21] Use of TnphoA and T7 RNA polymerase to study fimbrial proteins

Abstract

Publisher Summary This chapter focuses on two tools that have proved to be particularly useful for studying cloned-gene clusters responsible for fimbriation and adhesion of enteric bacteria. Pertinent techniques, concepts, strategies, precautions, and advantages of (1) region-directed Tn phoA (Tn5IS 50 L :: phoA ) mutagenesis and (2) in vivo expression of cloned polycistronic genes with a T7 RNA polymerase/promoter system are presented. Because of their versatile properties, ease in handling, and disposition to molecular manipulations, transposons are among the most frequently used mutagenic tools. Two types of studies, in particular, can benefit from the use of transposon mutagenesis. In the first, the method is used to identify fimbrial or adhesion genes by random mutagenesis of a pathogenic strain followed by screening (if possible, selecting) for nonfimbriated or nonadhesive mutants. This approach is then followed by cloning and further analysis of the mutated locus. In a second type of study, a gene cluster of interest is already cloned on a multicopy number plasmid to be analyzed in Escherichia coli in more detail by region-directed mutagenesis, as discussed in the chapter. Mutated alleles can then be introduced into the original pathogenic strain by marker-exchange experiments. In both types of studies, Tn phoA has demonstrated advantages over other transposons, as it can be used to identify exported proteins. Tn phoA is particularly well suited for the study of bacterial fimbrial proteins and adhesins, because such proteins typically cross bacterial membranes to interact with host molecules.