Construction and characterization of mutants impaired in the biosynthesis of the K88ab antigen

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Abstract
Plasmid pFM205 contains the genetic determinant for the K88ab antigen [of Escherichia coli] and is composed of a 4.3-megadalton DNA fragment derived from wild-type K88ab plasmid pRI8801 and cloning vehicle pBR322. The K88 DNA of pFM205 contains 5 genes, which code for polypeptides with apparent MW of 17,000, 26,000 (the K88ab subunit), 27,000, 27,500 and 81,000. All 5 polypeptides were synthesized as precursors .apprx. 2000 daltons larger than the mature polypeptides, indicating that they are transported across the cytoplasmic membrane by means of a signal sequence. A set of deletion derivatives of pFM205 was constructed, each containing a deletion in 1 of the 5 genes. In strains harboring derivatives of pFM205 containing a deletion in the gene for the 17,000- or 81,000-dalton polypeptide, the K88ab subunit was synthesized and transported to the outside of the cell. These strains did not adhere to brush borders or guinea pig erythrocytes, suggesting that the K88ab subunits were not assembled into normal fimbriae. Strains harboring plasmids containing a deletion in the gene for the 27,500-dalton polypeptide still adhered to brush borders and guinea pig erythrocytes, although very little K88ab antigen could be detected with an immunological assay. In strains harboring plasmids containing a deletion in the gene for the 27,000-dalton polypeptide, the K88ab subunit was synthesized but was probably subsequently degraded rapidly.