scholarly journals Genetic Evidence for a Molybdopterin-Containing Tellurate Reductase

2013 ◽  
Vol 79 (10) ◽  
pp. 3171-3175 ◽  
Author(s):  
Joanne Theisen ◽  
Gerben J. Zylstra ◽  
Nathan Yee
Keyword(s):  
Genetic Evidence ◽  
Biosynthesis Pathway ◽  
Wild Type ◽  
Transport Pathway ◽  
Content Type ◽  
E Coli ◽  
Reduction Activity ◽  
Mutant Strains ◽  
Molybdate Transport ◽  
K 12

ABSTRACTThe genetic identity and cofactor composition of the bacterial tellurate reductase are currently unknown. In this study, we examined the requirement of molybdopterin biosynthesis and molybdate transporter genes for tellurate reduction inEscherichia coliK-12. The results show that mutants deleted of themoaA,moaB,moaE, ormoggene in the molybdopterin biosynthesis pathway lost the ability to reduce tellurate. Deletion of themodBormodCgene in the molybdate transport pathway also resulted in complete loss of tellurate reduction activity. Genetic complementation by the wild-type sequences restored tellurate reduction activity in the mutant strains. These findings provide genetic evidence that tellurate reduction inE. coliinvolves a molybdoenzyme.

scholarly journals RpoS Contributes to Phagocyte Oxidase-Mediated Stress Resistance during Urinary Tract Infection by Escherichia coli CFT073

mBio ◽  
2013 ◽  
Vol 4 (1) ◽  
Author(s):  
Andrew J. Hryckowian ◽  
Rodney A. Welch
Keyword(s):  
Oxidative Stress ◽  
Urinary Tract ◽  
Wild Type ◽  
Upec Strain ◽  
Content Type ◽  
General Stress Response ◽  
E Coli ◽  
Phagocyte Oxidase ◽  
Strain Cft073 ◽  
K 12

ABSTRACTUropathogenicEscherichia coli(UPEC) is the most common causative agent of community-acquired urinary tract infection (UTI). In order to cause UTI, UPEC must endure stresses ranging from nutrient limitation to host immune components. RpoS (σS), the general stress response sigma factor, directs gene expression under a variety of inhibitory conditions. Our study ofrpoSin UPEC strain CFT073 began after we discovered anrpoS-frameshift mutation in one of our laboratory stocks of “wild-type” CFT073. We demonstrate that anrpoS-deletion mutation in CFT073 leads to a colonization defect during UTI of CBA/J mice at 48 hours postinfection (hpi). There is no difference between the growth rates of CFT073 and CFT073rpoSin urine. This indicates thatrpoSis needed for replication and survival in the host rather than being needed to address limitations imposed by urine nutrients. Consistent with previous observations inE. coliK-12, CFT073rpoSis more sensitive to oxidative stress than the wild type. We demonstrate that peroxide levels are elevated in voided urine from CFT073-infected mice compared to urine from mock-infected mice, which supports the notion that oxidative stress is generated by the host in response to UPEC. In mice that lack phagocyte oxidase, the enzyme complex expressed by phagocytes that produces superoxide, the competitive defect of CFT073rpoSin bladder colonization is lost. These results demonstrate that σSis important for UPEC survival under conditions of phagocyte oxidase-generated stress during UTI. Though σSaffects the pathogenesis of other bacterial species, this is the first work that directly implicates σSas important for UPEC pathogenesis.IMPORTANCEUPEC must cope with a variety of stressful conditions in the urinary tract during infection. RpoS (σS), the general stress response sigma factor, is known to direct the expression of many genes under a variety of stressful conditions in laboratory-adaptedE. coliK-12. Here, we show that σSis needed by the model UPEC strain CFT073 to cope with oxidative stress provided by phagocytes during infection. These findings represent the first report that implicates σSin the fitness of UPEC during infection and support the idea of the need for a better understanding of the effects of this global regulator of gene expression during UTI.

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 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 pqiABC and yebST, Putative mce Operons of Escherichia coli, Encode Transport Pathways and Contribute to Membrane Integrity

2016 ◽  
Vol 199 (1) ◽  
Author(s):  
Takayuki Nakayama ◽  
Qiu-Mei Zhang-Akiyama
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Unknown Function ◽  
Membrane Integrity ◽  
Transport Pathway ◽  
Transport Pathways ◽  
Content Type ◽  
E Coli ◽  
Outer Leaflet ◽  
Mutant Strains

ABSTRACT The membranes of single-cell organisms are crucial as the first line of defense. The outer membrane of Gram-negative bacteria is an asymmetric bilayer in which lipopolysaccharides (LPSs) and phospholipids are localized in the outer and inner leaflet, respectively. This asymmetry is important for membrane integrity. In Escherichia coli, the Mla transport pathway maintains this asymmetry by removing phospholipids from the outer leaflet. The MlaD component of this system is a mammalian cell entry (MCE) domain protein, and E. coli has two other MCE domain proteins of unknown function (PqiB and YebT). Here, we show that these two proteins are components of novel transport pathways that contribute to membrane integrity. The pqiAB operon is regulated by SoxS and RpoS. The yebST operon contains pqiAB homologues. Here, we found a third member of the pqi operon, ymbA (pqiC). A PqiB-PqiC complex bridges the inner and the outer membrane, and in other bacteria, pqiBC genes are located in operons together with transporter proteins. We show here that simultaneous deletion of pqiABC and yebST operons in an Δmla background rendered cells more sensitive to SDS-EDTA, and the SDS-EDTA sensitivity of mla mutants was rescued by additional copies of pqiABC. We also found that the yebST operon was induced by a defect in LPS molecules. In conclusion, PqiABC and YebST are novel transport pathways related to the Mla transport pathway and important for membrane integrity. IMPORTANCE Membranes of bacteria are crucial for stress resistance. The composition of the E. coli outer membrane is asymmetric, with asymmetry maintained by the Mla ABC transport pathway. We propose that the stress-inducible pqiABC operon and homologous yebST operon, both of previously unknown function, encode transport pathway proteins related to the Mla transport pathway. Deletion of these operons rendered cells more sensitive to membrane stress, and additional copies of pqiABC suppressed the SDS-EDTA sensitivity of mla mutant strains. We found that yebS′-′lacZ fusion was activated in mutant strains with defective LPS molecules.

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 Tailoring a Global Iron Regulon to a Uropathogen

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Rajdeep Banerjee ◽  
Erin Weisenhorn ◽  
Kevin J. Schwartz ◽  
Kevin S. Myers ◽  
Jeremy D. Glasner ◽  
...  
Keyword(s):  
Amino Acids ◽  
Urinary Tract ◽  
Amino Acid ◽  
Regulatory Network ◽  
Iron Homeostasis ◽  
Iron Limitation ◽  
Content Type ◽  
E Coli ◽  
General Stress ◽  
K 12

ABSTRACT Pathogenicity islands and plasmids bear genes for pathogenesis of various Escherichia coli pathotypes. Although there is a basic understanding of the contribution of these virulence factors to disease, less is known about variation in regulatory networks in determining disease phenotypes. Here, we dissected a regulatory network directed by the conserved iron homeostasis regulator, ferric uptake regulator (Fur), in uropathogenic E. coli (UPEC) strain CFT073. Comparing anaerobic genome-scale Fur DNA binding with Fur-dependent transcript expression and protein levels of the uropathogen to that of commensal E. coli K-12 strain MG1655 showed that the Fur regulon of the core genome is conserved but also includes genes within the pathogenicity/genetic islands. Unexpectedly, regulons indicative of amino acid limitation and the general stress response were also indirectly activated in the uropathogen fur mutant, suggesting that induction of the Fur regulon increases amino acid demand. Using RpoS levels as a proxy, addition of amino acids mitigated the stress. In addition, iron chelation increased RpoS to the same levels as in the fur mutant. The increased amino acid demand of the fur mutant or iron chelated cells was exacerbated by aerobic conditions, which could be partly explained by the O2-dependent synthesis of the siderophore aerobactin, encoded by an operon within a pathogenicity island. Taken together, these data suggest that in the iron-poor environment of the urinary tract, amino acid availability could play a role in the proliferation of this uropathogen, particularly if there is sufficient O2 to produce aerobactin. IMPORTANCE Host iron restriction is a common mechanism for limiting the growth of pathogens. We compared the regulatory network controlled by Fur in uropathogenic E. coli (UPEC) to that of nonpathogenic E. coli K-12 to uncover strategies that pathogenic bacteria use to overcome iron limitation. Although iron homeostasis functions were regulated by Fur in the uropathogen as expected, a surprising finding was the activation of the stringent and general stress responses in the uropathogen fur mutant, which was rescued by amino acid addition. This coordinated global response could be important in controlling growth and survival under nutrient-limiting conditions and during transitions from the nutrient-rich environment of the lower gastrointestinal (GI) tract to the more restrictive environment of the urinary tract. The coupling of the response of iron limitation to increased demand for amino acids could be a critical attribute that sets UPEC apart from other E. coli pathotypes.

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 The Type VI Secretion System Modulates Flagellar Gene Expression and Secretion in Citrobacter freundii and Contributes to Adhesion and Cytotoxicity to Host Cells

2015 ◽  
Vol 83 (7) ◽  
pp. 2596-2604 ◽  
Author(s):  
Liyun Liu ◽  
Shuai Hao ◽  
Ruiting Lan ◽  
Guangxia Wang ◽  
Di Xiao ◽  
...  
Keyword(s):  
Secretion System ◽  
Host Cells ◽  
Target Cells ◽  
Deletion Mutants ◽  
Citrobacter Freundii ◽  
Wild Type ◽  
Type Vi Secretion System ◽  
Content Type ◽  
Type Vi Secretion ◽  
Mutant Strains

The type VI secretion system (T6SS) as a virulence factor-releasing system contributes to virulence development of various pathogens and is often activated upon contact with target cells.Citrobacter freundiistrain CF74 has a complete T6SS genomic island (GI) that containsclpV,hcp-2, andvgrT6SS genes. We constructedclpV,hcp-2,vgr, and T6SS GI deletion mutants in CF74 and analyzed their effects on the transcriptome overall and, specifically, on the flagellar system at the levels of transcription and translation. Deletion of the T6SS GI affected the transcription of 84 genes, with 15 and 69 genes exhibiting higher and lower levels of transcription, respectively. Members of the cell motility class of downregulated genes of the CF74ΔT6SS mutant were mainly flagellar genes, including effector proteins, chaperones, and regulators. Moreover, the production and secretion of FliC were also decreased inclpV,hcp-2,vgr, or T6SS GI deletion mutants in CF74 and were restored upon complementation. In swimming motility assays, the mutant strains were found to be less motile than the wild type, and motility was restored by complementation. The mutant strains were defective in adhesion to HEp-2 cells and were restored partially upon complementation. Further, the CF74ΔT6SS, CF74ΔclpV, and CF74Δhcp-2mutants induced lower cytotoxicity to HEp-2 cells than the wild type. These results suggested that the T6SS GI in CF74 regulates the flagellar system, enhances motility, is involved in adherence to host cells, and induces cytotoxicity to host cells. Thus, the T6SS plays a wide-ranging role inC. freundii.

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.

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