Inhibiting Escherichia coli cytochrome c nitrite reductase: voltammetry reveals an enzyme equipped for action despite the chemical challenges it may face in vivo

2006 ◽  
Vol 34 (1) ◽  
pp. 133-135 ◽  
Author(s):  
J.D. Gwyer ◽  
D.J. Richardson ◽  
J.N. Butt
Keyword(s):  
Escherichia Coli ◽  
Cytochrome C ◽  
Nitrite Reductase ◽  
Large Family ◽  
Protein Film ◽  
E Coli ◽  
Protein Film Voltammetry ◽  
Cytochrome C Nitrite Reductase ◽  
Before And After

Escherichia coli cytochrome c nitrite reductase is one of a large family of homologous enzymes that are particularly prevalent in pathogenic enterobacteria. The enzymes are periplasmic and in vivo may find themselves challenged by molecules that could enhance or compromise their performance. In the present study, we describe protein film voltammetry in which the activity of E. coli cytochrome c nitrite reductase is challenged by the presence of a number of small molecules. These results are discussed in light of the environment(s) that the enzyme may face before and after colonization of a human host.

Cytochrome c nitrite reductase: from structural to physicochemical analysis

2005 ◽  
Vol 33 (1) ◽  
pp. 137-140 ◽  
Author(s):  
B. Burlat ◽  
J.D. Gwyer ◽  
S. Poock ◽  
T. Clarke ◽  
J.A. Cole ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cytochrome C ◽  
Structural Characterization ◽  
Nitrite Reductase ◽  
Structural Features ◽  
Physicochemical Analysis ◽  
Protein Film ◽  
Protein Film Voltammetry ◽  
Cytochrome C Nitrite Reductase

The recent structural characterization of the NrfA from Escherichia coli provides a framework to rationalize the spectroscopic and functional properties of this enzyme. Analyses by EPR and magnetic CD spectroscopies have been complemented by protein-film voltammetry and these are discussed in relation to the essential structural features of the enzyme.

scholarly journals The histidine of the c-type cytochrome CXXCH haem-binding motif is essential for haem attachment by the Escherichia coli cytochrome c maturation (Ccm) apparatus

2005 ◽  
Vol 389 (2) ◽  
pp. 587-592 ◽  
Author(s):  
James W. A. Allen ◽  
Nicholas Leach ◽  
Stuart J. Ferguson
Keyword(s):  
Escherichia Coli ◽  
Cytochrome C ◽  
Covalent Attachment ◽  
Binding Motif ◽  
Binding Motifs ◽  
Cytochrome C Maturation ◽  
E Coli ◽  
Cytochrome B562

c-type cytochromes are characterized by covalent attachment of haem to the protein by two thioether bonds formed between the haem vinyl groups and the cysteine sulphurs in a CXXCH peptide motif. In Escherichia coli and many other Gram-negative bacteria, this post-translational haem attachment is catalysed by the Ccm (cytochrome c maturation) system. The features of the apocytochrome substrate required and recognized by the Ccm apparatus are uncertain. In the present study, we report investigations of maturation of cytochrome b562 variants containing CXXCR, CXXCK or CXXCM haem-binding motifs. None of them showed any evidence for correct maturation by the Ccm system. However, we have determined, for each variant, that the proteins (i) were expressed in large amounts, (ii) could bind haem in vivo and/or in vitro and (iii) were not degraded in the cell. Together with previous observations, these results strongly suggest that the apocytochrome substrate feature recognized by the Ccm system is simply the two cysteine residues and the histidine of the CXXCH haem-binding motif. Using the same experimental approach, we have also investigated a cytochrome b562 variant containing the special CWSCK motif that binds the active-site haem of E. coli nitrite reductase NrfA. Whereas a CWSCH analogue was matured by the Ccm apparatus in large amounts, the CWSCK form was not detectably matured either by the Ccm system or by the dedicated Nrf biogenesis proteins, implying that the substrate recognition features for haem attachment in NrfA may be more extensive than the CWSCK motif.

scholarly journals Assessment of Probiotic Potential and Antagonistic Characteristics of Bifidobacterium bifidum Isolated from Infant Feces against Enteropathogenic Escherichia coli

2019 ◽  
Author(s):  
Muhammad Ashraf ◽  
Rehana Kanwal ◽  
Iahtasham Khan ◽  
Sadia Munir ◽  
Rizwan Aslam ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bifidobacterium Bifidum ◽  
Mice Model ◽  
E Coli ◽  
Beneficial Effects ◽  
Probiotic Potential ◽  
Before And After ◽  
Agar Well Diffusion Assay

Enteropathogenic microorganisms like Escherichia coli cause severe intestinal problems by disrupting the gut homeostasis. The live microorganisms, when given in adequate quantities provide several beneficial effects to the host are known as probiotics. One of the pronounced benefits conferred by probiotic is to antagonize the growth of enteropathogens competing for adherence to the intestinal epithelium. Bifidobacterium is the major genera of human especially infant are intestinal microbiota. In current study, Bifidobacterium bifidum was isolated from the infant stools and probiotic potential was assessed using prescribed tolerance tests against low pH, gastric juices and bile salts. Anti-infectious activity of probiotic Bifidobacterium bifidum against enteropathogenic E. coli was checked both in vitro and in vivo using agar well diffusion assay and mice model respectively. Mice feces were evaluated for both Bifidobacterium bifidum and E. coli counts in all groups and analyzed statistically. In vitro results showed Bifidobacterium bifidum possess marked antibacterial activity against E. coli. There was significant decrease in enteropathogenic E. coli burden in the mice group fed with Bifidobacterium bifidum before and after challenge. In conclusion, the endogenous Bifidobacterium bifidum have excellent probiotic potential and can be used prophylactic and treatment option against enteropathogens.

Escherichia coli Cytochrome c Nitrite Reductase NrfA

2008 ◽  
pp. 63-77 ◽  
Author(s):  
Thomas A. Clarke ◽  
Paul C. Mills ◽  
Susie R. Poock ◽  
Julea N. Butt ◽  
Myles R. Cheesman ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cytochrome C ◽  
Nitrite Reductase ◽  
Cytochrome C Nitrite Reductase

scholarly journals Characterization and in Vivo Cloning of prlC, a Suppressor of Signal Sequence Mutations in Escherichia coli K12

Genetics ◽  
1987 ◽  
Vol 116 (4) ◽  
pp. 513-521
Author(s):  
Nancy J Trun ◽  
Thomas J Silhavy
Keyword(s):  
Escherichia Coli ◽  
Genetic Mapping ◽  
Signal Sequence ◽  
Illegitimate Recombination ◽  
Mutant Phenotype ◽  
E Coli ◽  
Physical Location ◽  
Sequence Deletion ◽  
New Alleles

ABSTRACT The prlC gene of E. coli was originally identified as an allele, prlC1, which suppresses certain signal sequence mutations in the genes for several exported proteins. We have isolated six new alleles of prlC that also confer this phenotype. These mutations can be placed into three classes based on the degree to which they suppress the lamBsignal sequence deletion, lamBs78. Genetic mapping reveals that the physical location of the mutations in prlC correlates with the strength of the suppression, suggesting that different regions of the gene can be altered to yield a suppressor phenotype. We also describe an in vivo cloning procedure using λplacMu9H. The procedure relies on transposition and illegitimate recombination to generate a specialized transducing phage that carries prlC1. This method should be applicable to any gene for which there is a mutant phenotype.

scholarly journals Mechanistic insights into synergy between nalidixic acid and tetracycline against clinical isolates of Acinetobacter baumannii and Escherichia coli

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Amit Gaurav ◽  
Varsha Gupta ◽  
Sandeep K. Shrivastava ◽  
Ranjana Pathania
Keyword(s):  
Escherichia Coli ◽  
Acinetobacter Baumannii ◽  
Nalidixic Acid ◽  
Bacterial Infections ◽  
Clinical Isolates ◽  
Hospital Environment ◽  
Infection Model ◽  
Drug Resistant ◽  
E Coli

AbstractThe increasing prevalence of antimicrobial resistance has become a global health problem. Acinetobacter baumannii is an important nosocomial pathogen due to its capacity to persist in the hospital environment. It has a high mortality rate and few treatment options. Antibiotic combinations can help to fight multi-drug resistant (MDR) bacterial infections, but they are rarely used in the clinics and mostly unexplored. The interaction between bacteriostatic and bactericidal antibiotics are mostly reported as antagonism based on the results obtained in the susceptible model laboratory strain Escherichia coli. However, in the present study, we report a synergistic interaction between nalidixic acid and tetracycline against clinical multi-drug resistant A. baumannii and E. coli. Here we provide mechanistic insight into this dichotomy. The synergistic combination was studied by checkerboard assay and time-kill curve analysis. We also elucidate the mechanism behind this synergy using several techniques such as fluorescence spectroscopy, flow cytometry, fluorescence microscopy, morphometric analysis, and real-time polymerase chain reaction. Nalidixic acid and tetracycline combination displayed synergy against most of the MDR clinical isolates of A. baumannii and E. coli but not against susceptible isolates. Finally, we demonstrate that this combination is also effective in vivo in an A. baumannii/Caenorhabditis elegans infection model (p < 0.001)

scholarly journals Lysyl-tRNA synthetase from Escherichia coli K12. Chromatographic heterogeneity and the lysU-gene product

1987 ◽  
Vol 248 (1) ◽  
pp. 43-51 ◽  
Author(s):  
J Charlier ◽  
R Sanchez
Keyword(s):  
Escherichia Coli ◽  
Gene Product ◽  
Activity Ratio ◽  
Deae Cellulose ◽  
Trna Synthetase ◽  
Trna Synthetases ◽  
E Coli ◽  
Aminoacyl Trna Synthetases

In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene. During its purification from E. coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5′)tetraphospho(5′)adenosine (Ap4A) synthesis activities. Ap4A synthesis was measured by a new assay using DEAE-cellulose filters. The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage. Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected. LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not. Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock. Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1. LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+. These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product.

scholarly journals degS Is Necessary for Virulence and Is among Extraintestinal Escherichia coli Genes Induced in Murine Peritonitis

2003 ◽  
Vol 71 (6) ◽  
pp. 3088-3096 ◽  
Author(s):  
Peter Redford ◽  
Paula L. Roesch ◽  
Rodney A. Welch
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract Infection ◽  
Urinary Tract ◽  
Tract Infection ◽  
Luria Bertani ◽  
Broth Culture ◽  
Wild Type ◽  
E Coli ◽  
Virulence Attenuation

ABSTRACT Extraintestinal Escherichia coli strains cause meningitis, sepsis, urinary tract infection, and other infections outside the bowel. We examined here extraintestinal E. coli strain CFT073 by differential fluorescence induction. Pools of CFT073 clones carrying a CFT073 genomic fragment library in a promoterless gfp vector were inoculated intraperitoneally into mice; bacteria were recovered by lavage 6 h later and then subjected to fluorescence-activated cell sorting. Eleven promoters were found to be active in the mouse but not in Luria-Bertani (LB) broth culture. Three are linked to genes for enterobactin, aerobactin, and yersiniabactin. Three others are linked to the metabolic genes metA, gltB, and sucA, and another was linked to iha, a possible adhesin. Three lie before open reading frames of unknown function. One promoter is associated with degS, an inner membrane protease. Mutants of the in vivo-induced loci were tested in competition with the wild type in mouse peritonitis. Of the mutants tested, only CFT073 degS was found to be attenuated in peritoneal and in urinary tract infection, with virulence restored by complementation. CFT073 degS shows growth similar to that of the wild type at 37°C but is impaired at 43°C or in 3% ethanol LB broth at 37°C. Compared to the wild type, the mutant shows similar serum survival, motility, hemolysis, erythrocyte agglutination, and tolerance to oxidative stress. It also has the same lipopolysaccharide appearance on a silver-stained gel. The basis for the virulence attenuation is unclear, but because DegS is needed for σE activity, our findings implicate σE and its regulon in E. coli extraintestinal pathogenesis.

scholarly journals Biological Cost of Single and Multiple Norfloxacin Resistance Mutations in Escherichia coli Implicated in Urinary Tract Infections

2005 ◽  
Vol 49 (6) ◽  
pp. 2343-2351 ◽  
Author(s):  
Patricia Komp Lindgren ◽  
Linda L. Marcusson ◽  
Dorthe Sandvang ◽  
Niels Frimodt-Møller ◽  
Diarmaid Hughes
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Infection Model ◽  
Resistance Mutations ◽  
Topoisomerase Iv ◽  
E Coli ◽  
Tract Infections ◽  
Subsequent Selection

ABSTRACT Resistance to fluoroquinolones in urinary tract infection (UTIs) caused by Escherichia coli is associated with multiple mutations, typically those that alter DNA gyrase and DNA topoisomerase IV and those that regulate AcrAB-TolC-mediated efflux. We asked whether a fitness cost is associated with the accumulation of these multiple mutations. Mutants of the susceptible E. coli UTI isolate Nu14 were selected through three to five successive steps with norfloxacin. Each selection was performed with the MIC of the selected strain. After each selection the MIC was measured; and the regions of gyrA, gyrB, parC, and parE, previously associated with resistance mutations, and all of marOR and acrR were sequenced. The first selection step yielded mutations in gyrA, gyrB, and marOR. Subsequent selection steps yielded mutations in gyrA, parE, and marOR but not in gyrB, parC, or acrR. Resistance-associated mutations were identified in almost all isolates after selection steps 1 and 2 but in less than 50% of isolates after subsequent selection steps. Selected strains were competed in vitro, in urine, and in a mouse UTI infection model against the starting strain, Nu14. First-step mutations were not associated with significant fitness costs. However, the accumulation of three or more resistance-associated mutations was usually associated with a large reduction in biological fitness, both in vitro and in vivo. Interestingly, in some lineages a partial restoration of fitness was associated with the accumulation of additional mutations in late selection steps. We suggest that the relative biological costs of multiple mutations may influence the evolution of E. coli strains that develop resistance to fluoroquinolones.

A Comparison of Hand Washing Techniques To Remove Escherichia coli and Caliciviruses under Natural or Artificial Fingernails

2003 ◽  
Vol 66 (12) ◽  
pp. 2296-2301 ◽  
Author(s):  
CHIA-MIN LIN ◽  
FONE-MAO WU ◽  
HOI-KYUNG KIM ◽  
MICHAEL P. DOYLE ◽  
BARRY S. MICHAELS ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Tap Water ◽  
Hand Washing ◽  
Microbial Populations ◽  
Liquid Soap ◽  
E Coli ◽  
Hand Sanitizer ◽  
Before And After ◽  
Escherichia Coli Jm109 ◽  
Viral Indicators

Compared with other parts of the hand, the area beneath fingernails harbors the most microorganisms and is most difficult to clean. Artificial fingernails, which are usually long and polished, reportedly harbor higher microbial populations than natural nails. Hence, the efficacy of different hand washing methods for removing microbes from natural and artificial fingernails was evaluated. Strains of nonpathogenic Escherichia coli JM109 and feline calicivirus (FCV) strain F9 were used as bacterial and viral indicators, respectively. Volunteers with artificial or natural nails were artificially contaminated with ground beef containing E. coli JM109 or artificial feces containing FCV. Volunteers washed their hands with tap water, regular liquid soap, antibacterial liquid soap, alcohol-based hand sanitizer gel, regular liquid soap followed by alcohol gel, or regular liquid soap plus a nailbrush. The greatest reduction of inoculated microbial populations was obtained by washing with liquid soap plus a nailbrush, and the least reduction was obtained by rubbing hands with alcohol gel. Lower but not significantly different (P &gt; 0.05) reductions of E. coli and FCV counts were obtained from beneath artificial than from natural fingernails. However, significantly (P ≤ 0.05) higher E. coli and FCV counts were recovered from hands with artificial nails than from natural nails before and after hand washing. In addition, microbial cell numbers were correlated with fingernail length, with greater numbers beneath fingernails with longer nails. These results indicate that best practices for fingernail sanitation of food handlers are to maintain short fingernails and scrub fingernails with soap and a nailbrush when washing hands.

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