The genes responsible for the expression of type 1 fimbriae, produced by the majority of E. coli strains, have been cloned from an E. coli K12 strain. The "passenger" DNA from an initial cosmid clone was reduced in size and subcloned in pACYC184 and pBR322 vectors. A DNA fragment of around 8 kbp was found to be required for the biosynthesis of type 1 fimbriae. This was further studied by transposon-mediated insertional inactivation and by BAL31-mediated deletions. Four genes, designated fimA, B, C, and D were found to be involved in the synthesis of the fimbriae. They encoded proteins that in their processed form appeared with apparent molecular weights of 16.5 kd, 23 kd, 26 kd, and 89 kd, the 16.6 kd polypeptide being the fimbrial subunit. The order to the genes was found to be: fimB, fimA, fimC, and fimD, organized in three transcriptional units.
This study demonstrated that transposable elements in Pseudomonas cepacia could be inserted upstream of a poorly expressed gene and increase its expression more than 30-fold. Five elements, TnPcl, IS402, IS403, IS404, and IS405, were isolated by their ability to increase expression of the P-lactamase gene of the broad-host-range plasmid pRP1. Increased expression resulted only from insertion of these elements, suggesting that insertional activation is an important means of elevating gene expression in this organism. Four of the elements inserted between a Pstl site within the ,B-lactamase gene and a BamHI site located 375 base pairs upstream of its promoter. The element IS403 inserted distal to the BamHI site within the coding region for the gene tnpR, suggesting that insertional activation can act over greater than expected distances. In addition, the element IS402 activated the P-lactamase genes carried on plasmids pRP1 and pMR5 (temperature-sensitive pRP1) equally well in opposite orientations, demonstrating that insertional activation by this element occurs independent of its orientation.
The P fimbrial gene clusters encoding the serologically different F71, F72, F9, and Fll fimbriae were compared functionally. The results show that these gene clusters are closely related.
Construction and analysis of mutations in the hypervariable regions of the F71, F9 and F11 fimbrillin genes are described. The results show that mutations in the hypervariable regions of the fimbrillin can be made without abolishing the ability of these fimbrillins to assemble into filaments. The mutant fimbriae show binding patterns with specific monoclonal antibodies that differed from those of the corresponding wild‐type fimbriae. These results confirm that antigenic determinants of the P‐fimbriae are, at least partly, located in the hypervariable regions, and suggest that more than one antigenic determinant is involved in the F71 and F11 specificity.
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