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Nonenterotoxigenic porcine Escherichia coli strains belonging to the serogroup O45 have been associated with postweaning diarrhea in swine and adhere to intestinal epithelial cells in a characteristic attaching and effacing (A/E) pattern. O45 porcine enteropathogenic E. coli (PEPEC) strain 86-1390 induces typical A/E lesions in a pig ileal explant model. Using TnphoA transposon insertion mutagenesis on strain 86-1390, we found a mutant that did not induce A/E lesions. The insertion was identified in a gene designated paa (porcine A/E-associated gene). Sequence analysis of paa revealed an open reading frame of 753 bp encoding a 27.6-kDa protein which displayed 100, 51.8, and 49% homology with Paa of enterohemorrhagic E. coli O157:H7 strains (EDL933 and Sakai), PEB3 of Campylobacter jejuni, and AcfC of Vibrio cholerae, respectively. Chromosomal localization studies indicated that the region containing paa was inserted between the yciD and yciE genes at about 28.3 min of the E. coli K-12 chromosome. The presence of paa and eae sequences in the porcine O45 strains is highly correlated with the A/E phenotype. However, the observation that three eae-positive but paa-negative PEPEC O45 strains were A/E negative provides further evidence for the importance of the paa gene in the A/E activity of O45 strains. As well, the complementation of the paa mutant restored the A/E activity of the 86-1390 strain, showing the involvement of Paa in PEPEC pathogenicity. These observations suggest that Paa contributes to the early stages of A/E E. coli virulence.Attaching and effacing (A/E) Escherichia coli (AEEC) induces distinctive histopathological lesions on the intestinal mucosa, known as the A/E lesions. These lesions are characteristic of enteric pathogens such as enteropathogenic E. coli (EPEC), responsible for severe childhood diarrhea in developing countries (14, 38), enterohemorrhagic E. coli (EHEC), causing hemorrhagic colitis and hemolytic-uremic syndrome, a diarrheagenic E. coli strain of rabbits (RDEC-1), strains of Hafnia alvei isolated from children with diarrhea, and Citrobacter rodentium, causing transmissible colonic hyperplasia in mice (4,16,53). A/E lesions have also been associated with diarrhea in different animal species such as rabbits, calves, dogs, cats, lambs, pigs, and tamarins (8,9,22,32,37,55).A/E lesions result from intimate bacterial adherence to the apical surfaces of enterocytes and activation of several chromosomal gene products that interact with components of the host cell, leading to host cell protein phosphorylation, effacement of target brush borders, and disruption of the underlying actin cytoskeleton (11, 38). The genes are clustered in a chromosomal pathogenicity island called the locus of enterocyte effacement (LEE). Its location and size vary in different strains. In EPEC strain E2348/69 and EHEC O157:H7 strains, the LEE is inserted in the selC locus at about 82 min on the E. coli K-12 chromosome, but its size varies from 35 kb for EPEC to 43 kb for EHEC. In strains of serotype O26:H-, the...

The ileal in vitro organ culture (IVOC) model using tissues originating from colostrum-deprived newborn piglets has proven to be an effective way to study the attaching and effacing (A/E) phenotype of porcine enteropathogenic Escherichia coli (EPEC) ex vivo. The aim of this study was to investigate the role of intimin subtype and Tir in the adherence of EPEC and Shiga-toxin-producing E. coli (STEC), isolated from different animal species, to porcine intestinal IVOC. Moreover, the role of intimin in Tir-independent adherence of the human EPEC strain E2348/69 was investigated using intimin and Tir-deficient derivatives. Our results demonstrated that A/E E. coli strains (AEEC) from various animal species and humans induce the A/E phenotype in porcine ileal IVOC and that intimin subtype influences intestinal adherence and tropism of AEEC strains. We also showed that a tir mutant of EPEC strain E2348/69 demonstrates close adherence to the epithelial cells of porcine ileal IVOC segments, with microvillous effacement but with no evidence of actin polymerization or pedestal formation, and that intimin seems to be involved in this phenotype. Overall, this study provides further evidence for the existence of one or more host-cell-encoded intimin receptor(s) in the pig gut.Enteropathogenic Escherichia coli (EPEC) and Shiga-toxinproducing E. coli (STEC) are an important cause of enteric diseases in both humans and animals (42). EPEC are the most common bacterial cause of diarrhea in infants from developing countries, whereas STEC, especially those of serotype O157: H7, are important emerging pathogens causing food-borne infections leading to bloody diarrhea and hemolytic-uremic syndrome in developed countries. EPEC, and certain STEC, cause typical, intestinal attaching and effacing (A/E) lesions which are characterized by intimate bacterial adherence to intestinal epithelial cells, effacement of the brush border, Factin rearrangement, and formation of a pedestal of polymerized F-actin and other cytoskeletal elements underneath the adherence site. Most A/E phenotype elements are encoded on a 35.6-kb (EPEC) to 43-kb (STEC of the O157:H7 serotype) pathogenicity island called locus of enterocytes effacement (LEE) (39, 40). The LEE contains genes encoding an outer membrane adhesin termed intimin (eae gene), a type III secretion system machinery (Esc and Sep proteins), chaperones (Ces proteins), and translocator (EspA, EspB, and EspD) and effector (EspF, EspG, and Map) proteins, as well as the translocated intimin receptor (Tir). The function of several open reading frames is still not known.At least five distinct major intimin subtypes, designated ␣, ␤, ␦, ␥, and ε, have been identified to date (1, 43). Receptor binding activity of intimin is located in the C-terminal 280-amino-acid region (Int280) (22) that comprises three separate domains, two immunoglobulin-like domains, and a C-type lectin-like module (32). Intimin was shown to bind to Tir (13,24,33) and directly to uninfected host cells (2,12,22,24,41). The latter seems to ...

f Variovorax sp. strain WDL1, which mineralizes the phenylurea herbicide linuron, expresses a novel linuron-hydrolyzing enzyme, HylA, that converts linuron to 3,4-dichloroaniline (DCA). The enzyme is distinct from the linuron hydrolase LibA enzyme recently identified in other linuron-mineralizing Variovorax strains and from phenylurea-hydrolyzing enzymes (PuhA, PuhB) found in Gram-positive bacteria. The dimeric enzyme belongs to a separate family of hydrolases and differs in K m , temperature optimum, and phenylurea herbicide substrate range. Within the metal-dependent amidohydrolase superfamily, HylA and PuhA/PuhB belong to two distinct protein families, while LibA is a member of the unrelated amidase signature family. The hylA gene was identified in a draft genome sequence of strain WDL1. The involvement of hylA in linuron degradation by strain WDL1 is inferred from its absence in spontaneous WDL1 mutants defective in linuron hydrolysis and its presence in linuron-degrading Variovorax strains that lack libA. In strain WDL1, the hylA gene is combined with catabolic gene modules encoding the downstream pathways for DCA degradation, which are very similar to those present in Variovorax sp. SRS16, which contains libA. Our results show that the expansion of a DCA catabolic pathway toward linuron degradation in Variovorax can involve different but isofunctional linuron hydrolysis genes encoding proteins that belong to evolutionary unrelated hydrolase families. This may be explained by divergent evolution and the independent acquisition of the corresponding genetic modules.is a phenylurea herbicide widely used in agriculture to control germinating and newly emerging grasses and broad-leafed weeds. Biodegradation contributes largely to the dissipation of linuron in the environment. Several single bacterial strains (1, 2) and consortia (3, 4) that degrade (3, 5) or even mineralize and use linuron as the sole source of carbon, nitrogen, and energy have been reported (1-4). Bacterial degradation of linuron is initiated by amide hydrolysis of linuron to 3,4-dichloroaniline (DCA) and N,O-dimethylhydroxylamine (N,O-DMHA). In the case of linuron mineralization, DCA is further converted to water and carbon dioxide (Fig. 1). Bacteria belonging to the genus Variovorax appear to play a crucial role in linuron biodegradation. In linuron-degrading consortia, they are almost always responsible for at least the initial hydrolysis step in linuron degradation, and most linuron-mineralizing single-strain isolates are of the genus Variovorax (1, 2). The genetic basis of linuron degradation in the linuron-mineralizing Variovorax sp. strain SRS16 was recently elucidated (6) and involves three major catabolic gene modules. In strain SRS16, conversion of linuron to DCA is catalyzed by the hydrolase LibA, encoded by the libA gene. Further mineralization of DCA involves a multicomponent dioxygenase complex encoded by dcaQTA 1 A 2 BR, which degrades DCA to a chlorocatechol intermediate. The latter is further degraded by a modified ortho-cleava...

Dissipation kinetics of mesotrione, a new triketone herbicide, sprayed on soil from Limagne (Puy-de-Dôme, France) showed that the soil microflora were able to biotransform it. Bacteria from this soil were cultured in mineral salt solution supplemented with mesotrione as sole source of carbon for the isolation of mesotrione-degrading bacteria. The bacterial community structure of the enrichment cultures was analyzed by temporal temperature gradient gel electrophoresis (TTGE). The TTGE fingerprints revealed that mesotrione had an impact on bacterial community structure only at its highest concentrations and showed mesotrione-sensitive and mesotrione-adapted strains. Two adapted strains, identified as Bacillus sp. and Arthrobacter sp., were isolated by colony hybridization methods. Biodegradation assays showed that only the Bacillus sp. strain was able to completely and rapidly biotransform mesotrione. Among several metabolites formed, 2-amino-4-methylsulfonylbenzoic acid (AMBA) accumulated in the medium. Although sulcotrione has a chemical structure closely resembling that of mesotrione, the isolates were unable to degrade it.

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We have successfully used the major subunit ClpG of Escherichia coli CS31A fimbriae as an antigenic and immunogenic exposure-delivery vector for various heterologous peptides varying in nature and length. However, the ability of ClpG as a carrier to maintain in vitro and in vivo the native biological properties of passenger peptide has not yet been reported. To address this possibility, we genetically fused peptides containing all or part of the E. coli human heat-stable enterotoxin (STh) sequence to the amino or carboxyl ends of ClpG. Using antibodies to the ClpG and STh portions for detecting the hybrids; AMS (4-acetamido-4-maleimidylstilbene-2,2-disulfonate), a potent free thiol-trapping reagent, for determining the redox state of STh in the fusion; and the suckling mouse assay for enterotoxicity, we demonstrated that all ClpG-STh proteins were secreted in vitro and in vivo outside the E. coli cells in a heat-stable active oxidized (disulfidebonded) form. Indeed, in contrast to many earlier studies, blocking the natural NH 2 or COOH extremities of STh had, in all cases, no drastic effect on cell release and toxin activity. Only antigenicity of STh C-terminally extended with ClpG was strongly affected in a conformation-dependent manner. These results suggest that the STh activity was not altered by the chimeric structure, and therefore that, like the natural toxin, STh in the fusion had a spatial structure flexible enough to be compatible with secretion and enterotoxicity (folding and STh receptor recognition). Our study also indicates that disulfide bonds were essential for enterotoxicity but not for release, that spontaneous oxidation by molecular oxygen occurred in vitro in the medium, and that the E. coli cell-bound toxin activity in vivo resulted from an effective export processing of hybrids and not cell lysis. None of the ClpG-STh subunits formed hybrid CS31A-STh fimbriae at the cell surface of E. coli, and a strong decrease in the toxin activity was observed in the absence of CS31A helper proteins. In fact, chimeras translocated across the outer membrane as a free folded monomer once they were guided into the periplasm by the ClpG leader peptide through the CS31A-dependent secretory pathway. In summary, ClpG appears highly attractive as a carrier reporter protein for basic and applied research through the engineering of E. coli for culture supernatant delivery of an active cysteine-containing protein, such as the heat-stable enterotoxin.

The virulence genotype profile and presence of a pathogenicity island(s) (PAI) were studied in 18 strains of F165-positive Escherichia coli originally isolated from diseased calves or piglets. On the basis of their adhesion phenotypes and genotypes, these extraintestinal pathogenic strains were classified into three groups. The F165 fimbrial complex consists of at least two serologically and genetically distinct fimbriae: F165 1 and F165 2 . F165 1 is encoded by the foo operon (pap-like), and F165 2 is encoded by fot (sfa related). Strains in group 1 were foo and fot positive, strains in group 2 were foo and afa positive, and strains in group 3 were foo positive only. The strains were tested for the presence of virulence genes found mainly in extraintestinal pathogenic E. coli (ExPEC) strains. Although all the strains were positive for the papA variant encoding F11 fimbriae incD, traT, and papC, the prevalence of virulence genes commonly found in PAIs associated with ExPEC strains was highly variable, with strains of group 2 harboring most of the virulence genes tested. papG allele III was detected in all strains in group 1 and in one strain in group 3. All other strains were negative for the known alleles encoding PapG adhesins. The association of virulence genes with tRNA genes was characterized in these strains by using pulsed-field gel electrophoresis and DNA hybridization. The insertion site of the foo operon was found at the pheU tRNA locus in 16 of the 18 strains and at the selC tRNA locus in the other 2 strains. Furthermore, 8 of the 18 strains harbored a high-pathogenicity island which was inserted in either the asnT or the asnV/U tRNA locus. These results suggest the presence of one or more PAIs in septicemic strains from animals and the association of the foo operon with at least one of these islands. F165-positive strains share certain virulence traits with ExPEC, and most of them are pathogenic in piglets, as tested in experimental infections.Escherichia coli is a frequent cause of intestinal and extraintestinal diseases in humans and animals. Typical extraintestinal infections include urinary tract infections, newborn meningitis, polyserositis, and septicemia. All these groups of pathogenic E. coli strains have been called extraintestinal pathogenic E. coli (ExPEC). The recognized virulence factors of ExPEC include diverse adhesins (e.g., P fimbriae, S/F1C fimbriae, F165 fimbriae, Afa/Dr adhesins, and type 1 fimbriae), toxins (e.g., hemolysin, cytotoxic necrotizing factor, and cytolethal distending toxin), surface antigens (e.g., group II and group III capsules and lipopolysaccharide), invasins (e.g., an invasin responsible for invasion of brain endothelium [IbeA, also called Ibe10]), iron uptake systems (e.g., the aerobactin system), and secretion systems (e.g., type III secretion systems). These virulence factors facilitate colonization and invasion of the host, avoidance or disruption of host defense mechanisms, injury to host tissues, and/or stimulation of a noxious host inflammatory response (...