Rapid and high resolution genotyping of all Escherichia coli serotypes using 10 genomic repeat-containing loci

Bacterial cytokinesis is mediated by the Z-ring, which is formed by the prokaryotic tubulin homolog FtsZ. Recent data indicate that the Z-ring is composed of small patches of FtsZ protofilaments that travel around the bacterial cell by treadmilling. Treadmilling involves a switch from a relaxed (R) state, favored for monomers, to a tense (T) conformation, which is favored upon association into filaments. The R conformation has been observed in numerous monomeric FtsZ crystal structures and the T conformation in Staphylococcus aureus FtsZ crystallized as assembled filaments. However, while Escherichia coli has served as a main model system for the study of the Z-ring and the associated divisome, a structure has not yet been reported for E. coli FtsZ. To address this gap, structures were determined of the E. coli FtsZ mutant FtsZ(L178E) with GDP and GTP bound to 1.35 and 1.40 Å resolution, respectively. The E. coli FtsZ(L178E) structures both crystallized as straight filaments with subunits in the R conformation. These high-resolution structures can be employed to facilitate experimental cell-division studies and their interpretation in E. coli.

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Quantifying the interplay between rapid bacterial evolution within the mouse intestine and transmission between hosts

10.1101/2020.12.04.412072 ◽

2020 ◽

Author(s):

Kimberly S. Vasquez ◽

Lisa Willis ◽

Nate Cira ◽

Katharine M. Ng ◽

Miguel F. Pedro ◽

...

Keyword(s):

Escherichia Coli ◽

Population Genetics ◽

High Resolution ◽

Evolutionary Dynamics ◽

Day Treatment ◽

Whole Genome ◽

Germ Free ◽

Selection Dynamics ◽

Mouse Intestine ◽

Ciprofloxacin Resistance

SummaryDue to limitations on high-resolution strain tracking, selection dynamics during gut-microbiota colonization and transmission between hosts remain mostly mysterious. Here, we introduced hundreds of barcoded Escherichia coli strains into germ-free mice and quantified strain-level dynamics and metagenomic changes. Mutants involved in motility and utilization of abundant metabolites were reproducibly selected within days. Even with rapid selection, coprophagy enforced similar barcode distributions across co-housed mice. Whole-genome sequencing of hundreds of isolates quantified evolutionary dynamics and revealed linked alleles. A population-genetics model predicted substantial fitness advantages for certain mutants and that migration accounted for ~10% of the resident microbiota each day. Treatment with ciprofloxacin demonstrated the interplay between selection and transmission. While initial colonization was mostly uniform, in two mice a bottleneck reduced diversity and selected for ciprofloxacin resistance in the absence of drug. These findings highlight the interplay between environmental transmission and rapid, deterministic selection during evolution of the intestinal microbiota.

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Identification of Common Subpopulations of Non-Sorbitol-Fermenting, β-Glucuronidase-Negative Escherichia coli O157:H7 from Bovine Production Environments and Human Clinical Samples

Applied and Environmental Microbiology ◽

10.1128/aem.70.11.6846-6854.2004 ◽

2004 ◽

Vol 70 (11) ◽

pp. 6846-6854 ◽

Cited By ~ 94

Author(s):

Zhijie Yang ◽

Joy Kovar ◽

Jaehyoung Kim ◽

Joseph Nietfeldt ◽

David R. Smith ◽

...

Keyword(s):

Escherichia Coli ◽

High Resolution ◽

Recent Common Ancestor ◽

Clinical Samples ◽

Genome Scanning ◽

Large Sets ◽

Marker Test ◽

Production Environments ◽

Different Populations ◽

Phylogenetic Lineages

ABSTRACT Non-sorbitol-fermenting, β-glucuronidase-negative Escherichia coli O157:H7 strains are regarded as a clone complex, and populations from different geographical locations are believed to share a recent common ancestor. Despite their relatedness, high-resolution genotyping methods can detect significant genome variation among different populations. Phylogenetic analysis of high-resolution genotyping data from these strains has shown that subpopulations from geographically unlinked continents can be divided into two primary phylogenetic lineages, termed lineage I and lineage II, and limited studies of the distribution of these lineages suggest there could be differences in their propensity to cause disease in humans or to be transmitted to humans. Because the genotyping methods necessary to discriminate the two lineages are tedious and subjective, these methods are not particularly suited for studying the large sets of strains that are required to systematically evaluate the ecology and transmission characteristics of these lineages. To overcome this limitation, we have developed a lineage-specific polymorphism assay (LSPA) that can readily distinguish between the lineage I and lineage II subpopulations. In the studies reported here, we describe the development of a six-marker test (LSPA-6) and its validation in a side-by-side comparison with octamer-based genome scanning. Analysis of over 1,400 O157:H7 strains with the LSPA-6 demonstrated that five genotypes comprise over 91% of the strains, suggesting that these subpopulations may be widespread.

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Cloning of trg, a gene for a sensory transducer in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.152.1.372-383.1982 ◽

1982 ◽

Vol 152 (1) ◽

pp. 372-383

Author(s):

S Harayama ◽

P Engström ◽

H Wolf-Watz ◽

T Iino ◽

G L Hazelbauer

Keyword(s):

Escherichia Coli ◽

High Resolution ◽

Sodium Dodecyl Sulfate ◽

Structural Gene ◽

Preliminary Analysis ◽

Sodium Dodecyl ◽

Polyacrylamide Gels ◽

Physiological Functions ◽

The Common ◽

Dependent Modification

Clones of trg, a gene which codes for a chemotactic transducer, were isolated linked to ColE1 and pBR322 vectors. Studies with the hybrid plasmids demonstrated unequivocally that trg is the structural gene for methyl-accepting chemotaxis protein III. The Trg protein was found to be structurally complex, electrophoresing as a series of seven bands on high-resolution sodium dodecyl sulfate-polyacrylamide gels. The multiplicity of bands is a function of the activity of cheR, which codes for a methyltransferase, and of cheB, which codes for a demethylase. It appears that Trg, a quantitatively minor transducer, resembles the two major transducer proteins, Tsr and Tar, in that all three are multiply methylated and also multiply modified in a second way which requires an active cheB gene. However, preliminary analysis of the Trg protein indicated that it is significantly less related structurally to the Tsr or Tar protein than those two transducers are to each other. This implies that the features of multiple methylation and cheB-dependent modification are likely to be critical for the common physiological functions in chemotactic excitation and adaptation performed by all three transducers.

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Predictive Fluorescent Amplified-Fragment Length Polymorphism Analysis of Escherichia coli: High-Resolution Typing Method with Phylogenetic Significance

Journal of Clinical Microbiology ◽

10.1128/jcm.37.5.1274-1279.1999 ◽

1999 ◽

Vol 37 (5) ◽

pp. 1274-1279 ◽

Cited By ~ 42

Author(s):

Catherine Arnold ◽

Lou Metherell ◽

Geraldine Willshaw ◽

Anthony Maggs ◽

John Stanley

Keyword(s):

Escherichia Coli ◽

High Resolution ◽

Length Polymorphism ◽

Fragment Length ◽

Fragment Length Polymorphism ◽

Amplified Fragment Length Polymorphism ◽

Polymorphism Analysis ◽

Enzyme Electrophoresis ◽

Typing Method ◽

E Coli

The fluorescent amplified-fragment length polymorphism (FAFLP) assay potentially amplifies a unique set of genome fragments from each bacterial clone. It uses stringently hybridizing primers which carry a fluorescent label. Precise fragment sizing is achieved by the inclusion of an internal size standard in every lane. Therefore, a unique genotype identifier(s) can be found in the form of fragments of precise size or sizes, and these can be generated reproducibly. In order to evaluate the potential of FAFLP as an epidemiological typing method with a valid phylogenetic basis, we applied it to 87 strains ofEscherichia coli. These comprised the EcoR collection, which has previously been classified by multilocus enzyme electrophoresis (MLEE) and which represents the genetic diversity of the species E. coli, plus 15 strains of the clinically important serogroup O157. FAFLP with an unlabelled nonselectiveEcoRI primer (Eco+0) and a labelled selectiveMseI primer (Mse+TA) gave strain-specific profiles. Fragments of identical sizes (in base pairs) were assumed to be identical, and the genetic distances between the strains were calculated. A phylogenetic tree derived from measure of distance correlated closely with the MLEE groupings of the EcoR collection and placed the verocytotoxin-producing O157 strains on an outlier branch. Our data indicate that FAFLP is suitable for epidemiological investigation of E. coli infection, providing well-defined and reproducible identifiers of genotype for each strain. Since FAFLP objectively samples the whole genome, each strain or isolate can be assigned a place within the broad context of the whole species and can also be subjected to a high-resolution comparison with closely related strains to investigate epidemiological clonality.

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High Resolution Melt Assays to Detect and Identify Vibrio parahaemolyticus, Bacillus cereus, Escherichia coli, and Clostridioides difficile Bacteria

Microorganisms ◽

10.3390/microorganisms8040561 ◽

2020 ◽

Vol 8 (4) ◽

pp. 561

Author(s):

Allison C. Bender ◽

Jessica A. Faulkner ◽

Katherine Tulimieri ◽

Thomas H. Boise ◽

Kelly M. Elkins

Keyword(s):

Escherichia Coli ◽

High Resolution ◽

Bacillus Cereus ◽

Vibrio Parahaemolyticus ◽

Bacterial Species ◽

Human Pathogens ◽

High Resolution Melt ◽

E Coli ◽

Clostridioides Difficile ◽

Sensitivity Specificity

Over one hundred bacterial species have been determined to comprise the human microbiota in a healthy individual. Bacteria including Escherichia coli, Bacillus cereus, Clostridioides difficile, and Vibrio parahaemolyticus are found inside of the human body and B. cereus and E. coli are also found on the skin. These bacteria can act as human pathogens upon ingestion of contaminated food or water, if they enter an open wound, or antibiotics, and environment or stress can alter the microbiome. In this study, we present new polymerase chain reaction (PCR) high-resolution melt (HRM) assays to detect and identify the above microorganisms. Amplified DNA from C. difficile, E. coli, B. cereus, and V. parahaemolyticus melted at 80.37 ± 0.45 °C, 82.15 ± 0.37 °C, 84.43 ± 0.50 °C, and 86.74 ± 0.65 °C, respectively. A triplex PCR assay was developed to simultaneously detect and identify E. coli, B. cereus, and V. parahaemolyticus, and cultured microorganisms were successfully amplified, detected, and identified. The assays demonstrated sensitivity, specificity, reproducibility, and robustness in testing.

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