Cloning and expression of the phospholipase D gene from Corynebacterium pseudotuberculosis in Escherichia coli

Abstract

A toxic phospholipase D (PLD) is putatively involved in pathogenesis of Corynebacterium pseudotuberculosis infections. We report here the cloning and expression of the PLD gene (pld) in Escherichia coli. A cosmid library of DNA from C. pseudotuberculosis biovar ovis isolate Whetten 1 was constructed and screened for PLD-producing recombinants by plating them on LB agar containing sheep erythrocytes and equi factors. One recombinant, designated pCpO1, yielded a gene product which displayed synergistic hemolytic and sphingomyelinase D activities, both of which are characteristic of PLD. Subcloning into pUC19 yielded a recombinant, pCpO50, which contained a 1.8-kilobase insert. Analysis of supernatant fluids and cell extracts of cultures of E. coli(pCpO50) revealed sphingomyelinase activity and a protein of about 31,000 Mr, neither of which were detected in E. coli(pUC19). The 31-kilodalton protein also reacted with antibodies in serum from a sheep naturally infected with C. pseudotuberculosis, serum which also contained PLD-neutralizing antibodies. When Southern blots of BamHI digests of DNA from biovar ovis and biovar equi isolates of C. pseudotuberculosis were probed with pCpO50, bands of 4.8 and 1.9 kilobases, respectively, were seen, suggesting that the genome organization of pld is different for isolates from the two biovars.