpT-mediated 3HPP transport was driven by the proton motive force. The change of Ala-272 of MhpT to a histidine, surprisingly, resulted in enhanced transport activity, and strain W3110ΔmhpTcontaining the MhpT A272H mutation had a slightly higher growth rate than the wild-type strain at pH 8.2. Hence, we demonstrated that MhpT is a specific 3HPP transporter and vital forE. coliK-12 W3110 growth on this substrate under basic conditions.

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

2015 ◽  
Vol 82 (1) ◽  
pp. 244-254 ◽  
Author(s):  
Manuel S. Godoy ◽  
Pablo I. Nikel ◽  
José G. Cabrera Gomez ◽  
M. Julia Pettinari
Keyword(s):  
Escherichia Coli ◽  
Type Strain ◽  
Wild Type Strain ◽  
Oxygen Availability ◽  
Biochemical Network ◽  
Metabolic Effects ◽  
Null Mutant ◽  
Wild Type ◽  
Content Type ◽  
E Coli

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.

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

2015 ◽  
Vol 197 (23) ◽  
pp. 3658-3665 ◽  
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.

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

2014 ◽  
Vol 82 (12) ◽  
pp. 5056-5068 ◽  
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.

scholarly journals UDP-N-Acetylmuramic Acid l-Alanine Ligase (MurC) Inhibition in atolCMutant Escherichia coli Strain Leads to Cell Death

2014 ◽  
Vol 58 (10) ◽  
pp. 6165-6171 ◽  
Author(s):  
Vaishali Humnabadkar ◽  
K. R. Prabhakar ◽  
Ashwini Narayan ◽  
Sreevalli Sharma ◽  
Supreeth Guptha ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Efflux Pump ◽  
Coli Strain ◽  
Cellular Activity ◽  
Wild Type ◽  
Compound A ◽  
Content Type ◽  
E Coli ◽  
In The Wild ◽  
High Concentrations

ABSTRACTThe Mur ligases play an essential role in the biosynthesis of bacterial peptidoglycan and hence are attractive antibacterial targets. A screen of the AstraZeneca compound library led to the identification of compound A, a pyrazolopyrimidine, as a potent inhibitor ofEscherichia coliandPseudomonas aeruginosaMurC. However, cellular activity againstE. coliorP. aeruginosawas not observed. Compound A was active against efflux pump mutants of both strains. Experiments using anE. colitolCmutant revealed accumulation of the MurC substrate and a decrease in the level of product upon treatment with compound A,indicating inhibition of MurC enzyme in these cells. Such a modulation was not observed in theE. coliwild-type cells. Further, overexpression of MurC in theE. colitolCmutant led to an increase in the compound A MIC by ≥16-fold, establishing a correlation between MurC inhibition and cellular activity. In addition, estimation of the intracellular compound A level showed an accumulation of the compound over time in thetolCmutant strain. A significant compound A level was not detected in the wild-typeE. colistrain even upon treatment with high concentrations of the compound. Therefore, the lack of MIC and absence of MurC inhibition in wild-typeE. coliwere possibly due to suboptimal compound concentration as a consequence of a high efflux level and/or poor permeativity of compound A.

scholarly journals Expression of Hemin Receptor Molecule ChuA Is Influenced by RfaH in Uropathogenic Escherichia coliStrain 536

2001 ◽  
Vol 69 (3) ◽  
pp. 1924-1928 ◽  
Author(s):  
Gábor Nagy ◽  
Ulrich Dobrindt ◽  
Maren Kupfer ◽  
Levente Emödy ◽  
Helge Karch ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Membrane Protein ◽  
Type Strain ◽  
Wild Type Strain ◽  
Regulatory Protein ◽  
Wild Type ◽  
Receptor Molecule ◽  
E Coli ◽  
Protein Levels ◽  
In The Wild

ABSTRACT The outer membrane protein ChuA responsible for hemin utilization has been recently identified in several pathogenic Escherichia coli strains. We report that the regulatory protein RfaH influences ChuA expression in the uropathogenic E. colistrain 536. In an rfaH mutant, the chuAtranscript as well as the ChuA protein levels were significantly decreased in comparison with those in the wild-type strain. Within thechuA gene, a consensus motif known as the JUMPStart (just upstream of many polysaccharide associated gene starts) sequence was found, which is shared by RfaH-affected operons. Furthermore, the presence of two different subclasses of thechuA determinant and their distribution in E. coli pathogroups are described.

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

2001 ◽  
Vol 183 (17) ◽  
pp. 5187-5197 ◽  
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.

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