Cloned Diphtheria Toxin Fragment A is Expressed from the Tox Promoter and Exported to the Periplasm by the SecA Apparatus of Escherichia Coli K12

The diphtheria toxin fragment A-related polypeptide encoded by the recombinant plasmid pDT201 (Leong, D., Coleman, K. D., and Murphy, J. R. (1983) Science 220, 515-517) and expressed in Escherichia coli K12 was found to have an electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels corresponding to 28,500 daltons. Immunoprecipitation experiments revealed that the mature form of the fragment A-related polypeptide was exported to the periplasmic compartment of E. coli K12 harboring plasmid pDT201. The polypeptide was sensitive to trypsin "nicking." Following treatment an Mr = 24,000 fragment was released which co-electrophoresed with fragment A of diphtheria toxin. The precursor form (Mr = 31,000) of the fragment A-related polypeptide was found to accumulate in the cytoplasmic fraction of the temperature-sensitive secretion-defective strain of E. coli K12 under nonpermissive conditions. We further demonstrate that a 263-base pair HaeIII fragment of pDT201 which carries the putative diphtheria tox promoter has promoter activity in E. coli K12.