Physical mapping of repetitive extragenic palindromic sequences in Escherichia coli and phylogenetic distribution among Escherichia coli strains and other enteric bacteria.

Three enzymes are required for N-acetylglucosamine (NAG) utilization in Escherichia coli: enzyme IInag (gene nagE), N-acetylglucosamine-6-phosphate deacetylase (gene nagA), and glucosamine-6-phosphate isomerase (gene nagB). The three genes are located near 16 min on the E. coli chromosome. A strain of E. coli, KPN9, incapable of utilizing N-acetylglucosamine, was used to screen a genomic library of E. coli for a complementing recombinant colicin E1 plasmid that allowed for growth on N-acetylglucosamine. Plasmid pLC5-21 was found to contain all three known nag genes on a 5.7-kilobase (5.7-kb) fragment of DNA. The products of these nag genes were identified by complementation of E. coli strains with mutations in nagA, nagB, and nagE. The gene products from the 5.7-kb fragment were identified by [35S]methionine-labelled maxicells and autoradiography of sodium dodecyl sulphate – polyacrylamide electrophoresis gels. The gene products had the following relative masses (Mrs: nagE, 62 000; nagA, 45 000; nagB, 29 000. In addition, another product of Mr 44 000 was detected. The genes have been sequenced to reveal an additional open reading frame (nagC), a putative catabolite activator protein binding site that may control nagB and nagE, putative rho-independent terminator sites for nagB and nagE, and sequence homologies for RNA polymerase binding sites preceding each of the open reading frames, except for nagA. The calculated molecular weights (MWs) of the gene products derived from the sequence are as follows: nagA, 40 954; nagB, 29 657; nagC, 44 664; nagE, 68 356. No role is known for nagC, although a number of regulatory roles appear to be plausible. No obvious transcriptional termination site distal to nagC was found and another open reading frame begins after nagC. This gene, nagD, was isolated separately from pLC5-21, and the sequence revealed a protein with a calculated MW of 27 181. The nagD gene is followed by repetitive extragenic palindromic sequences. The nag genes appear to be organized in an operon: [Formula: see text]Key words: N-acetylglucosamine, N-acetylglucosamine-6-P deacetylase, glucosamine-6-P isomerase, repetitive extragenic palindromic sequences, catabolite repression.

Download Full-text

Genetic and physical mapping of the mcrA (rglA) and mcrB (rglB) loci of Escherichia coli K-12.

Genetics ◽

10.1093/genetics/122.2.279 ◽

1989 ◽

Vol 122 (2) ◽

pp. 279-296 ◽

Cited By ~ 4

Author(s):

E A Raleigh ◽

R Trimarchi ◽

H Revel

Keyword(s):

Escherichia Coli ◽

Physical Mapping ◽

Gene Order ◽

Enteric Bacteria ◽

Genetic And Physical Mapping ◽

Specific Restriction ◽

K 12

Abstract We have genetically analyzed, cloned and physically mapped the modified cytosine-specific restriction determinants mcrA (rglA) and mcrB (rglB) of Escherichia coli K-12. The independently discovered Rgl and Mcr restriction systems are shown to be identical by three criteria: 1) mutants with the RglA- or RglB- phenotypes display the corresponding McrA- or McrB- phenotypes, and vice versa; 2) the gene(s) for RglA and McrA reside together at one locus, while gene(s) for RglB and McrB are coincident at a different locus; and 3) RglA+ and RglB+ recombinant clones complement for the corresponding Mcr-deficient lesions. The mcrA (rglA) gene(s) is on the excisable element e14, just clockwise of purB at 25 min. The mcrB (rglB) gene(s), at 99 min, is in a cluster of restriction functions that includes hsd and mrr, determinants of host-specific restriction (EcoK) and methyladenine-specific restriction respectively. Gene order is mcrB-hsdS-hsdM-hsdR-mrr-serB. Possible models for the acqusition of these restriction determinants by enteric bacteria are discussed.

Download Full-text

Genetic but No Phenotypic Associations between Biocide Tolerance and Antibiotic Resistance in Escherichia coli from German Broiler Fattening Farms

Microorganisms ◽

10.3390/microorganisms9030651 ◽

2021 ◽

Vol 9 (3) ◽

pp. 651

Author(s):

Alice Roedel ◽

Szilvia Vincze ◽

Michaela Projahn ◽

Uwe Roesler ◽

Caroline Robé ◽

...

Keyword(s):

Escherichia Coli ◽

Antibiotic Resistance ◽

Acquired Resistance ◽

Antibiotic Resistance Genes ◽

Animal Husbandry ◽

Metal Resistance ◽

Resistant Bacteria ◽

Virulence Determinants ◽

Phylogenetic Distribution ◽

Resistance Determinants

Biocides are frequently applied as disinfectants in animal husbandry to prevent the transmission of drug-resistant bacteria and to control zoonotic diseases. Concerns have been raised, that their use may contribute to the selection and persistence of antimicrobial-resistant bacteria. Especially, extended-spectrum β-lactamase- and AmpC β-lactamase-producing Escherichia coli have become a global health threat. In our study, 29 ESBL-/AmpC-producing and 64 NON-ESBL-/AmpC-producing E.coli isolates from three German broiler fattening farms collected in 2016 following regular cleaning and disinfection were phylogenetically characterized by whole genome sequencing, analyzed for phylogenetic distribution of virulence-associated genes, and screened for determinants of and associations between biocide tolerance and antibiotic resistance. Of the 30 known and two unknown sequence types detected, ST117 and ST297 were the most common genotypes. These STs are recognized worldwide as pandemic lineages causing disease in humans and poultry. Virulence determinants associated with extraintestinal pathogenic E.coli showed variable phylogenetic distribution patterns. Isolates with reduced biocide susceptibility were rarely found on the tested farms. Nine isolates displayed elevated MICs and/or MBCs of formaldehyde, chlorocresol, peroxyacetic acid, or benzalkonium chloride. Antibiotic resistance to ampicillin, trimethoprim, and sulfamethoxazole was most prevalent. The majority of ESBL-/AmpC-producing isolates carried blaCTX-M (55%) or blaCMY-2 (24%) genes. Phenotypic biocide tolerance and antibiotic resistance were not interlinked. However, biocide and metal resistance determinants were found on mobile genetic elements together with antibiotic resistance genes raising concerns that biocides used in the food industry may lead to selection pressure for strains carrying acquired resistance determinants to different antimicrobials.

Download Full-text

Functional Analysis of Genes Comprising the Locus of Heat Resistance in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.01400-17 ◽

2017 ◽

Vol 83 (20) ◽

Cited By ~ 10

Author(s):

Ryan Mercer ◽

Oanh Nguyen ◽

Qixing Ou ◽

Lynn McMullen ◽

Michael G. Gänzle

Keyword(s):

Escherichia Coli ◽

Heat Shock ◽

Heat Shock Proteins ◽

Heat Resistance ◽

Growth Phase ◽

Genomic Island ◽

Enteric Bacteria ◽

Content Type ◽

E Coli ◽

Shock Proteins

ABSTRACT The locus of heat resistance (LHR) is a 15- to 19-kb genomic island conferring exceptional heat resistance to organisms in the family Enterobacteriaceae, including pathogenic strains of Salmonella enterica and Escherichia coli. The complement of LHR-comprising genes that is necessary for heat resistance and the stress-induced or growth-phase-induced expression of LHR-comprising genes are unknown. This study determined the contribution of the seven LHR-comprising genes yfdX1 GI, yfdX2, hdeD GI, orf11, trx GI, kefB, and psiE GI by comparing the heat resistances of E. coli strains harboring plasmid-encoded derivatives of the different LHRs in these genes. (Genes carry a subscript “GI” [genomic island] if an ortholog of the same gene is present in genomes of E. coli.) LHR-encoded heat shock proteins sHSP20, ClpKGI, and sHSPGI are not sufficient for the heat resistance phenotype; YfdX1, YfdX2, and HdeD are necessary to complement the LHR heat shock proteins and to impart a high level of resistance. Deletion of trx GI, kefB, and psiE GI from plasmid-encoded copies of the LHR did not significantly affect heat resistance. The effect of the growth phase and the NaCl concentration on expression from the putative LHR promoter p2 was determined by quantitative reverse transcription-PCR and by a plasmid-encoded p2:GFP promoter fusion. The expression levels of exponential- and stationary-phase E. coli cells were not significantly different, but the addition of 1% NaCl significantly increased LHR expression. Remarkably, LHR expression in E. coli was dependent on a chromosomal copy of evgA. In conclusion, this study improved our understanding of the genes required for exceptional heat resistance in E. coli and factors that increase their expression in food. IMPORTANCE The locus of heat resistance (LHR) is a genomic island conferring exceptional heat resistance to several foodborne pathogens. The exceptional level of heat resistance provided by the LHR questions the control of pathogens by current food processing and preparation techniques. The function of LHR-comprising genes and their regulation, however, remain largely unknown. This study defines a core complement of LHR-encoded proteins that are necessary for heat resistance and demonstrates that regulation of the LHR in E. coli requires a chromosomal copy of the gene encoding EvgA. This study provides insight into the function of a transmissible genomic island that allows otherwise heat-sensitive enteric bacteria, including pathogens, to lead a thermoduric lifestyle and thus contributes to the detection and control of heat-resistant enteric bacteria in food.

Download Full-text

Multiplex Polymerase Chain Reaction Assay for Identification of Enterotoxigenic Escherichia Coli Strains

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063870101300405 ◽

2001 ◽

Vol 13 (4) ◽

pp. 308-311 ◽

Cited By ~ 22

Author(s):

Jacek Osek

Keyword(s):

Escherichia Coli ◽

Polymerase Chain Reaction ◽

Multiplex Polymerase Chain Reaction ◽

Direct Determination ◽

Enteric Bacteria ◽

Enterotoxigenic Escherichia Coli ◽

Chain Reaction ◽

Gram Negative ◽

E Coli ◽

Polymerase Chain

A multiplex polymerase chain reaction (PCR) system was developed for identification of enterotoxigenic Escherichia coli (ETEC) strains and to differentiate them from other gram negative enteric bacteria. This test simultaneously amplifies heat-labile (LTI) and heat-stable (STI and STII) toxin sequences and the E. coli-specific universal stress protein ( uspA). The specificity of the method was validated by single PCR tests performed with the reference E. coli and non- E. coli strains and with bacteria isolated from pig feces. The multiplex PCR allowed the rapid and specific identification of enterotoxin-positive E. coli and may be used as a method for direct determination of ETEC and to differentiate them from other E. coli and gram-negative enteric isolates.

Download Full-text

Bacterial repetitive extragenic palindromic sequences are DNA targets for Insertion Sequence elements

BMC Genomics ◽

10.1186/1471-2164-7-62 ◽

2006 ◽

Vol 7 (1) ◽

Cited By ~ 60

Author(s):

Raquel Tobes ◽

Eduardo Pareja

Keyword(s):

Insertion Sequence ◽

Sequence Elements ◽

Palindromic Sequences ◽

Insertion Sequence Elements ◽

Repetitive Extragenic Palindromic

Download Full-text

The Escherichia coli K-12 NarL and NarP Proteins Insulate the nrf Promoter from the Effects of Integration Host Factor

Journal of Bacteriology ◽

10.1128/jb.00975-06 ◽

2006 ◽

Vol 188 (21) ◽

pp. 7449-7456 ◽

Cited By ~ 21

Author(s):

Douglas F. Browning ◽

David J. Lee ◽

Alan J. Wolfe ◽

Jeffrey A. Cole ◽

Stephen J. W. Busby

Keyword(s):

Escherichia Coli ◽

Response Regulator ◽

Regulatory Region ◽

Enteric Bacteria ◽

Integration Host Factor ◽

Host Factor ◽

Receptor Protein ◽

E Coli ◽

Serovar Typhimurium ◽

K 12

ABSTRACT The Escherichia coli K-12 nrf operon promoter can be activated fully by the FNR protein (regulator of fumarate and nitrate reduction) binding to a site centered at position −41.5. FNR-dependent transcription is suppressed by integration host factor (IHF) binding at position −54, and this suppression is counteracted by binding of the NarL or NarP response regulator at position −74.5. The E. coli acs gene is transcribed from a divergent promoter upstream from the nrf operon promoter. Transcription from the major acsP2 promoter is dependent on the cyclic AMP receptor protein and is modulated by IHF and Fis binding at multiple sites. We show that IHF binding to one of these sites, located at position −127 with respect to the nrf promoter, has a positive effect on nrf promoter activity. This activation is dependent on the face of the DNA helix, independent of IHF binding at other locations, and found only when NarL/NarP are not bound at position −74.5. Binding of NarL/NarP appears to insulate the nrf promoter from the effects of IHF. The acs-nrf regulatory region is conserved in other pathogenic E. coli strains and related enteric bacteria but differs in Salmonella enterica serovar Typhimurium.

Download Full-text

Use of the polymerase chain reaction for physical mapping of Escherichia coli genes.

Journal of Bacteriology ◽

10.1128/jb.173.17.5253-5255.1991 ◽

1991 ◽

Vol 173 (17) ◽

pp. 5253-5255 ◽

Cited By ~ 6

Author(s):

J Versalovic ◽

T Koeuth ◽

E R McCabe ◽

J R Lupski

Keyword(s):

Escherichia Coli ◽

Polymerase Chain Reaction ◽

Physical Mapping ◽

Chain Reaction ◽

Polymerase Chain

Download Full-text

Typing of avian pathogenic Escherichia coli strains by REP-PCR

Pesquisa Veterinária Brasileira ◽

10.1590/s0100-736x2006000200002 ◽

2006 ◽

Vol 26 (2) ◽

pp. 69-73 ◽

Cited By ~ 4

Author(s):

Marcelo Brocchi ◽

Alessandra Ferreira ◽

Marcelo Lancellotti ◽

Eliana G. Stehling ◽

Tatiana A. Campos ◽

...

Keyword(s):

Escherichia Coli ◽

Clonal Structure ◽

Avian Pathogenic Escherichia Coli ◽

Chain Reaction ◽

Discriminating Power ◽

Pathogenic Escherichia Coli ◽

Molecular Sizes ◽

Polymerase Chain ◽

Clonal Variability ◽

Repetitive Extragenic Palindromic

In the present study the repetitive extragenic palindromic (REP) polymerase chain reaction (PCR) technique was used to establish the clonal variability of 49 avian Escherichia coli (APEC) strains isolated from different outbreak cases of septicemia (n=24), swollen head syndrome (n=14) and omphalitis (n=11). Thirty commensal strains isolated from poultry with no signs of these illnesses were used as control strains. The purified DNA of these strains produced electrophoretic profiles ranging from 0 to 15 bands with molecular sizes varying from 100 bp to 6.1 kb, allowing the grouping of the 79 strains into a dendrogram containing 49 REP-types. Although REP-PCR showed good discriminating power it was not able to group the strains either into specific pathogenic classes or to differentiate between pathogenic and non-pathogenic strains. On the contrary, we recently demonstrated that other techniques such as ERIC-PCR and isoenzyme profiles are appropriate to discriminate between commensal and APEC strains and also to group these strains into specific pathogenic classes. In conclusion, REP-PCR seems to be a technique neither efficient nor universal for APEC strains discrimination. However, the population clonal structure obtained with the use of REP-PCR must not be ignored particularly if one takes into account that the APEC pathogenic mechanisms are not completely understood yet.

Download Full-text

Identifying Host Sources of Fecal Pollution: Diversity of Escherichia coli in Confined Dairy and Swine Production Systems

Applied and Environmental Microbiology ◽

10.1128/aem.71.10.5992-5998.2005 ◽

2005 ◽

Vol 71 (10) ◽

pp. 5992-5998 ◽

Cited By ~ 33

Author(s):

Zexun Lu ◽

David Lapen ◽

Andrew Scott ◽

Angela Dang ◽

Edward Topp

Keyword(s):

Escherichia Coli ◽

Dairy Farm ◽

Sampling Strategy ◽

Farming Systems ◽

Microbial Source Tracking ◽

Fecal Pollution ◽

Source Tracking ◽

E Coli ◽

Repetitive Extragenic Palindromic Pcr ◽

Repetitive Extragenic Palindromic

ABSTRACT Repetitive extragenic palindromic PCR fingerprinting of Escherichia coli is one microbial source tracking approach for identifying the host source origin of fecal pollution in aquatic systems. The construction of robust known-source libraries is expensive and requires an informed sampling strategy. In many types of farming systems, waste is stored for several months before being released into the environment. In this study we analyzed, by means of repetitive extragenic palindromic PCR using the enterobacterial repetitive intergenic consensus primers and comparative analysis using the Bionumerics software, collections of E. coli obtained from a dairy farm and from a swine farm, both of which stored their waste as a slurry in holding tanks. In all fecal samples, obtained from either barns or holding tanks, the diversity of the E. coli populations was underrepresented by collections of 500 isolates. In both the dairy and the swine farms, the diversity of the E. coli community was greater in the manure holding tank than in the barn, when they were sampled on the same date. In both farms, a comparison of stored manure samples collected several months apart suggested that the community composition changed substantially in terms of the detected number, absolute identity, and relative abundance of genotypes. Comparison of E. coli populations obtained from 10 different locations in either holding tank suggested that spatial variability in the E. coli community should be accounted for when sampling. Overall, the diversity in E. coli populations in manure slurry storage facilities is significant and likely is problematic with respect to library construction for microbial source tracking applications.

Download Full-text