scholarly journals Glucose-6-phosphate dehydrogenase and ferredoxin-NADP(H) reductase contribute to damage repair during the soxRS response of Escherichia coli

Microbiology ◽  
2006 ◽  
Vol 152 (4) ◽  
pp. 1119-1128 ◽  
Author(s):  
Mariana Giró ◽  
Néstor Carrillo ◽  
Adriana R. Krapp
Keyword(s):  
Escherichia Coli ◽  
Survival Rates ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
Iron Sulfur Clusters ◽  
Iron Sulfur ◽  
Oxidative Challenge ◽  
Glucose 6 Phosphate Dehydrogenase

The NADP(H)-dependent enzymes glucose-6-phosphate dehydrogenase (G6PDH) and ferredoxin(flavodoxin)-NADP(H) reductase (FPR), encoded by the zwf and fpr genes, respectively, are committed members of the soxRS regulatory system involved in superoxide resistance in Escherichia coli. Exposure of E. coli cells to the superoxide propagator methyl viologen (MV) led to rapid accumulation of G6PDH, while FPR was induced after a lag period of several minutes. Bacteria expressing G6PDH from a multicopy plasmid accumulated higher NADPH levels and displayed a protracted soxRS response, whereas FPR build-up had the opposite effects. Inactivation of either of the two genes resulted in enhanced sensitivity to MV killing, while further increases in the cellular content of FPR led to higher survival rates under oxidative conditions. In contrast, G6PDH accumulation over wild-type levels of expression failed to increase MV tolerance. G6PDH and FPR could act concertedly to deliver reducing equivalents from carbohydrates, via NADP+, to the FPR acceptors ferredoxin and/or flavodoxin. To evaluate whether this electron-transport system could mediate reductive repair reactions, the pathway was reconstituted in vitro from purified components; the reconstituted system was found to be functional in reactivation of oxidatively damaged iron–sulfur clusters of hydro-lyases such as aconitase and 6-phosphogluconate dehydratase. Recovery of these activities after oxidative challenge was faster and more extensive in transformed bacteria overexpressing FPR than in wild-type cells, indicating that the reductase could sustain hydro-lyase repair in vivo. However, FPR-deficient mutants were still able to fix iron–sulfur clusters at significant rates, suggesting that back-up routes for ferredoxin and/or flavodoxin reduction might be called into action to rescue inactivated enzymes when FPR is absent.

scholarly journals Fimbria-Encoding GeneyadCHas a Pleiotropic Effect on Several Biological Characteristics and Plays a Role in Avian Pathogenic Escherichia coli Pathogenicity

2015 ◽  
Vol 84 (1) ◽  
pp. 187-193 ◽  
Author(s):  
Renu Verma ◽  
Thaís Cabrera Galvão Rojas ◽  
Renato Pariz Maluta ◽  
Janaína Luisa Leite ◽  
Livia Pilatti Mendes da Silva ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Soft Agar ◽  
Pleiotropic Effect ◽  
Biological Characteristics ◽  
Wild Type ◽  
Content Type ◽  
Pathogenic Escherichia Coli

The extraintestinal pathogen termed avian pathogenicEscherichia coli(APEC) is known to cause colibacillosis in chickens. The molecular basis of APEC pathogenesis is not fully elucidated yet. In this work, we deleted a component of the Yad gene cluster (yadC) in order to understand the role of Yad in the pathogenicity of the APEC strain SCI-07.In vitro, the transcription level ofyadCwas upregulated at 41°C and downregulated at 22°C. TheyadCexpressionin vivowas more pronounced in lungs than in spleen, suggesting a role in the early steps of the infection. Chicks infected with the wild-type and mutant strains presented, respectively, 80% and 50% mortality rates. The ΔyadCstrain presented a slightly decreased ability to adhere to HeLa cells with or without thed-mannose analog compared with the wild type. Real-time PCR (RT-PCR) assays showed thatfimHwas downregulated (P< 0.05) andcsgAandecpAwere slightly upregulated in the mutant strain, showing thatyadCmodulates expression of other fimbriae. Bacterial internalization studies showed that the ΔyadCstrain had a lower number of intracellular bacteria recovered from Hep-2 cells and HD11 cells than the wild-type strain (P< 0.05). Motility assays in soft agar demonstrated that the ΔyadCstrain was less motile than the wild type (P< 0.01). Curiously, flagellum-associated genes were not dramatically downregulated in the ΔyadCstrain. Taken together, the results show that the fimbrial adhesin Yad contributes to the pathogenicity and modulates different biological characteristics of the APEC strain SCI-07.

scholarly journals Mutational Analysis of Residues in PriA and PriC Affecting Their Ability To Interact with SSB in Escherichia coli K-12

2020 ◽  
Vol 202 (23) ◽  
Author(s):  
Anastasiia N. Klimova ◽  
Steven J. Sandler
Keyword(s):  
Escherichia Coli ◽  
Mutational Analysis ◽  
Wild Type ◽  
Content Type ◽  
Growth Defects ◽  
C Terminus ◽  
K 12 ◽  
And Function

ABSTRACT Escherichia coli PriA and PriC recognize abandoned replication forks and direct reloading of the DnaB replicative helicase onto the lagging-strand template coated with single-stranded DNA-binding protein (SSB). Both PriA and PriC have been shown by biochemical and structural studies to physically interact with the C terminus of SSB. In vitro, these interactions trigger remodeling of the SSB on ssDNA. priA341(R697A) and priC351(R155A) negated the SSB remodeling reaction in vitro. Plasmid-carried priC351(R155A) did not complement priC303::kan, and priA341(R697A) has not yet been tested for complementation. Here, we further studied the SSB-binding pockets of PriA and PriC by placing priA341(R697A), priA344(R697E), priA345(Q701E), and priC351(R155A) on the chromosome and characterizing the mutant strains. All three priA mutants behaved like the wild type. In a ΔpriB strain, the mutations caused modest increases in SOS expression, cell size, and defects in nucleoid partitioning (Par−). Overproduction of SSB partially suppressed these phenotypes for priA341(R697A) and priA344(R697E). The priC351(R155A) mutant behaved as expected: there was no phenotype in a single mutant, and there were severe growth defects when this mutation was combined with ΔpriB. Analysis of the priBC mutant revealed two populations of cells: those with wild-type phenotypes and those that were extremely filamentous and Par− and had high SOS expression. We conclude that in vivo, priC351(R155A) identified an essential residue and function for PriC, that PriA R697 and Q701 are important only in the absence of PriB, and that this region of the protein may have a complicated relationship with SSB. IMPORTANCE Escherichia coli PriA and PriC recruit the replication machinery to a collapsed replication fork after it is repaired and needs to be restarted. In vitro studies suggest that the C terminus of SSB interacts with certain residues in PriA and PriC to recruit those proteins to the repaired fork, where they help remodel it for restart. Here, we placed those mutations on the chromosome and tested the effect of mutating these residues in vivo. The priC mutation completely abolished function. The priA mutations had no effect by themselves. They did, however, display modest phenotypes in a priB-null strain. These phenotypes were partially suppressed by SSB overproduction. These studies give us further insight into the reactions needed for replication restart.

scholarly journals In Vivo Titration of Mitomycin C Action by Four Escherichia coli Genomic Regions on Multicopy Plasmids

2001 ◽  
Vol 183 (7) ◽  
pp. 2259-2264 ◽  
Author(s):  
Yan Wei ◽  
Amy C. Vollmer ◽  
Robert A. LaRossa
Keyword(s):  
Escherichia Coli ◽  
Mitomycin C ◽  
Transcriptional Activation ◽  
Efflux Pump ◽  
Wild Type ◽  
Potent Inducer ◽  
E Coli ◽  
Genomic Regions

ABSTRACT Mitomycin C (MMC), a DNA-damaging agent, is a potent inducer of the bacterial SOS response; surprisingly, it has not been used to select resistant mutants from wild-type Escherichia coli. MMC resistance is caused by the presence of any of four distinctE. coli genes (mdfA, gyrl, rob, andsdiA) on high-copy-number vectors. mdfAencodes a membrane efflux pump whose overexpression results in broad-spectrum chemical resistance. The gyrI (also called sbmC) gene product inhibits DNA gyrase activity in vitro, while the rob protein appears to function in transcriptional activation of efflux pumps. SdiA is a transcriptional activator of ftsQAZ genes involved in cell division.

scholarly journals Cnu, a Novel oriC-Binding Protein of Escherichia coli

2005 ◽  
Vol 187 (20) ◽  
pp. 6998-7008 ◽  
Author(s):  
Myung Suk Kim ◽  
Sung-Hun Bae ◽  
Sang Hoon Yun ◽  
Hee Jung Lee ◽  
Sang Chun Ji ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid Identity ◽  
Replication Initiation ◽  
In Vitro Assays ◽  
Wild Type ◽  
Genetic Method ◽  
Dna Replication Initiation ◽  
Pair Sequence

ABSTRACT We have found, using a newly developed genetic method, a protein (named Cnu, for oriC-binding nucleoid-associated) that binds to a specific 26-base-pair sequence (named cnb) in the origin of replication of Escherichia coli, oriC. Cnu is composed of 71 amino acids (8.4 kDa) and shows extensive amino acid identity to a group of proteins belonging to the Hha/YmoA family. Cnu was previously discovered as a protein that, like Hha, complexes with H-NS in vitro. Our in vivo and in vitro assays confirm the results and further suggest that the complex formation with H-NS is involved in Cnu/Hha binding to cnb. Unlike the hns mutants, elimination of either the cnu or hha gene did not disturb the growth rate, origin content, and synchrony of DNA replication initiation of the mutants compared to the wild-type cells. However, the cnu hha double mutant was moderately reduced in origin content. The Cnu/Hha complex with H-NS thus could play a role in optimal activity of oriC.

scholarly journals Contributions of O Island 48 to Adherence of Enterohemorrhagic Escherichia coli O157:H7 to Epithelial Cells In Vitro and in Ligated Pig Ileal Loops

2009 ◽  
Vol 75 (18) ◽  
pp. 5779-5786 ◽  
Author(s):  
Xianhua Yin ◽  
Roger Wheatcroft ◽  
James R. Chambers ◽  
Bianfang Liu ◽  
Jing Zhu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Cluster Formation ◽  
Gene Clusters ◽  
Enterohemorrhagic Escherichia Coli ◽  
Wild Type ◽  
The Difference ◽  
Large Clusters

ABSTRACT O island 48 (OI-48) of Escherichia coli consists of three functional gene clusters that encode urease, tellurite resistance (Ter), and putative adhesins Iha and AIDA-1. The functions of these clusters in enterohemorrhagic E. coli (EHEC) O157:H7 infection are unknown. Deletion mutants for these three regions were constructed and evaluated for their ability to adhere to epithelial cells in vitro and in ligated pig ileal loops. Deletion of the Ter gene cluster reduced the ability of the organism to adhere to and form large clusters on IPEC-J2 and HEp-2 cells. Complementation of the mutation by introducing the wild-type ter genes restored adherence and large-cluster formation. Tests in ligated pig ileal loops showed a decrease in colonization by the Ter-negative mutant, but the difference was not significant compared to colonization by the wild type (26.4% ± 21.2% versus 40.1% ± 19.1%; P = 0.168). The OI-48 aidA gene deletion had no effect on adherence in vitro or in vivo. Deletion of the iha and ureC genes had no effect on adherence in vitro but significantly reduced the colonization of EHEC O157:H7 in the ligated pig intestine. These data suggest that Ter, Iha, and urease may contribute to EHEC O157:H7 pathogenesis by promoting adherence of the pathogen to the host intestinal epithelium.

scholarly journals Increased Pho Regulon Activation Correlates with Decreased Virulence of an Avian Pathogenic Escherichia coli O78 Strain

2010 ◽  
Vol 78 (12) ◽  
pp. 5324-5331 ◽  
Author(s):  
Nicolas Bertrand ◽  
Sébastien Houle ◽  
Guillaume LeBihan ◽  
Édith Poirier ◽  
Charles M. Dozois ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Regulatory System ◽  
Rabbit Serum ◽  
Infection Model ◽  
Pho Regulon ◽  
Wild Type ◽  
Mutant Strains ◽  
Pst System

ABSTRACT Avian pathogenic Escherichia coli (APEC) strains are associated with respiratory infections, septicemia, cellulitis, peritonitis, and other conditions, since colibacillosis manifests in many ways. The Pho regulon is jointly controlled by the two-component regulatory system PhoBR and by the phosphate-specific transport (Pst) system. To determine the specific roles of the PhoBR regulon and the Pst system in the pathogenesis of the APEC O78 strain χ7122, different phoBR and pst mutant strains were tested in vivo in chickens and in vitro for virulence traits. Mutations resulting in constitutive activation of the Pho regulon rendered strains more sensitive than the wild type to hydrogen peroxide and to the bactericidal effects of rabbit serum. In addition, production of type 1 fimbriae was also impaired in these strains. Using a chicken competitive infection model, all PhoB constitutive mutants were outcompeted by the wild-type parent, including strains containing a functional Pst system. Cumulative inactivation of the Pst system and the PhoB regulator resulted in a restoration of virulence. In addition, loss of the PhoB regulator alone did not affect virulence in the chicken infection model. Interestingly, the level of attenuation of the mutant strains correlated directly with the level of activation of the Pho regulon. Overall, results indicate that activation of the Pho regulon rather than phosphate transport by the Pst system plays a major role in the attenuation of the APEC O78 strain χ7122.

scholarly journals Cross-subunit catalysis and a new phenomenon of recessive resurrection in Escherichia coli RNase E

2019 ◽  
Vol 48 (2) ◽  
pp. 847-861 ◽  
Author(s):  
Nida Ali ◽  
Jayaraman Gowrishankar
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Rna Binding ◽  
Initial Step ◽  
Dominant Negative ◽  
Rnase E ◽  
Wild Type ◽  
Catalytic Active Site

Abstract RNase E is a 472-kDa homo-tetrameric essential endoribonuclease involved in RNA processing and turnover in Escherichia coli. In its N-terminal half (NTH) is the catalytic active site, as also a substrate 5′-sensor pocket that renders enzyme activity maximal on 5′-monophosphorylated RNAs. The protein's non-catalytic C-terminal half (CTH) harbours RNA-binding motifs and serves as scaffold for a multiprotein degradosome complex, but is dispensable for viability. Here, we provide evidence that a full-length hetero-tetramer, composed of a mixture of wild-type and (recessive lethal) active-site mutant subunits, exhibits identical activity in vivo as the wild-type homo-tetramer itself (‘recessive resurrection’). When all of the cognate polypeptides lacked the CTH, the active-site mutant subunits were dominant negative. A pair of C-terminally truncated polypeptides, which were individually inactive because of additional mutations in their active site and 5′-sensor pocket respectively, exhibited catalytic function in combination, both in vivo and in vitro (i.e. intragenic or allelic complementation). Our results indicate that adjacent subunits within an oligomer are separately responsible for 5′-sensing and cleavage, and that RNA binding facilitates oligomerization. We propose also that the CTH mediates a rate-determining initial step for enzyme function, which is likely the binding and channelling of substrate for NTH’s endonucleolytic action.

scholarly journals Validation of a Noninvasive, Real-Time Imaging Technology Using Bioluminescent Escherichia coli in the Neutropenic Mouse Thigh Model of Infection

2001 ◽  
Vol 45 (1) ◽  
pp. 129-137 ◽  
Author(s):  
H. L. Rocchetta ◽  
C. J. Boylan ◽  
J. W. Foley ◽  
P. W. Iversen ◽  
D. L. LeTourneau ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Antimicrobial Agents ◽  
Viable Cell ◽  
Multicopy Plasmid ◽  
In Vivo Evaluation ◽  
Therapeutic Success ◽  
Cell Counts

ABSTRACT A noninvasive, real-time detection technology was validated for qualitative and quantitative antimicrobial treatment applications. Thelux gene cluster of Photorhabdus luminescenswas introduced into an Escherichia coli clinical isolate, EC14, on a multicopy plasmid. This bioluminescent reporter bacterium was used to study antimicrobial effects in vitro and in vivo, using the neutropenic-mouse thigh model of infection. Bioluminescence was monitored and measured in vitro and in vivo with an intensified charge-coupled device (ICCD) camera system, and these results were compared to viable-cell determinations made using conventional plate counting methods. Statistical analysis demonstrated that in the presence or absence of antimicrobial agents (ceftazidime, tetracycline, or ciprofloxacin), a strong correlation existed between bioluminescence levels and viable cell counts in vitro and in vivo. Evaluation of antimicrobial agents in vivo could be reliably performed with either method, as each was a sound indicator of therapeutic success. Dose-dependent responses could also be detected in the neutropenic-mouse thigh model by using either bioluminescence or viable-cell counts as a marker. In addition, the ICCD technology was examined for the benefits of repeatedly monitoring the same animal during treatment studies. The ability to repeatedly measure the same animals reduced variability within the treatment experiments and allowed equal or greater confidence in determining treatment efficacy. This technology could reduce the number of animals used during such studies and has applications for the evaluation of test compounds during drug discovery.

scholarly journals Why Does Escherichia coli Grow More Slowly on Glucosamine than on N-Acetylglucosamine? Effects of Enzyme Levels and Allosteric Activation of GlcN6P Deaminase (NagB) on Growth Rates

2005 ◽  
Vol 187 (9) ◽  
pp. 2974-2982 ◽  
Author(s):  
Laura I. Álvarez-Añorve ◽  
Mario L. Calcagno ◽  
Jacqueline Plumbridge
Keyword(s):  
Escherichia Coli ◽  
Growth Rates ◽  
Sugar Transport ◽  
Point Mutations ◽  
Sole Source ◽  
Wild Type ◽  
Phosphotransferase System ◽  
Allosteric Activation

ABSTRACT Wild-type Escherichia coli grows more slowly on glucosamine (GlcN) than on N-acetylglucosamine (GlcNAc) as a sole source of carbon. Both sugars are transported by the phosphotransferase system, and their 6-phospho derivatives are produced. The subsequent catabolism of the sugars requires the allosteric enzyme glucosamine-6-phosphate (GlcN6P) deaminase, which is encoded by nagB, and degradation of GlcNAc also requires the nagA-encoded enzyme, N-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase. We investigated various factors which could affect growth on GlcN and GlcNAc, including the rate of GlcN uptake, the level of induction of the nag operon, and differential allosteric activation of GlcN6P deaminase. We found that for strains carrying a wild-type deaminase (nagB) gene, increasing the level of the NagB protein or the rate of GlcN uptake increased the growth rate, which showed that both enzyme induction and sugar transport were limiting. A set of point mutations in nagB that are known to affect the allosteric behavior of GlcN6P deaminase in vitro were transferred to the nagB gene on the Escherichia coli chromosome, and their effects on the growth rates were measured. Mutants in which the substrate-induced positive cooperativity of NagB was reduced or abolished grew even more slowly on GlcN than on GlcNAc or did not grow at all on GlcN. Increasing the amount of the deaminase by using a nagC or nagA mutation to derepress the nag operon improved growth. For some mutants, a nagA mutation, which caused the accumulation of the allosteric activator GlcNAc6P and permitted allosteric activation, had a stronger effect than nagC. The effects of the mutations on growth in vivo are discussed in light of their in vitro kinetics.

scholarly journals In Vivo Demonstration of FNR Dimers in Response to Lower O2 Availability

2007 ◽  
Vol 189 (7) ◽  
pp. 2930-2932 ◽  
Author(s):  
Adrian J. Jervis ◽  
Jeffrey Green
Keyword(s):  
Escherichia Coli ◽  
Transcription Factor ◽  
Current Model ◽  
In Vitro Studies ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
Iron Sulfur ◽  
Two Hybrid

ABSTRACT Escherichia coli FNR is an O2-sensing transcription factor. In vitro studies indicate that anaerobic iron-sulfur cluster acquisition promotes FNR dimerization. Here, two-hybrid assays show that iron-sulfur cluster-dependent FNR dimers are formed in vivo in response to lower O2 availability, consistent with the current model of FNR activation.

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