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The kinetic binding characteristics of four Bacillus thuringiensis CryI insecticidal crystal proteins to a Cry-binding protein, purified from Manduca sexta brush-border vesicles, were analyzed by an optical biosensor. This 120-kilodalton binding protein, previously determined to be aminopeptidase N, was converted to a 115-kilodalton water-soluble form by removing the attached glycosylphosphatidylinositol anchor with phospholipase C. The solubilized form recognized the three major subclasses of CryIA toxins but not CryIC even though all four CryI proteins are toxic to larvae of M. sexta. CryIA(a) and CryIA(b) toxins bound to a single site on the solubilized aminopeptidase N molecule whereas CryIA(c) bound to two distinct sites. Apparent kinetic rate constants were determined for each binding reaction. All three CryIA toxins exhibited moderately fast on rates (approximately 10(-5) M-1 s-1) and a slow reversible off rate (approximately 10(-3) s-1). Although the second CryIA(c)-binding site retained a moderately fast association rate, it was characterized by a rate of dissociation from the amino-peptidase an order of magnitude faster than observed for the other CryIA-binding sites. CryIA(c) binding to both sites was strongly inhibited in the presence of N-acetylgalactosamine (IC50 = 5 mM) but not N-acetylglucosamine, mannose, or glucose. CryIA(a) and CryIA(b) binding were unaffected in the presence of the same sugars. Our results serve to illustrate both the complexity and the diverse nature of toxin interactions with Cry-binding proteins.

A total of 112 Escherichia coli O149:K91 strains isolated from pigs with diarrhea in Quebec, Canada, between 1978 and 2000 were characterized for their genotypic antimicrobial resistance profiles. Tests for resistance to 10 antimicrobial agents were conducted. Resistance to tetracycline and sulfonamides was found to be the most frequent, but resistance to cefotaxime and ceftiofur was absent. An increase in the number of isolates resistant to at least three antimicrobials was observed over time. The distribution of 28 resistance genes covering six antimicrobial families (beta-lactams, aminoglycosides, phenicols, tetracycline, trimethoprim, and sulfonamides) was assessed by colony hybridization. Significant differences in the distributions of tetracycline [tet(A), tet(B), tet(C)], trimethoprim (dhfrI, dhfrV, dhfrXIII), and sulfonamide (sulI, sulII) resistance genes were observed during the study period (

The role of atypical enteropathogenic Escherichia coli (EPEC) in childhood diarrhea is controversial. The aim of the present study was to search for genes linked with diarrhea in atypical EPEC strains from a case-control study among Norwegian children. Using DNA microarray analysis, genomic DNAs from strains isolated from children with (n ‫؍‬ 37) and without (n ‫؍‬ 20) diarrhea were hybridized against 242 different oligonucleotide probes specific for 182 virulence genes or markers from all known E. coli pathotypes. PCR was performed to test the strains for seven putative virulence genes not included in the microarray panel. The OI-122 gene efa1/lifA was the gene with the strongest statistical association with diarrhea (P ‫؍‬ 0.0008). Other OI-122 genes (set/ent, nleB, and nleE) and genes with other locations (lpfA, paa, ehxA, and ureD) were also associated with diarrheal disease. The phylogenetic marker gene yjaA was negatively associated with diarrhea (P ‫؍‬ 0.0004). Atypical EPEC strains could be classified in two main virulence groups based on their content of OI-122, lpfA, and yjaA genes. Among children with diarrhea, atypical EPEC isolates belonging to virulence group I (OI-122 and lpfA positive, yjaA negative) were the most common, while the majority of isolates from healthy children were classified as virulence group II strains (OI-122 negative, lpfA and yjaA positive; P < 0.001). In conclusion, using DNA microarray analysis to determine the virulence gene profile of atypical EPEC isolates, several genes were found to be significantly associated with diarrhea. Based on their composition of virulence genes, the majority of strains could be classified in two virulence groups, of which one was seen mainly in children with diarrhea.

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