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Toxin-antitoxin (TA) systems are plasmid- or chromosome-encoded protein complexes composed of a stable toxin and a short-lived inhibitor of the toxin. In cultures of Escherichia coli, transcription of toxin-antitoxin genes was induced in a nondividing subpopulation of bacteria that was tolerant to bactericidal antibiotics. Along with transcription of known toxin-antitoxin operons, transcription of mqsR and ygiT, two adjacent genes with multiple TA-like features, was induced in this cell population. Here we show that mqsR and ygiT encode a toxin-antitoxin system belonging to a completely new family which is represented in several groups of bacteria. The mqsR gene encodes a toxin, and ectopic expression of this gene inhibits growth and induces rapid shutdown of protein synthesis in vivo. ygiT encodes an antitoxin, which protects cells from the effects of MqsR. These two genes constitute a single operon which is transcriptionally repressed by the product of ygiT. We confirmed that transcription of this operon is induced in the ampicillin-tolerant fraction of a growing population of E. coli and in response to activation of the HipA toxin. Expression of the MqsR toxin does not kill bacteria but causes reversible growth inhibition and elongation of cells. doi: 10.1128/JB.01266-09

Double exposure Shiga toxigenic Escherichia coli causes severe gastrointestinal disease, in part mediated by subtilase cytotoxin. The B subunit of this toxin is now shown to have high affinity for glycans containing N-glycolylneuraminic acid, a saccharide that is not synthesized by humans. Instead it is ingested as part of the diet, in red meat and dairy products and subsequently incorporated into intestinal and kidney tissue. Ironically, red meat and dairy products, rich sources of N-glycolylneuraminic acid, are also the foods most commonly contaminated with the toxic bacteria. So through dietary choices, humans may both expose themselves to a pathogen and simultaneously become more susceptible to it as their tissues are sensitized to a key virulence factor. Shiga toxigenic Escherichia coli causes severe gastrointestinal disease, which is in part mediated by subtilase cytotoxin. The B subunit of this toxin is now shown to have high affinity to glycans containing N-glycolylneuraminic acid, a saccharide that is not synthesized by humans. Instead it is ingested by dietary intake of red meat and dairy products and subsequently incorporated into intestinal and kidney tissue. AB.sub.5 toxins comprise an A subunit that corrupts essential eukaryotic cell functions, and pentameric B subunits that direct target-cell uptake after binding surface glycans. Subtilase cytotoxin (SubAB) is an AB.sub.5 toxin secreted by Shiga toxigenic Escherichia coli (STEC).sup.1, which causes serious gastrointestinal disease in humans.sup.2. SubAB causes haemolytic uraemic syndrome-like pathology in mice.sup.3 through SubA-mediated cleavage of BiP/GRP78, an essential endoplasmic reticulum chaperone.sup.4. Here we show that SubB has a strong preference for glycans terminating in the sialic acid N-glycolylneuraminic acid (Neu5Gc), a monosaccharide not synthesized in humans. Structures of SubB-Neu5Gc complexes revealed the basis for this specificity, and mutagenesis of key SubB residues abrogated in vitro glycan recognition, cell binding and cytotoxicity. SubAB specificity for Neu5Gc was confirmed using mouse tissues with a human-like deficiency of Neu5Gc and human cell lines fed with Neu5Gc. Despite lack of Neu5Gc biosynthesis in humans, assimilation of dietary Neu5Gc creates high-affinity receptors on human gut epithelia and kidney vasculature. This, and the lack of Neu5Gc-containing body fluid competitors in humans, confers susceptibility to the gastrointestinal and systemic toxicities of SubAB. Ironically, foods rich in Neu5Gc are the most common source of STEC contamination. Thus a bacterial toxin's receptor is generated by metabolic incorporation of an exogenous factor derived from food., Author(s): Emma Byres [sup.1] , Adrienne W. Paton [sup.2] , James C. Paton [sup.2] , Jonas C. Löfling [sup.3] , David F. Smith [sup.4] , Matthew C. J. Wilce [sup.1] [...]

We compared 61 Shiga toxin-producing Escherichia coli (STEC) serogroups from 448 food isolates with 71 STEC serogroups from 1,447 isolates from patients in Germany. Two thirds (41/61), representing 72% of [...]

The locus of enterocyte effacement (LEE) and genomic O island 122 (OI-122) are pathogenicity islands in verocytotoxin-producing Escherichia coli (VTEC) serotypes that are associated with outbreaks and serious disease. Composed of three modules, OI-122 may occur as 'complete' (with all three modules) or 'incomplete' (with one or two modules) in different strains. OI-122 encodes two non-LEE effector (Nle) molecules that are secreted by the LEE type III secretion system, and LEE and OI-122 are cointegrated in some VTEC strains. Thus, they are functionally linked, but little is known about the patterns of acquisition of these codependent islands. To examine this, we conducted a population genetics analysis, using multilocus sequence typing (MLST), with 72 VTEC strains (classified into seropathotypes A to E) and superimposed on the results the LEE and OI-122 contents of these organisms. The wide distribution of LEE and OI-122 modules among MLST clonal groups corroborates the hypothesis that there has been lateral transfer of both pathogenicity islands. Sequence analysis of a pagC-like gene in OI-122 module 1 also revealed two nonsynonymous single-nucleotide polymorphisms that could help discriminate a subset of seropathotype C strains and determine the presence of the LEE. A nonsense mutation was found in this gene in five less virulent strains, consistent with a decaying or inactive gene. The modular nature of OI-122 could be explained by the acquisition of modules by lateral transfer, either singly or as a group, and by degeneration of genes within modules. Correlations between clonal group, seropathotype, and LEE and OI-122 content provide insight into the role of genomic islands in VTEC evolution.

The apoptotic activity of a MazF, a toxin from the bacteria Escherichia coli, in mammal cells is studied. It is demonstrated that MazF breaks down the cellular mRNA by displacing anti-apoptotic proteins MCL-1 and BCL-[X.sub.L], mediated by proapoptotic BAK and its regulatorBH3-only protein NBK/BIK.

The natural prevalence of the Escherichia coli O157-specific efbE gene, Shiga toxin 1 ([stx.sub.1]), Shiga toxin 2 ([stx.sub.2]) and encoding intimin eaeA in manure from organic and low-input conventional Dutch farms is examined. The nonlinear biphasic patterns of the survival curve are because of the changing nutrient status of the manure over time, instead of the presence of subpopulations differing in the level of resistance.

Toxin-antitoxin (TA) systems are widely represented on mobile genetic elements as well as in bacterial chromosomes. TA systems encode a toxin and an antitoxin neutralizing it. We have characterized a homolog of the ccd TA system of the F plasmid ([ccd.sub.F]) located in the chromosomal backbone of the pathogenic O157:H7 Escherichia coli strain ([ccd.sub.O157]). The [ccd.sub.F] and the [ccd.sub.O157] systems coexist in O157:H7 isolates, as these pathogenic strains contain an F-related virulence plasmid carrying the [ccd.sub.F] system. We have shown that the chromosomal [ccd.sub.O157] system encodes functional toxin and antitoxin proteins that share properties with their plasmidic homologs: the CcdB[O.sub.157] toxin targets the DNA gyrase, and the CcdA[O.sub.157] antitoxin is degraded by the Lon protease. The [ccd.sub.O157] chromosomal system is expressed in its natural context, although promoter activity analyses revealed that its expression is weaker than that of [ccd.sub.F] [ccd.sub.O157] is unable to mediate postsegregational killing when cloned in an unstable plasmid, supporting the idea that chromosomal TA systems play a role(s) other than stabilization in bacterial physiology. Our cross-interaction experiments revealed that the chromosomal toxin is neutralized by the plasmidic antitoxin while the plasmidic toxin is not neutralized by the chromosomal antitoxin, whether expressed ectopically or from its natural context. Moreover, the ccdr system is able to mediate postsegregational killing in an E. coli strain harboring the [ccd.sub.O157] system in its chromosome. This shows that the plasmidic [ccd.sub.F] system is functional in the presence of its chromosomal counterpart.

Toxin-antitoxin loci belonging to the yefM-yoeB family are located in the chromosome or in some plasmids of several bacteria. We cloned the yefM-yoeB locus of Streptococcus pneumoniae, and these genes encode bona fide antitoxin (Yef[M.sub.Spn]) and toxin (Yoe[B.sub.Spn]) products. We showed that overproduction of Yoe[B.sub.Spn], is toxic to Escherichia coli cells, leading to severe inhibition of cell growth and to a reduction in cell viability; this toxicity was more pronounced in an E. coli B strain than in two E. coli K-12 strains. The Yoe[B.sub.Spn]-mediated toxicity could be reversed by the cognate antitoxin, Yef[M.sub.Spn], but not by overproduction of the E. coli YefM antitoxin. The pneumococcal proteins were purified and were shown to interact with each other both in vitro and in vivo. Far-UV circular dichroism analyses indicated that the pneumococcal antitoxin was partially, but not totally, unfolded and was different than its E. coli counterpart. Molecular modeling showed that the toxins belonging to the family were homologous, whereas the antitoxins appeared to be specifically designed for each bacterial locus; thus, the toxin-antitoxin interactions were adapted to the different bacterial environmental conditions. Both structural features, folding and the molecular modeled structure, could explain the lack of cross-complementation between the pneumococcal and E. coli antitoxins.

Haemolysin E (HlyE) is a novel pore-forming toxin first identified in Escherichia coli K-12. Analysis of the 3-D structure of HlyE led to the proposal that a unique hydrophobic [beta]-hairpin structure (the [beta]-tongue, residues 177-203) interacts with the lipid bilayer in target membranes. In seeming contradiction to this, the hlyE sequence from a pathogenic E. coli strain (JM4660) that lacks all other haemolysins has been reported to encode an Arg residue at position 188 that was difficult to reconcile with the proposed role of the [beta]-tongue. Here it is shown that the JM4660 hlyE sequence encodes Gly, not Arg, at position 188 and that substitution of Gly188 by Arg in E. coli K-12 HlyE abolishes activity, emphasizing the importance of the head domain in HlyE function. Nevertheless, 76 other amino acid substitutions were confirmed compared to the HlyE protein of E. coli K-12. The JM4660 HlyE protein was dimeric, suggesting a mechanism for improving toxin solubility, and it lysed red blood cells from many species by forming 36-41 [Angstrom] diameter pores. However, the haemolytic phenotype of JM4660 was found to be unstable due to defects in HlyE export, indicating that export of active HlyE is not an intrinsic property of the protein but requires additional components. TnphoA mutagenesis of hlyE shows that secretion from the cytoplasm to the periplasm does not require the carboxyl-terminal region of HlyE. Finally, disruption of genes associated with cell envelope function, including tatC, impairs HlyE export, indicating that outer membrane integrity is important for effective HlyE secretion.

The genes that produce Shiga toxin in Escherichia coli O91:H21 are similar to genes on plasmids from Shigella flexneri and enteropathogenic and Shiga toxin-producing strains of E. coli. They are also similar to the enterohemorrhagic E. coli reference strain EDL933. Shiga toxin-producing strains of E. coli can cause disease in humans, including hemorrhagic colitis and hemolytic-uremic syndrome.

A study was conducted on Escherichia coli subunit toxins called verotoxins (VT) and the molecular basis for the binding of the receptor of the VT1 holotoxin, globotriaosylceramide (Gb3) by the VT1 B-subunit. The experiment demonstrates that a fluorescent derivative of Gb3 possessing a coumarin probe in place of the acyl group normally attached to the Gb3 ceramide moiety is useful for monitoring interactions between VT1B and its receptor.

A mathematical model representing the dynamics of a colicinogenic and a colicin-sensitive population propagated under serial transfer culture conditions is presented. Also, a series of in vitro invasion experiments utilizing six representatives of the E colicin group were conducted, including the estimation of the growth rates and colicinogenic properties of the strains. Findings revealed that the properties of various colicinogenic strains cannot be employed to elucidate on the extensive variation in the relative abundance of different colicins in natural populations of bacteria.

The ligand-gated outer membrane porin FepA of Escherichia coli functions as the receptor of the siderophore ferric enterobactin. The ability of seven analogs of enterobactin to supply iron through FepA was characterized by quantitatively measuring the binding and transport of their 59Fe complexes. Findings disagreed with the view that chirality of the iron complex affects its recognition by FepA and illustrated the need for an unsubstituted catecholate coordination center for binding to the outer membrane protein.

A study was conducted on an automated fluorescence-based polymerase chain reaction (PCR) system to determine whether it is effective for identifying Shiga toxin-producing Escherichia coli. The system, a model Ag-9600 AmpliSensor analyzer, was tested on 53 naturally contaminated meat and 48 raw milk samples. Results indicate that the automated PCR-based system can be used for detecting all serotypes of STEC in food and clinical samples.

Colicins are toxins encoded by plasmids that are formed by and are active against Escherichia coli and similar bacteria. Their structure is composed of a domain for the recognition of a specific receptor, a domain for translocation and a domain for the lethal activity. Colicins can cross the formidable physical barrier of the outer membrane either through the passive diffusion pathway or the energy-dependent pathway. In addition to porins, a group of proteins called Tol is needed for colicin import.

Verotoxins are a class of bacterial toxins whose morphology constitutes an enzymatic A subunit where the toxic activity resides and is linked to a B oligomer that binds to specific cell-surface carbohydrate receptors. The solution structure of this carbohydrate B subunit of verotoxin VT-1 from the enterohemmorrhagic Escherichia coli is determined in relation to the trimethylsilylethyl derivative of the trisaccharide moiety of Gb3. The solution structure was obtained using nuclear magnetic resonance methods and is described.

Escherichia coli pathogens are responsible for a wide number of diseases, including kidney infection and diarrhoea. The enteropathogenic (CNF2) and uropathogenic (CNF1) strains of E. coli cause diseases in animals and humans respectively and produce the virulent toxins designated cytotoxic necrotizing factors (CNF). Studies show that CNF1 deamidates a glutamine residue at position 63 of Rho resulting in the constitutive activation of Rho and inducing the actin stress fibres to reorganize.

Hemolysin of Escherichia coli is activated by fatty acylation of the protoxin, directed by the putative acyl transferase HlyC and by acyl carrier protein (ACP). Mass spectrometry and Edman degradation of proteolytic products from mature toxin activated in vitro with tritium-labeled acylACP revealed two fatty-acylated internal lysine residues, lysine 564 and lysine 690. Resistance of the acylation to chemical treatments suggested that fatty acid was amide linked. Substitution of the two lysines confirmed that they were the only sites of acylation and showed that although each was acylated in the absence of the other, both sites were required for in vivo toxin activity., Hemolysin (HlyA) secreted by pathogenic E. coli binds to mammalian cell membranes, disrupting cellular activities and causing cell lysis by pore formation (1, 2). The toxin is made as an [...]

The concept of interconversions between atypical enteropathogenic Escherichia coli (aEPEC) O26 and enterohemorrhagic Escherichia coli (EHEC) O26 involving loss and gain of Shiga toxins (Stx)-encoding bacteriophages is examined.

The survival rates of each acid resistance (AR) mechanism is quantified separately in clinical isolates representing three groups of Shiga toxin-producing Escherichia coli (STEC) cones and six commensal strains from ECOR group A. Members of the STEC clones are not significantly more acid resistance than the commensal strains when analyzed using any individual AR mechanism.

Shiga toxin gene and the shiga-toxigenic Escherichia coli (STEC) prevalences were substantially related with the animal production type and the season. The three groups of dairy, feedlot and the cow-calf consisting of four farms in each group were checked for the type of production in the state of Washington.

A study on electron transport in supramolecular architectures reports on a new interfacial assembly that incorporates the high redox concentration in three-dimensional polymers and the controllable structure and functionality of the two-dimensional monolayer systems. This new interface uses structurally defines redox liposomes engineered with biomolecular recognition capability by combining cell surface receptor DM1 into the bilayer membrane.

Shiga-toxin-producing Escherichia coli (STEC), of which E. coli O157:H7 is one, are discussed. Other E. coli serotypes, less well known than O157:H7, are also important. Emergence of new STECs may make a problem already vexing regulators and consumers worse. Use of irradiation and other nonthermal food processing modes may be helpful. Testing methods should be improved. Steps taken in advance of emergence of new STECs may save lives later. Shiga-like toxin genes may spread to E coli strains other than O157:H7.