Analysis of the membrane localised transport protein, haemolysin B.

The Escherichia coli haemolysin operon consists of four contiguous genes, hlyCABD on an approximately 7.5Kbp length of DNA. The 107KDa HlyA haemolysin is activated by the cytoplasmically localised 20KDa HlyC polypeptide and secreted to the culture medium by the cell envelope localised HlyB and HlyD (53KDa) polypeptides. Two proteins corresponding to HlyB have previously been identified with apparent molecular weights of 46KDa and 66KDa. Both the HlyB and HlyD polypeptides appear to be expressed at extremely low levels in vivo. Interestingly, the HlyB polypeptide appears to be a member of a novel family of ATP-dependent membrane localised transport proteins spanning the phylogenetic scale. In order to investigate the structure and function of the HlyB polypeptide(s) attempts were made to amplify the expression of hlyB, following the DNA sequencing of the E.coli LE2001 hlyB gene, using a variety of plasmid vectors designed for the high level expression of cloned inserts in order to isolate sufficient HlyB to raise antisera. These experiments identified potential levels of control in the regulation of hlyB expression that, although present in the original pathogenic strain, E.coli LE2001, appeared absent or attenuated in the plasmid subclones of hlyB. However, no over-expressed HlyB polypeptide(s) were identified. Therefore, antibodies were raised against synthetic penta-peptides selected from regions within HlyB and against LacZ-HlyB fusion proteins. Experiments using the anti-HlyB antisera resulted in the immunoprecipitation of HlyB from radiolabelled E.coli minicells and the identification of a 46KDa polypeptide expressed in the original wild-type pathogenic strain, E.coli LE2001. Further experiments demonstrated that the HlyA toxin was also secreted transiently during the growth of E.coli LE2001 and that the in vivo level of hlyB mRNA was restricted to a similar "secretion window". Therefore, the regulation of hlyB expression was considered to be a suitable candidate for the control of HlyA secretion during growth.