Comparison of outer membrane protein and lipopolysaccharide profiles of enterotoxigenic Escherichia coli strains isolet in Säo Paulo, Brazil

The outer membrane protein (OMP) and lipopolysaccharide (LPS) patterns of 12 strains of serogroups of enterotoxigenic E. coli frequntly isolated in Säo Paulo city werte determined by fractionation techniques and by sodium dodecyl sulfate-plyacrylamide gel electrophoresis (SDS-PAGE). Five O6, three O78 and four O128 serogroup isolates of different serotypes (flagellar antigens) and virulence factors (toxins and colonization factor antigens) showed a high degree of variability in their OMP pattern and at least nine groups could be identified. The analysis of LPS aptterns by SDS-PAGE showed a homogenous profile for the O6 strains and some minor differences for the O128 and 078 strains. The oresented data indicate that analysis of OMP and LPS by SDS-PAGE may further improve the discriminating ability of extensively used serological techniques or the detection of virulence factors and could be a useful tool in epidemiological studies of enterotoxigenic E. coli (ETEC) strains from this area

The N-terminal amino acid sequence is essential for foot-and-mouth disease virus replicase activity

Foot-and-mouth disease virus replicase was expressed by fusing its cDNA to the OmpA signal peptide coding sequence present in the pIN-III ompA series vectors. Two constructions were developed to express either a full-lenghtt or truncated enzyme lacking the 20 aminoacids at the N-terminal en. Bacterial extr5acts expressing the recombinant proteins were submitted to SDS-PAGE and the presence of the replicase was revealed by immunoblotting. The truncated form exhibited a higher mobility and the relative positions of the proteins show that the signal peptide was removed. The biological activity of these two molecules was tested using a poly(A)-dep[endent oligo(U)-primed poly(U)-polymer4ase assay. The full-lenght replicase is active. The aminoterminal truncated wnzyme had 0.02% activity o9f the intact5 one. This result indicates the importaqnce of the twenty N-terminal amino acids for the activity of FMDV RNA dependent RNMA polymerase

In vitro activities of a recombinant foot-and-mouth disease virus replicase expressed in Escherichia coli

The replicase gene of foot-and-mouth disease virus (FMDV) was expressed in Escherichia coli under the control of a tac promoter. The recombinant enzyme was purified by inclusion body precipitation, elution, and poly(U) Sepharose chromatography. The enzyme exhibits poly(A)-dependent oligo(U)-primed poly(U) polymerase activity. The specific activity of the purified replicase is 1.3 x 10 5. The recombinant replicase synthesizes RNA using FMDV RNA as template, as well as heterologous RNAs, such as globin RNA and synthetic RNAs, polyadenylated or not. In all polymerization reactions, RNA products twice the size of the template are formed, both in the presence and absence of an oligo(U) primer. The enzyme is also capable of incorporating [alpha32P]UTP in all RNAs tested except the viral template. This activity does not seem to be related to the primer independent polymerization activity. The products from polymerization reactions were characterized by hybridization. In the absence of primer they consist of the template and a complementary strand covalently attached, while in the presence of primer they consist of two complementary strands synthesized de novo. We propose mechanisms of RNA synthesis by the recombinant FMDV replicase in the absence and presence of primer. These mechanisms are discussed in terms of models for in vitro RNA synthesis of other piconaviruses

Expression of an active foot-and-mouth disease virus RNA polymerase in Escherichia coli

The expression of a native form of the foot-and-mouth disease virus RNA polymerase was obtained. Two oligonucleotides of 66 base pairs were used to renuild the 5' end of the gene and to introduce the first methionine codon. The expression of the active polymerase in E. coli was achieved by inserting the gene before the tac promoter of the pKK223-3 plasmid