scholarly journals Transcription Activation at Escherichia coli FNR-Dependent Promoters by the Gonococcal FNR Protein: Effects of a Novel S18F Substitution and Comparisons with the Corresponding Substitution in E. coli FNR

2003 ◽  
Vol 185 (16) ◽  
pp. 4734-4747 ◽  
Author(s):  
Tim Overton ◽  
Eleanor G. F. Reid ◽  
Robin Foxall ◽  
Harry Smith ◽  
Stephen J. W. Busby ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Neisseria Gonorrhoeae ◽  
Transcription Activation ◽  
The Other ◽  
E Coli ◽  
Iron Sulfur ◽  
Fnr Protein ◽  
Iron Sulfur Center ◽  
After Error

ABSTRACT The Neisseria gonorrhoeae genome encodes a homologue of the Escherichia coli FNR protein (the fumarate and nitrate reductase regulator). Despite its similarity to E. coli FNR, the gonococcal FNR only partially complemented an E. coli fnr mutation. After error-prone PCR mutagenesis of the gonococcal fnr gene, we identified four mutant fnr derivatives carrying the same S18F substitution, and we showed that the mutant FNR could activate transcription from a range of class I and class II FNR-dependent promoters in E. coli. Prompted by the similarities between gonococcal and E. coli FNR, we made changes in gonococcal fnr that created substitutions that are equivalent to previously characterized substitutions in E. coli FNR. First, our experiments showed that cysteine, C116, in the gonococcal FNR, equivalent to C122 in E. coli FNR, is essential, presumably because, as in E. coli FNR, it binds to an iron-sulfur center. Second, the L22H and D148A substitutions in gonococcal FNR were made. These changes are equivalent to the L28H and D154A changes in E. coli FNR, which had been shown to increase FNR activity in the presence of oxygen. We show that the effects of these substitutions in gonococcal FNR are distinct from those of the S18F substitution. Similarly, substitutions in the putative activating regions of gonococcal FNR were made. We show that the activity of gonococcal FNR in E. coli can be increased by transplanting certain activating regions from E. coli FNR. The effects of these substitutions are additive to those due to S18F. From these data, we conclude that the effects of the S18F substitution in gonococcal FNR are distinct from the effects of the other substitutions. S18 is immediately adjacent to one of three N-terminal cysteine residues that coordinate the iron-sulfur center, and thus the S18F substitution is most likely to stabilize this center. Support for this came from complementary experiments in which we created the S24F substitution in E. coli FNR, which is equivalent to the S18F substitution in gonococcal FNR. Our results show that the S24F substitution changes the activity of E. coli FNR and that the changes are distinct from those due to previously characterized substitutions.

scholarly journals Fnr-, NarP- and NarL-Dependent Regulation of Transcription Initiation from the Haemophilus influenzae Rd napF (Periplasmic Nitrate Reductase) Promoter in Escherichia coli K-12

2005 ◽  
Vol 187 (20) ◽  
pp. 6928-6935 ◽  
Author(s):  
Valley Stewart ◽  
Peggy J. Bledsoe
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Haemophilus Influenzae ◽  
Control Region ◽  
Binding Sites ◽  
Transcription Initiation ◽  
Transcriptional Control ◽  
Class I ◽  
E Coli ◽  
Fnr Protein

ABSTRACT Periplasmic nitrate reductase (napFDAGHBC operon product) functions in anaerobic respiration. Transcription initiation from the Escherichia coli napF operon control region is activated by the Fnr protein in response to anaerobiosis and by the NarQ-NarP two-component regulatory system in response to nitrate or nitrite. The binding sites for the Fnr and phospho-NarP proteins are centered at positions −64.5 and −44.5, respectively, with respect to the major transcription initiation point. The E. coli napF operon is a rare example of a class I Fnr-activated transcriptional control region, in which the Fnr protein binding site is located upstream of position −60. To broaden our understanding of napF operon transcriptional control, we studied the Haemophilus influenzae Rd napF operon control region, expressed as a napF-lacZ operon fusion in the surrogate host E. coli. Mutational analysis demonstrated that expression required binding sites for the Fnr and phospho-NarP proteins centered at positions −81.5 and −42.5, respectively. Transcription from the E. coli napF operon control region is activated by phospho-NarP but antagonized by the orthologous protein, phospho-NarL. By contrast, expression from the H. influenzae napF-lacZ operon fusion in E. coli was stimulated equally well by nitrate in both narP and narL null mutants, indicating that phospho-NarL and -NarP are equally effective regulators of this promoter. Overall, the H. influenzae napF operon control region provides a relatively simple model for studying synergistic transcription by the Fnr and phospho-NarP proteins acting from class I and class II locations, respectively.

scholarly journals Structural genes for nitrate-inducible formate dehydrogenase in Escherichia coli K-12.

Genetics ◽  
1990 ◽  
Vol 125 (4) ◽  
pp. 691-702 ◽  
Author(s):  
B L Berg ◽  
V Stewart
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Formate Dehydrogenase ◽  
Recombinant Dna ◽  
Structural Genes ◽  
Respiratory Pathway ◽  
E Coli ◽  
Formate Oxidation ◽  
Operon Expression ◽  
K 12

Abstract Formate oxidation coupled to nitrate reduction constitutes a major anaerobic respiratory pathway in Escherichia coli. This respiratory chain consists of formate dehydrogenase-N, quinone, and nitrate reductase. We have isolated a recombinant DNA clone that likely contains the structural genes, fdnGHI, for the three subunits of formate dehydrogenase-N. The fdnGHI clone produced proteins of 110, 32 and 20 kDa which correspond to the subunit sizes of purified formate dehydrogenase-N. Our analysis indicates that fdnGHI is organized as an operon. We mapped the fdn operon to 32 min on the E. coli genetic map, close to the genes for cryptic nitrate reductase (encoded by the narZ operon). Expression of phi(fdnG-lacZ) operon fusions was induced by anaerobiosis and nitrate. This induction required fnr+ and narL+, two regulatory genes whose products are also required for the anaerobic, nitrate-inducible activation of the nitrate reductase structural gene operon, narGHJI. We conclude that regulation of fdnGHI and narGHJI expression is mediated through common pathways.

scholarly journals Escherichia coli O157:H7 SURVIVAL IN TRADITIONAL AND LOW LACTOSE YOGURT DURING FERMENTATION AND COOLING PERIODS

2017 ◽  
Vol 18 (0) ◽  
Author(s):  
Camila Sampaio Cutrim ◽  
Raphael Ferreira de Barros ◽  
Robson Maia Franco ◽  
Marco Antonio Sloboda Cortez
Keyword(s):  
Escherichia Coli ◽  
The Other ◽  
Lactose Hydrolysis ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Gas Formation ◽  
Whole Milk ◽  
Milk Contamination ◽  
Different Types ◽  
Fermentation Period

Abstract The purpose of this study was to evaluate the behavior of E. coli O157:H7 during lactose hydrolysis and fermentation of traditional and low lactose yogurt. It also aimed to verify E. coli O157:H7 survival after 12 h of storage at 4 ºC ±1 ºC. Two different types of yogurts were prepared, two with whole milk and two with pre-hydrolyzed whole milk; in both groups one yogurt was inoculated with E. coli O157:H7 and the other one was not inoculated. The survival of E. coli and pH of yogurt were determined during fermentation and after 12-h refrigeration. The results showed that E. coli O157:H7 was able to grow during the fermentation period (from 4.34 log CFU.mL-1 to 6.13 log CFU.mL-1 in traditional yogurt and 4.34 log CFU.mL-1 to 6.16 log CFU.mL-1 in low lactose yogurt). The samples with E. coli O157:H7 showed gas formation and syneresis. Thus, E. coli O157:H7 was able to survive and grow during fermentation of traditional and low lactose yogurts affecting the manufacture technology. Moreover, milk contamination by E. coli before LAB addition reduces the growth of L. bulgaricus and S. thermophilus especially when associated with reduction of lactose content.

scholarly journals Functional Studies of [FeFe] Hydrogenase Maturation in an Escherichia coli Biosynthetic System

2006 ◽  
Vol 188 (6) ◽  
pp. 2163-2172 ◽  
Author(s):  
Paul W. King ◽  
Matthew C. Posewitz ◽  
Maria L. Ghirardi ◽  
Michael Seibert
Keyword(s):  
Escherichia Coli ◽  
Catalytic Domain ◽  
Expression System ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
E Coli ◽  
Functional Studies ◽  
Hydrogenase Maturation ◽  
Iron Sulfur ◽  
And Function

ABSTRACT Maturation of [FeFe] hydrogenases requires the biosynthesis and insertion of the catalytic iron-sulfur cluster, the H cluster. Two radical S-adenosylmethionine (SAM) proteins proposed to function in H cluster biosynthesis, HydEF and HydG, were recently identified in the hydEF-1 mutant of the green alga Chlamydomonas reinhardtii (M. C. Posewitz, P. W. King, S. L. Smolinski, L. Zhang, M. Seibert, and M. L. Ghirardi, J. Biol. Chem. 279:25711-25720, 2004). Previous efforts to study [FeFe] hydrogenase maturation in Escherichia coli by coexpression of C. reinhardtii HydEF and HydG and the HydA1 [FeFe] hydrogenase were hindered by instability of the hydEF and hydG expression clones. A more stable [FeFe] hydrogenase expression system has been achieved in E. coli by cloning and coexpression of hydE, hydF, and hydG from the bacterium Clostridium acetobutylicum. Coexpression of the C. acetobutylicum maturation proteins with various algal and bacterial [FeFe] hydrogenases in E. coli resulted in purified enzymes with specific activities that were similar to those of the enzymes purified from native sources. In the case of structurally complex [FeFe] hydrogenases, maturation of the catalytic sites could occur in the absence of an accessory iron-sulfur cluster domain. Initial investigations of the structure and function of the maturation proteins HydE, HydF, and HydG showed that the highly conserved radical-SAM domains of both HydE and HydG and the GTPase domain of HydF were essential for achieving biosynthesis of active [FeFe] hydrogenases. Together, these results demonstrate that the catalytic domain and a functionally complete set of Hyd maturation proteins are fundamental to achieving biosynthesis of catalytic [FeFe] hydrogenases.

The HiPIP from the acidophilic Acidithiobacillus ferrooxidans is correctly processed and translocated in Escherichia coli, in spite of the periplasm pH difference between these two micro-organisms

Microbiology ◽  
2005 ◽  
Vol 151 (5) ◽  
pp. 1421-1431 ◽  
Author(s):  
Patrice Bruscella ◽  
Laure Cassagnaud ◽  
Jeanine Ratouchniak ◽  
Gaël Brasseur ◽  
Elisabeth Lojou ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Acidithiobacillus Ferrooxidans ◽  
Biochemical Properties ◽  
Gene Encoding ◽  
Iron Sulfur Cluster ◽  
E Coli ◽  
Iron Sulfur ◽  
Sulfur Protein ◽  
Tat System ◽  
Micro Organisms

The gene encoding a putative high-potential iron–sulfur protein (HiPIP) from the strictly acidophilic and chemolithoautotrophic Acidithiobacillus ferrooxidans ATCC 33020 has been cloned and sequenced. This potential HiPIP was overproduced in the periplasm of the neutrophile and heterotroph Escherichia coli. As shown by optical and EPR spectra and by electrochemical studies, the recombinant protein has all the biochemical properties of a HiPIP, indicating that the iron–sulfur cluster was correctly inserted. Translocation of this protein in the periplasm of E. coli was not detected in a ΔtatC mutant, indicating that it is dependent on the Tat system. The genetic organization of the iro locus in strains ATCC 23270 and ATCC 33020 is different from that found in strains Fe-1 and BRGM. Indeed, in A. ferrooxidans ATCC 33020 and ATCC 23270 (the type strain), iro was not located downstream from purA but was instead downstream from petC2, encoding cytochrome c 1 from the second A. ferrooxidans cytochrome bc 1 complex. These findings underline the genotypic heterogeneity within the A. ferrooxidans species. The results suggest that Iro transfers electrons from a cytochrome bc 1 complex to a terminal oxidase, as proposed for the HiPIP in photosynthetic bacteria.

scholarly journals  Faecal shedding of verotoxigenic Escherichia coli in cattle in the Czech Republic

2011 ◽  
Vol 56 (No. 4) ◽  
pp. 149-155 ◽  
Author(s):  
P. Alexa ◽  
L. Konstantinova ◽  
Z. Sramkova-Zajacova
Keyword(s):  
Escherichia Coli ◽  
Czech Republic ◽  
Dairy Cattle ◽  
Age Groups ◽  
Immunomagnetic Separation ◽  
The Other ◽  
Somatic Antigen ◽  
The Czech Republic ◽  
E Coli ◽  
One Year

A survey to estimate the prevalence of verotoxigenic E. coli (VTEC) or enterohaemorrhagic E. coli (EHEC) in rectal swabs from healthy dairy cattle aged three weeks, three months and one year was conducted in three herds from the Czech Republic. Screening for the presence of the stx1, stx2 and eaeA genes in faecal swab cultures was performed by PCR, and in positive samples, isolated colonies were examined. Immunomagnetic separation was used for the isolation of the VTEC serogroup O157 from samples. VTEC were detected in animals from all three herds under study. In the group of 3-week-old calves, VTEC were only detected in samples collected in the summer months. However, in the other age-groups, VTEC were detected in both the summer and winter months. EHEC shedding was observed in 30 to 100% of the total samples collected from cattle aged three months and one year in the summer months, and in 30 to 60% of samples taken in the winter months. EHEC strains of serogroup O157 were detected in two herds. The range of verotoxins shed by VTEC isolates of serogroup O157 differed between herds. Besides serogroup O157, additional EHEC belonging to the antigen groups O26, O103, O128 and O153 have been identified, and in some of them, no somatic antigen was detected.

scholarly journals Using a Chemical Genetic Screen to Enhance Our Understanding of the Antimicrobial Properties of Gallium against Escherichia coli

Genes ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 34 ◽  
Author(s):  
Natalie Gugala ◽  
Kate Chatfield-Reed ◽  
Raymond J. Turner ◽  
Gordon Chua
Keyword(s):  
Escherichia Coli ◽  
Cell Envelope ◽  
Antimicrobial Properties ◽  
Iron Sulfur Cluster ◽  
Nucleotide Biosynthesis ◽  
E Coli ◽  
Chemical Genetic ◽  
Iron Sulfur ◽  
Insight Into ◽  
Mutant Collection

The diagnostic and therapeutic agent gallium offers multiple clinical and commercial uses including the treatment of cancer and the localization of tumors, among others. Further, this metal has been proven to be an effective antimicrobial agent against a number of microbes. Despite the latter, the fundamental mechanisms of gallium action have yet to be fully identified and understood. To further the development of this antimicrobial, it is imperative that we understand the mechanisms by which gallium interacts with cells. As a result, we screened the Escherichia coli Keio mutant collection as a means of identifying the genes that are implicated in prolonged gallium toxicity or resistance and mapped their biological processes to their respective cellular system. We discovered that the deletion of genes functioning in response to oxidative stress, DNA or iron–sulfur cluster repair, and nucleotide biosynthesis were sensitive to gallium, while Ga resistance comprised of genes involved in iron/siderophore import, amino acid biosynthesis and cell envelope maintenance. Altogether, our explanations of these findings offer further insight into the mechanisms of gallium toxicity and resistance in E. coli.

scholarly journals Extraintestinal Pathogenic and Antimicrobial-Resistant Escherichia coli, Including Sequence Type 131 (ST131), from Retail Chicken Breasts in the United States in 2013

2017 ◽  
Vol 83 (6) ◽  
Author(s):  
James R. Johnson ◽  
Stephen B. Porter ◽  
Brian Johnston ◽  
Paul Thuras ◽  
Sarah Clock ◽  
...  
Keyword(s):  
United States ◽  
Escherichia Coli ◽  
Virulence Genes ◽  
Meat Products ◽  
The United States ◽  
Sequence Type ◽  
The Other ◽  
Antibiotic Resistant ◽  
Content Type ◽  
E Coli

ABSTRACT Chicken meat products are hypothesized to be vehicles for transmitting antimicrobial-resistant and extraintestinal pathogenic Escherichia coli (ExPEC) to consumers. To reassess this hypothesis in the current era of heightened concerns about antimicrobial use in food animals, we analyzed 175 chicken-source E. coli isolates from a 2013 Consumer Reports national survey. Isolates were screened by PCR for ExPEC-defining virulence genes. The 25 ExPEC isolates (12% of 175) and a 2:1 randomly selected set of 50 non-ExPEC isolates were assessed for their phylogenetic/clonal backgrounds and virulence genotypes for comparison with their resistance profiles and the claims on the retail packaging label (“organic,” “no antibiotics,” and “natural”). Compared with the findings for non-ExPEC isolates, the group of ExPEC isolates had a higher prevalence of phylogroup B2 isolates (44% versus 4%; P < 0.001) and a lower prevalence of phylogroup A isolates (4% versus 30%; P = 0.001), a higher prevalence of multiple individual virulence genes, higher virulence scores (median, 11 [range, 4 to 16] versus 8 [range, 1 to 14]; P = 0.001), and higher resistance scores (median, 4 [range, 0 to 8] versus 3 [range, 0 to 10]; P < 0.001). All five isolates of sequence type 131 (ST131) were ExPEC (P = 0.003), were as extensively resistant as the other isolates tested, and had higher virulence scores than the other isolates tested (median, 12 [range, 11 to 13] versus 8 [range, 1 to 16]; P = 0.005). Organic labeling predicted lower resistance scores (median, 2 [range, 0 to 3] versus 4 [range, 0 to 10]; P = 0.008) but no difference in ExPEC status or virulence scores. These findings document a persisting reservoir of extensively antimicrobial-resistant ExPEC isolates, including isolates from ST131, in retail chicken products in the United States, suggesting a potential public health threat. IMPORTANCE We found that among Escherichia coli isolates from retail chicken meat products purchased across the United States in 2013 (many of these isolates being extensively antibiotic resistant), a minority had genetic profiles suggesting an ability to cause extraintestinal infections in humans, such as urinary tract infection, implying a risk of foodborne disease. Although isolates from products labeled “organic” were less extensively antibiotic resistant than other isolates, they did not appear to be less virulent. These findings suggest that retail chicken products in the United States, even if they are labeled “organic,” pose a potential health threat to consumers because they are contaminated with extensively antibiotic-resistant and, presumably, virulent E. coli isolates.

Different responses to nitrate and nitrite by the model organism Escherichia coli and the human pathogen Neisseria gonorrhoeae

2006 ◽  
Vol 34 (1) ◽  
pp. 111-114 ◽  
Author(s):  
R.N. Whitehead ◽  
J.A. Cole
Keyword(s):  
Escherichia Coli ◽  
Neisseria Gonorrhoeae ◽  
Model Organism ◽  
Anaerobic Growth ◽  
Human Pathogen ◽  
Regulate Gene Expression ◽  
E Coli ◽  
Regulatory Systems ◽  
Two Component ◽  
Nitrate And Nitrite

The ability of Escherichia coli to use both nitrate and nitrite as terminal electron acceptors during anaerobic growth is mediated by the dual-acting two-component regulatory systems NarX-NarL and NarQ-NarP. In contrast, Neisseria gonorrhoeae responds only to nitrite: it expresses only NarQ-NarP. We have shown that although N. gonorrhoeae NarQ can phosphorylate E. coli NarL and NarP, the N. gonorrhoeae NarP is unable to regulate gene expression in E. coli. Mutagenesis experiments have revealed residues in E. coli NarQ that are essential for nitrate and nitrite sensing. Chimaeric proteins revealed domains of NarQ that are important for ligand sensing.

scholarly journals NikAB- or NikB-Dependent Intracellular Recombination between Tandemly Repeated oriT Sequences of Plasmid R64 in Plasmid or Single-Stranded Phage Vectors

2003 ◽  
Vol 185 (13) ◽  
pp. 3871-3877 ◽  
Author(s):  
Nobuhisa Furuya ◽  
Teruya Komano
Keyword(s):  
Escherichia Coli ◽  
Dna Transfer ◽  
The Other ◽  
E Coli ◽  
Core Sequence ◽  
Bacterial Plasmid ◽  
Plasmid R64 ◽  
Dna Processing ◽  
Origin Of Transfer

ABSTRACT The origin of transfer (oriT) of a bacterial plasmid plays a key role in both the initiation and termination of conjugative DNA transfer. We have previously shown that a conjugation-dependent recombination between the tandem R64 oriT sequences cloned into pHSG398 occurred, resulting in the deletion of the intervening sequence during DNA transfer. In this study, we tandemly cloned two oriT sequences of IncI1 plasmid R64 into pUC18. Specific recombination between the two oriT sequences in pUC18 was observed within Escherichia coli cells harboring mini-R64. This recombination was found to be independent of both the recA gene and conjugative DNA transfer. The R64 genes nikA and nikB, required for conjugal DNA processing, were essential for this recombination. Although a fully active 92-bp oriT sequence was required at one site for the recombination, the 44-bp oriT core sequence was sufficient at the other site. Furthermore, when two oriT sequences were tandemly cloned into the single-stranded phage vector M13 and propagated within E. coli cells, recombination between the two oriT sequences was observed, depending on the nikB gene. These results suggest that the R64 relaxase protein NikB can execute cleavage and rejoining of single-stranded oriT DNA within E. coli cells, whereas such a reaction in double-stranded oriT DNA requires collaboration of the two relaxosome proteins, NikA and NikB.

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