scholarly journals Genotype and Phenotypes of an Intestine-Adapted Escherichia coli K-12 Mutant Selected by Animal Passage for Superior Colonization

2011 ◽  
Vol 79 (6) ◽  
pp. 2430-2439 ◽  
Author(s):  
Andrew J. Fabich ◽  
Mary P. Leatham ◽  
Joe E. Grissom ◽  
Graham Wiley ◽  
Hongshing Lai ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Carbon Sources ◽  
Genomic Analysis ◽  
Metabolic Efficiency ◽  
Spontaneous Mutant ◽  
Wild Type ◽  
Content Type ◽  
E Coli ◽  
Expression Of Genes ◽  
K 12

ABSTRACTWe previously isolated a spontaneous mutant ofEscherichia coliK-12, strain MG1655, following passage through the streptomycin-treated mouse intestine, that has colonization traits superior to the wild-type parent strain (M. P. Leatham et al., Infect. Immun.73:8039–8049, 2005). This intestine-adapted strain (E. coliMG1655*) grew faster on several different carbon sources than the wild type and was nonmotile due to deletion of theflhDgene. We now report the results of several high-throughput genomic analysis approaches to further characterizeE. coliMG1655*. Whole-genome pyrosequencing did not reveal any changes on its genome, aside from the deletion at theflhDClocus, that could explain the colonization advantage ofE. coliMG1655*. Microarray analysis revealed modest yet significant induction of catabolic gene systems across the genome in bothE. coliMG1655* and an isogenicflhDmutant constructed in the laboratory. Catabolome analysis with Biolog GN2 microplates revealed an enhanced ability of bothE. coliMG1655* and the isogenicflhDmutant to oxidize a variety of carbon sources. The results show that intestine-adaptedE. coliMG1655* is more fit than the wild type for intestinal colonization, because loss of FlhD results in elevated expression of genes involved in carbon and energy metabolism, resulting in more efficient carbon source utilization and a higher intestinal population. Hence, mutations that enhance metabolic efficiency confer a colonization advantage.

scholarly journals Conversion of Norepinephrine to 3,4-Dihdroxymandelic Acid in Escherichia coli Requires the QseBC Quorum-Sensing System and the FeaR Transcription Factor

2017 ◽  
Vol 200 (1) ◽  
Author(s):  
Sasikiran Pasupuleti ◽  
Nitesh Sule ◽  
Michael D. Manson ◽  
Arul Jayaraman
Keyword(s):  
Escherichia Coli ◽  
Transcription Factor ◽  
Quorum Sensing ◽  
Aromatic Amines ◽  
Response Regulator ◽  
Content Type ◽  
E Coli ◽  
Expression Of Genes ◽  
K 12 ◽  
Cognate Response Regulator

ABSTRACTThe detection of norepinephrine (NE) as a chemoattractant byEscherichia colistrain K-12 requires the combined action of the TynA monoamine oxidase and the FeaB aromatic aldehyde dehydrogenase. The role of these enzymes is to convert NE into 3,4-dihydroxymandelic acid (DHMA), which is a potent chemoattractant sensed by the Tsr chemoreceptor. These two enzymes must be induced by prior exposure to NE, and cells that are exposed to NE for the first time initially show minimal chemotaxis toward it. The induction of TynA and FeaB requires the QseC quorum-sensing histidine kinase, and the signaling cascade requires new protein synthesis. Here, we demonstrate that the cognate response regulator for QseC, the transcription factor QseB, is also required for induction. The related quorum-sensing kinase QseE appears not to be part of the signaling pathway, but its cognate response regulator, QseF, which is also a substrate for phosphotransfer from QseC, plays a nonessential role. The promoter of thefeaRgene, which encodes a transcription factor that has been shown to be essential for the expression oftynAandfeaB, has two predicted QseB-binding sites. One of these sites appears to be in an appropriate position to stimulate transcription from the P1promoter of thefeaRgene. This study unites two well-known pathways: one for expression of genes regulated by catecholamines (QseBC) and one for expression of genes required for metabolism of aromatic amines (FeaR, TynA, and FeaB). This cross talk allowsE. colito convert the host-derived and chemotactically inert NE into the potent bacterial chemoattractant DHMA.IMPORTANCEThe chemotaxis ofE. coliK-12 to norepinephrine (NE) requires the conversion of NE to 3,4-dihydroxymandleic acid (DHMA), and DHMA is both an attractant and inducer of virulence gene expression for a pathogenic enterohemorrhagicE. coli(EHEC) strain. The induction of virulence by DHMA and NE requires QseC. The results described here show that the cognate response regulator for QseC, QseB, is also required for conversion of NE into DHMA. Production of DHMA requires induction of a pathway involved in the metabolism of aromatic amines. Thus, the QseBC sensory system provides a direct link between virulence and chemotaxis, suggesting that chemotaxis to host signaling molecules may require that those molecules are first metabolized by bacterial enzymes to generate the actual chemoattractant.

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 Regulation ofwaaHby PhoB during PiStarvation Promotes Biofilm Formation byEscherichia coliO157:H7

2019 ◽  
Vol 201 (18) ◽  
Author(s):  
Philippe Vogeleer ◽  
Antony T. Vincent ◽  
Samuel M. Chekabab ◽  
Steve J. Charette ◽  
Alexey Novikov ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Ecological Niches ◽  
Pho Regulon ◽  
Starvation Stress ◽  
Content Type ◽  
E Coli ◽  
Expression Of Genes ◽  
K 12 ◽  
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. This activates the phosphate-specific transport (Pst) system that contains a high-affinity Pitransporter. In the Δpstmutant, PhoB is constitutively activated and regulates the expression of genes in the Pho regulon. Here, we show that Pistarvation and deletion of thepstsystem enhanceE. coliO157:H7 biofilm formation. Among differentially expressed genes of EDL933 grown under Pistarvation conditions and in the Δpstmutant, we have found that a member of the PhoB regulon,waaH, predicted to encode a glycosyltransferase, was highly expressed. Interestingly, WaaH contributed to biofilm formation ofE. coliO157:H7 during both Pistarvation and in the Δpstmutant. In the Δpstmutant, the presence ofwaaHwas associated with lipopolysaccharide (LPS) R3 core type modifications, whereas inE. coliO157:H7,waaHoverexpression had no effect on LPS structure during Pistarvation. Therefore,waaHparticipates inE. coliO157:H7 biofilm formation during Pistarvation, but its biochemical role remains to be clarified. This study highlights the importance of the Pistarvation stress response to biofilm formation, which may contribute to the persistence ofE. coliO157:H7 in the environment.IMPORTANCEEnterohemorrhagicEscherichia coliO157:H7 is a human pathogen that causes bloody diarrhea that can result in renal failure. Outside of mammalian hosts,E. coliO157:H7 survives for extended periods of time in nutrient-poor environments, likely as part of biofilms. InE. coliK-12, the levels of free extracellular Piaffect biofilm formation; however, it was unknown whether Piinfluences biofilm formation byE. coliO157:H7. Our results show that upon Pistarvation, PhoB activateswaaHexpression, which favors biofilm formation byE. coliO157:H7. These findings suggest that WaaH is a target for controlling biofilm formation. Altogether, our work demonstrates how adaptation to Pistarvation allowsE. coliO157:H7 to occupy different ecological niches.

scholarly journals YbcL of Uropathogenic Escherichia coli Suppresses Transepithelial Neutrophil Migration

2012 ◽  
Vol 80 (12) ◽  
pp. 4123-4132 ◽  
Author(s):  
Megan E. Lau ◽  
Jennifer A. Loughman ◽  
David A. Hunstad
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Immune Modulation ◽  
Neutrophil Migration ◽  
Single Amino Acid ◽  
Amino Acid Difference ◽  
Wild Type ◽  
Content Type ◽  
E Coli ◽  
K 12

ABSTRACTUropathogenicEscherichia coli(UPEC) strains suppress the acute inflammatory response in the urinary tract to ensure access to the intracellular uroepithelial niche that supports the propagation of infection. Our understanding of this initial cross talk between host and pathogen is incomplete. Here we report the identification of a previously uncharacterized periplasmic protein, YbcL, encoded by UPEC that contributes to immune modulation in the urinary tract by suppressing acute neutrophil migration. In contrast to wild-type UPEC, an isogenic strain lackingybcLexpression (UTI89 ΔybcL) failed to suppress transepithelial polymorphonuclear leukocyte (PMN) migrationin vitro, a defect complemented by expressingybcLepisomally. YbcL homologs are present in manyE. coligenomes; expression of the YbcL variant encoded by nonpathogenicE. coliK-12 strain MG1655 (YbcLMG) failed to complement the UTI89 ΔybcLdefect, whereas expression of the UPEC YbcL variant (YbcLUTI) in MG1655 conferred the capacity for suppressing PMN migration. This phenotypic difference was due to a single amino acid difference (V78T) between the two YbcL homologs, and a majority of clinical UPEC strains examined were found to encode the suppressive YbcL variant. Purified YbcLUTIprotein suppressed PMN migration in response to live or killed MG1655, and YbcLUTIwas detected in the supernatant during UPEC infection of bladder epithelial cells or PMNs. Lastly, early PMN influx to murine bladder tissue was augmented uponin vivoinfection with UTI89 ΔybcLcompared with wild-type UPEC. Our findings demonstrate a role for UPEC YbcL in suppression of the innate immune response during urinary tract infection.

scholarly journals Functional Analysis ofycfRandycfQin Escherichia coli O157:H7 Linked to Outbreaks of Illness Associated with Fresh Produce

2011 ◽  
Vol 77 (12) ◽  
pp. 3952-3959 ◽  
Author(s):  
Kaiping Deng ◽  
Siyun Wang ◽  
Xiaoqian Rui ◽  
Wei Zhang ◽  
Mary Lou Tortorello
Keyword(s):  
Escherichia Coli ◽  
Fresh Produce ◽  
The United States ◽  
Sublethal Concentration ◽  
Wild Type ◽  
Chloramphenicol Resistance ◽  
Content Type ◽  
E Coli ◽  
K 12 ◽  
Postharvest Processing

ABSTRACTFresh produce has been associated with multiple outbreaks of illness caused byEscherichia coliO157:H7. The mechanism ofE. coliO157:H7 survival through postharvest processing of fresh produce needs to be understood to help develop more effective interventions. In our recent transcriptomic study of strain Sakai, an isolate from the 1996 sprout outbreak in Japan, and strain TW14359, an isolate from the 2006 spinach outbreak in the United States, we showed thatycfRwas the most significantly upregulated gene in response to chlorine-based oxidative stress. YcfR is known to be a multiple stress resistance protein and a biofilm regulator inE. coliK-12 strains; however, its role in the pathogenicE. coliO157:H7 has not been clearly defined. In this study,ycfRwas replaced with a chloramphenicol resistance cassette oriented in two different directions to construct polar and nonpolarycfR::catmutants of Sakai and TW14359. Chlorine resistance and survival on spinach leaf surfaces were assessed in the wild-type strains and theycfRmutants. Both polar and nonpolarycfRmutants of Sakai showed significantly less chlorine resistance than their parent strain. In contrast, deletion ofycfRin TW14359 did not change chlorine resistance, indicating thatycfRin these two outbreak-relatedE. coliO157:H7 strains may function differently. In addition, after a 24-h incubation on spinach leaves in a sublethal concentration of chlorine, the Sakai nonpolarycfRmutant exhibited lower survival compared to the wild type. The results suggest a role forycfRin survival of Sakai during chlorine exposure. We also found that the upstreamycfQ, which is annotated as a DNA-binding regulator, acted as a repressor ofycfR. These findings suggest that gene regulation may be a mechanism by whichE. coliO157:H7 strain Sakai could survive in the postharvest processing environment.

scholarly journals Metabolic Engineering of a Novel Muconic Acid Biosynthesis Pathway via 4-Hydroxybenzoic Acid in Escherichia coli

2015 ◽  
Vol 81 (23) ◽  
pp. 8037-8043 ◽  
Author(s):  
Sudeshna Sengupta ◽  
Sudhakar Jonnalagadda ◽  
Lakshani Goonewardena ◽  
Veeresh Juturu
Keyword(s):  
Escherichia Coli ◽  
Metabolic Engineering ◽  
Carbon Sources ◽  
Raw Material ◽  
Hydroxybenzoic Acid ◽  
Muconic Acid ◽  
Content Type ◽  
E Coli ◽  
Catechol 1,2 Dioxygenase ◽  
K 12

ABSTRACTcis,cis-Muconic acid (MA) is a commercially important raw material used in pharmaceuticals, functional resins, and agrochemicals. MA is also a potential platform chemical for the production of adipic acid (AA), terephthalic acid, caprolactam, and 1,6-hexanediol. A strain ofEscherichia coliK-12, BW25113, was genetically modified, and a novel nonnative metabolic pathway was introduced for the synthesis of MA from glucose. The proposed pathway converted chorismate from the aromatic amino acid pathway to MA via 4-hydroxybenzoic acid (PHB). Three nonnative genes,pobA,aroY, andcatA, coding for 4-hydroxybenzoate hydrolyase, protocatechuate decarboxylase, and catechol 1,2-dioxygenase, respectively, were functionally expressed inE. colito establish the MA biosynthetic pathway.E. colinative genesubiC,aroFFBR,aroE, andaroLwere overexpressed and the genesptsH,ptsI,crr, andpykFwere deleted from theE. coligenome in order to increase the precursors of the proposed MA pathway. The final engineeredE. colistrain produced nearly 170 mg/liter of MA from simple carbon sources in shake flask experiments. The proposed pathway was proved to be functionally active, and the strategy can be used for future metabolic engineering efforts for production of MA from renewable sugars.

scholarly journals Molecular Control of Sucrose Utilization in Escherichia coli W, an Efficient Sucrose-Utilizing Strain

2012 ◽  
Vol 79 (2) ◽  
pp. 478-487 ◽  
Author(s):  
Suriana Sabri ◽  
Lars K. Nielsen ◽  
Claudia E. Vickers
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Good Growth ◽  
Knockout Mutant ◽  
Sucrose Transport ◽  
Molecular Control ◽  
Content Type ◽  
E Coli ◽  
K 12 ◽  
Sucrose Utilization

ABSTRACTSucrose is an industrially important carbon source for microbial fermentation. Sucrose utilization inEscherichia coli, however, is poorly understood, and most industrial strains cannot utilize sucrose. The roles of the chromosomally encoded sucrose catabolism (csc) genes inE. coliW were examined by knockout and overexpression experiments. At low sucrose concentrations, thecscgenes are repressed and cells cannot grow. Removal of either the repressor protein (cscR) or the fructokinase (cscK) gene facilitated derepression. Furthermore, combinatorial knockout ofcscRandcscKconferred an improved growth rate on low sucrose. The invertase (cscA) and sucrose transporter (cscB) genes are essential for sucrose catabolism inE. coliW, demonstrating that no other genes can provide sucrose transport or inversion activities. However,cscKis not essential for sucrose utilization. Fructose is excreted into the medium by thecscK-knockout strain in the presence of high sucrose, whereas at low sucrose (when carbon availability is limiting), fructose is utilized by the cell. Overexpression ofcscA,cscAK, orcscABcould complement the WΔcscRKABknockout mutant or confer growth on a K-12 strain which could not naturally utilize sucrose. However, phenotypic stability and relatively good growth rates were observed in the K-12 strain only when overexpressingcscAB, and full growth rate complementation in WΔcscRKABalso requiredcscAB. Our understanding of sucrose utilization can be used to improveE. coliW and engineer sucrose utilization in strains which do not naturally utilize sucrose, allowing substitution of sucrose for other, less desirable carbon sources in industrial fermentations.

scholarly journals First-Time Isolation and Characterization of a Bacteriophage Encoding the Shiga Toxin 2c Variant, Which Is Globally Spread in Strains of Escherichia coli O157

2004 ◽  
Vol 72 (12) ◽  
pp. 7030-7039 ◽  
Author(s):  
Eckhard Strauch ◽  
Christoph Schaudinn ◽  
Lothar Beutin
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Phage Lambda ◽  
Wild Type ◽  
Chromosomal Locus ◽  
O157 Strain ◽  
Diagnostic Pcr ◽  
E Coli ◽  
Isolation And Characterization ◽  
K 12

ABSTRACT A bacteriophage encoding the Shiga toxin 2c variant (Stx2c) was isolated from the human Escherichia coli O157 strain CB2851 and shown to form lysogens on the E. coli K-12 laboratory strains C600 and MG1655. Production of Stx2c was found in the wild-type E. coli O157 strain and the K-12 lysogens and was inducible by growing bacteria in the presence of ciprofloxacin. Phage 2851 is the first reported viable bacteriophage which carries an stx 2c gene. Electron micrographs of phage 2851 showed particles with elongated hexagonal heads and long flexible tails resembling phage lambda. Sequence analysis of an 8.4-kb region flanking the stx 2c gene and other genetic elements revealed a mosaic gene structure, as found in other Stx phages. Phage 2851 showed lysis of E. coli K-12 strains lysogenic for Stx phages encoding Stx1 (H19), Stx2 (933W), Stx (7888), and Stx1c (6220) but showed superinfection immunity with phage lambda, presumably originating from the similarity of the cI repressor proteins of both phages. Apparently, phage 2851 integrates at a different chromosomal locus than Stx2 phage 933W and Stx1 phage H19 in E. coli, explaining why Stx2c is often found in combination with Stx1 or Stx2 in E. coli O157 strains. Diagnostic PCR was performed to determine gene sequences specific for phage 2851 in wild-type E. coli O157 strains producing Stx2c. The phage 2851 q and o genes were frequently detected in Stx2c-producing E. coli O157 strains, indicating that phages related to 2851 are associated with Stx2c production in strains of E. coli O157 that were isolated in different locations and time periods.

scholarly journals Increased Survival of Antibiotic-Resistant Escherichia coli inside Macrophages

2012 ◽  
Vol 57 (1) ◽  
pp. 189-195 ◽  
Author(s):  
Migla Miskinyte ◽  
Isabel Gordo
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Bacterial Infections ◽  
Resistance Mutation ◽  
Fitness Cost ◽  
Resistance Mutations ◽  
Content Type ◽  
E Coli ◽  
Fitness Effects ◽  
K 12

ABSTRACTMutations causing antibiotic resistance usually incur a fitness cost in the absence of antibiotics. The magnitude of such costs is known to vary with the environment. Little is known about the fitness effects of antibiotic resistance mutations when bacteria confront the host's immune system. Here, we study the fitness effects of mutations in therpoB,rpsL, andgyrAgenes, which confer resistance to rifampin, streptomycin, and nalidixic acid, respectively. These antibiotics are frequently used in the treatment of bacterial infections. We measured two important fitness traits—growth rate and survival ability—of 12Escherichia coliK-12 strains, each carrying a single resistance mutation, in the presence of macrophages. Strikingly, we found that 67% of the mutants survived better than the susceptible bacteria in the intracellular niche of the phagocytic cells. In particular, allE. colistreptomycin-resistant mutants exhibited an intracellular advantage. On the other hand, 42% of the mutants incurred a high fitness cost when the bacteria were allowed to divide outside of macrophages. This study shows that single nonsynonymous changes affecting fundamental processes in the cell can contribute to prolonged survival ofE. coliin the context of an infection.

scholarly journals The Complete Genome of the Atypical Enteropathogenic Escherichia coli Archetype Isolate E110019 Highlights a Role for Plasmids in Dissemination of the Type III Secreted Effector EspT

2019 ◽  
Vol 87 (10) ◽  
Author(s):  
Tracy H. Hazen ◽  
David A. Rasko
Keyword(s):  
Escherichia Coli ◽  
Complete Genome ◽  
Genomic Analysis ◽  
Host Cells ◽  
Comparative Genomic ◽  
Content Type ◽  
E Coli ◽  
Type Iii ◽  
Severe Diarrhea ◽  
Typical Epec

ABSTRACT Enteropathogenic Escherichia coli (EPEC) is a leading cause of moderate to severe diarrhea among young children in developing countries, and EPEC isolates can be subdivided into two groups. Typical EPEC (tEPEC) bacteria are characterized by the presence of both the locus of enterocyte effacement (LEE) and the plasmid-encoded bundle-forming pilus (BFP), which are involved in adherence and translocation of type III effectors into the host cells. Atypical EPEC (aEPEC) bacteria also contain the LEE but lack the BFP. In the current report, we describe the complete genome of outbreak-associated aEPEC isolate E110019, which carries four plasmids. Comparative genomic analysis demonstrated that the type III secreted effector EspT gene, an autotransporter gene, a hemolysin gene, and putative fimbrial genes are all carried on plasmids. Further investigation of 65 espT-containing E. coli genomes demonstrated that different espT alleles are associated with multiple plasmids that differ in their overall gene content from the E110019 espT-containing plasmid. EspT has been previously described with respect to its role in the ability of E110019 to invade host cells. While other type III secreted effectors of E. coli have been identified on insertion elements and prophages of the chromosome, we demonstrated in the current study that the espT gene is located on multiple unique plasmids. These findings highlight a role of plasmids in dissemination of a unique E. coli type III secreted effector that is involved in host invasion and severe diarrheal illness.

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