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 pCpOl, 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, pCpO5O, which contained a 1.8-kilobase insert. Analysis of supernatant fluids and cell extracts of cultures of E. coli(pCpO5O) 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 pid is different for isolates from the two biovars. * Corresponding author. t Arizona Technical Paper no. 4274, a contribution of the Arizona Agricultural Experiment Station, with the approval of the director.

Intranuclear sphingomyelin is associated with transcriptionally active chromatin and plays a role in nuclear integrity

“…Very similar effect on the cellular structures was induced by microinjection of phospholipase D, which hydrolyses the phosphate bonds of PL and also of SM to give the corresponding phosphatidic acid. However, this phospholipase reaction does not give rise to ceramide (Songer et al, 1990; Liscovitch et al, 2000). SMase is the enzyme that hydrolyses SM producing ceramide and sphingosine.…”

Identification and role in virulence of putative iron acquisition genes from Corynebacterium pseudotuberculosis

“…100 Wg ml 31 ampicillin or 30 Wg ml 31 chloramphenicol (Cm) were added, as appropriate. All C. pseudotuberculosis strains used in this study were derivatives of the caprine isolate, Whetten 1 [5] and were grown on brain heart infusion (BHI) agar supplemented with 5% bovine blood, at 37³C. Liquid cultures of C. pseudotuberculosis were grown in BHI broth supplemented with 5% fetal calf serum at 37³C with shaking.…”

“…The nucleotide sequence of a 4256-bp region downstream of the C. pseudotuberculosis Whetten 1 pld gene was determined from appropriate subclones of the pld-encoding cosmid pCpO1 [5], including pCpO48 [5] and pCpO51s [2]. Sequencing was performed on both strands, crossing all restriction sites, on a 377A DNA sequencer (Applied Biosystems Inc.) at the Automated DNA Sequencing Service at the University of Arizona.…”

Targeted mutagenesis of the phospholipase D gene results in decreased virulence ofCorynebacterium pseudotuberculosis

“…respectiveiy. Bands of 4.8 and 1.6kb are marked and correspond to fragments previously associated with the gene encoding PLD(Songer at al.. 1990).…”