Cloning of the chromosomal determinants encoding hemolysin production and mannose-resistant hemagglutination in Escherichia coli

Abstract

The chromosomal hemolysin determinants from E. coli strains belonging to the 4 O-serotypes O4, O6, O18 and O75 were cloned. The hemolysin-producing clones were isolated from gene banks of these strains which were constructed by inserting partial Sau3A fragments of chromosomal DNA into the cosmid pJC74. The hemolytic cosmid clones were relatively stable. The inserts were further subcloned as SalI fragments in a pACYC184 or as BamHI-SalI fragments in a recombinant plasmid (pANN202) containing cistron C (hlyC) of the plasmid-encoded hemolysin determinant. Detailed restriction maps of each of these determinants were constructed, and it was found that, despite sharing overall homology, the determinants exhibited minor specific differences in their structure. These appeared to be restricted to cistron A (hlyA), which is the structural gene for hemolysin. In the gene banks of 2 of these hemolytic strains, clones which carried the genetic determinants for the mannose-resistant hemagglutination antigens Vb and VIc were also identified. Both of these fimbrial antigens were expressed in the E. coli K-12 clones to an extent similar to that observed in the wild-type strains. These recombinant cosmids were rather unstable, and, in the absence of selection, segregated at a high frequency.