Beta-turn formation in the processing region is important for efficient maturation of Escherichia coli maltose-binding protein by signal peptidase I in vivo.

Signal peptidase I (also called leader peptidase) is the endopeptidase that removes the signal peptides of most secreted proteins during or after translocation in Escherichia coli. Precursor recognition is contingent in part on the presence of small, uncharged residues in the -3 and -1 positions relative to the cleavage site, and may also depend on the structure of the processing region. Most precursor processing regions include residues likely to form a beta-turn. Mutations were introduced into the processing region of maltose-binding protein (MBP) that altered the prediction of beta-turn formation in this region. MBP species with a decreased probability of beta-turn formation were processed slowly or not at all, whereas MBP species with an increased probability of beta-turn formation were processed efficiently. Mutations altering the prediction of beta-turn formation in the MBP processing region were also made in cis to a proline in the +1 position. Cleavage at the normal processing site is blocked by proline in the +1 position; this MBP species, MBP27-P, inhibits processing of other proteins by signal peptidase I. Decreasing the probability of beta-turn formation in the processing region of MBP27-P eliminated the inhibition of signal peptidase I, and these MBP27-P derivatives remained unprocessed, suggesting that the formation of a beta-turn in the MBP processing region was necessary for recognition by signal peptidase I. Increasing the probability of beta-turn formation in cis to proline at +1 in MBP did not alter recognition of the protein by the processing enzyme. The results presented here are consistent with the hypothesis that the efficiency of recognition and processing by signal peptidase I is increased by the formation of a beta-turn in the processing region of the MBP signal peptide

Prevalence and Characterization of Non-O157 Shiga Toxin-Producing \u3ci\u3eEscherichia coli\u3c/i\u3e on Carcasses in Commercial Beef Cattle Processing Plants

Beef carcass sponge samples collected from July to August 1999 at four large processing plants in the United States were surveyed for the presence of non-O157 Shiga toxin-producing Escherichia coli (STEC). Twenty-eight (93%) of 30 single-source lots surveyed included at least one sample containing non-O157 STEC. Of 334 carcasses sampled prior to evisceration, 180 (54%) were found to harbor non-O157 STEC. Non-O157 STEC isolates were also recovered from 27 (8%) of 326 carcasses sampled after the application of antimicrobial interventions. Altogether, 361 non-O157 STEC isolates, comprising 41 different O serogroups, were recovered. O serogroups that previously have been associated with human disease accounted for 178 (49%) of 361 isolates. Although 40 isolates (11%) carried a combination of virulence factor genes (enterohemorrhagic E. coli hlyA, eae, and at least one stx gene) frequently associated with STEC strains causing severe human disease, only 12 of these isolates also belonged to an O serogroup previously associated with human disease. Combining previously reported data on O157-positive samples (R. O. Elder, J. E. Keen, G. R. Siragusa, G. A. Barkocy- Gallagher, M. Koohmaraie, and W. W. Laegreid, Proc. Natl. Acad. Sci. USA 97:2999–3003, 2000) with these data regarding non-O157-positive samples indicated total STEC prevalences of 72 and 10% in preevisceration and postprocessing beef carcass samples, respectively, showing that the interventions used by the beefprocessing industry effected a sevenfold reduction in carcass contamination by STEC

Prevalence and Characterization of Non-O157 Shiga Toxin-Producing Escherichia coli on Carcasses in Commercial Beef Cattle Processing Plants

Beef carcass sponge samples collected from July to August 1999 at four large processing plants in the United States were surveyed for the presence of non-O157 Shiga toxin-producing Escherichia coli (STEC). Twenty-eight (93%) of 30 single-source lots surveyed included at least one sample containing non-O157 STEC. Of 334 carcasses sampled prior to evisceration, 180 (54%) were found to harbor non-O157 STEC. Non-O157 STEC isolates were also recovered from 27 (8%) of 326 carcasses sampled after the application of antimicrobial interventions. Altogether, 361 non-O157 STEC isolates, comprising 41 different O serogroups, were recovered. O serogroups that previously have been associated with human disease accounted for 178 (49%) of 361 isolates. Although 40 isolates (11%) carried a combination of virulence factor genes (enterohemorrhagic E. coli hlyA, eae, and at least one stx gene) frequently associated with STEC strains causing severe human disease, only 12 of these isolates also belonged to an O serogroup previously associated with human disease. Combining previously reported data on O157-positive samples (R. O. Elder, J. E. Keen, G. R. Siragusa, G. A. Barkocy-Gallagher, M. Koohmaraie, and W. W. Laegreid, Proc. Natl. Acad. Sci. USA 97:2999-3003, 2000) with these data regarding non-O157-positive samples indicated total STEC prevalences of 72 and 10% in preevisceration and postprocessing beef carcass samples, respectively, showing that the interventions used by the beef-processing industry effected a sevenfold reduction in carcass contamination by STEC

Genotypic Analyses of \u3ci\u3eEscherichia coli\u3c/i\u3e O157:H7 and O157 Nonmotile Isolates Recovered from Beef Cattle and Carcasses at Processing Plants in the Midwestern States of the United States

Escherichia coli O157:H7 and O157 nonmotile isolates (E. coli O157) previously were recovered from feces, hides, and carcasses at four large Midwestern beef processing plants (R. O. Elder, J. E. Keen, G. R. Siragusa, G. A. Barkocy-Gallagher, M. Koohmaraie, and W. W. Laegreid, Proc. Natl. Acad. Sci. USA 97:2999–3003, 2000). The study implied relationships between cattle infection and carcass contamination within single-source lots as well as between preevisceration and postprocessing carcass contamination, based on prevalence. These relationships now have been verified based on identification of isolates by genomic fingerprinting. E. coli O157 isolates from all positive samples were analyzed by pulsed-field gel electrophoresis of genomic DNA after digestion with XbaI. Seventy-seven individual subtypes (fingerprint patterns) grouping into 47 types were discerned among 343 isolates. Comparison of the fingerprint patterns revealed three clusters of isolates, two of which were closely related to each other. Remarkably, isolates carrying both Shiga toxin genes and nonmotile isolates largely fell into specific clusters. Within lots analyzed, 68.2% of the postharvest (carcass) isolates matched preharvest (animal) isolates. For individual carcasses, 65.3 and 66.7% of the isolates recovered postevisceration and in the cooler, respectively, matched those recovered preevisceration. Multiple isolates were analyzed from some carcass samples and were found to include strains with different genotypes. This study suggests that most E. coli O157 carcass contamination originates from animals within the same lot and not from cross-contamination between lots. In addition, the data demonstrate that most carcass contamination occurs very early during processing