The structure of cloned hemolysin DNA from plasmid pHly185

Abstract

The complete DNA segment coding for hemolysis on Escherichia coli plasmid, pHly185, on to the vector pBR322 has been cloned. The recombinant plasmid, pJS204, coded the production of externalized hemolysin and contained four contiguous EcoRI fragments from the original plasmid. A restriction map of pJS204 was generated with six endonucleases. Deletion mutations were constructed from the recombinant plasmid by excision with either BamHI and PstI and religation. The BamHI deletion removed a 3680-bp portion from the recombinant plasmid and prevented the synthesis of active hemolytic activity. The PstI-generated deletion removed 7800 bp from the opposite end of the Hly sequence and resulted in a plasmid coding for the production of active intracellular hemolytic activity but contained no information for the export of hemolysin. We examined the ability of the deletion mutants of pJS204 to complement each other, and Tn5 insertion mutations in the hly region of the parent plasmid, pHly185. The complementation studies indicated the presence of three separate cistrons able to complement in trans in a recA E. coli host. Two cistrons were found to be required for synthesis and one cistron for export of the hemolysin. The presence of two promoters and the directions of transcription for the synthesis of the hly gene products was also inferred from the complementation studies.