ARCANOBACTERIUM-HAEMOLYTICUM PHOSPHOLIPASE-D IS GENETICALLY AND FUNCTIONALLY SIMILAR TO CORYNEBACTERIUM-PSEUDOTUBERCULOSIS PHOSPHOLIPASE-D

Arcanobacterium haemolyticum, a pathogen of the human upper respirator y tract and other systems, has been reported to produce soluble toxins , including a phospholipase D (PLD). We confirmed production of PLD by this organism and cloned and sequenced pld. Arcanobacterial PLD (PLD- A) was found to be a protein of almost-equal-to 31.5 kDa with a pI of almost-equal-to 9.4. Cosmid cloning, followed by subcloning into phage mid pBluescriptIISK+, yielded Escherichia coli(pAh140), a recombinant with a gene product corresponding to PLD-A. Evidence of PLD activity w as found by three assays in supernatant fluid of cultures of E. coli(p Ah140) and A. haemolyticum, but not in E. coli(pBluescriptIISK+). Expe riments to determine if this protein was secreted were not conducted, but previous work with PLD from Corynebacterium pseudotuberculosis sug gested that the presence of the enzyme in culture supernatant fluids w as due to lysis of E. coli rather than to active transport. Antibodies in polyclonal sera from goats immunized with native or recombinant PL D-A neutralized native and recombinant PLD-A activity, and antibodies against native or recombinant PLD from C. pseudotuberculosis (PLD-P) p artially neutralized native and recombinant PLD-A. Antibodies prepared against recombinant PLD-A labelled both recombinant and native PLD-A in Western blots (immunoblots) and dot blots, but antibodies against P LD-P did not. Sequencing of the insert in pAh140 revealed an open read ing frame of 930 bp coding for 309 amino acids, including a putative s ignal sequence of 26 amino acids (3.2 kDa, determined on the basis of homology with the 24-amino-acid signal sequence of pld from C. pseudot uberculosis bv. ovis) and the mature PLD protein (31.5 kDa). Sequence comparisons of coding regions revealed 65% DNA homology with pld genes from C. pseudotuberculosis and Corynebacterium ulcerans. Comparison o f amino acid sequences revealed 64% homology of PLD-A both with PLD-P and with PLD produced by C. ulcerans.