Biomaterial Database

Cloning and expression of the phospholipase D gene from Corynebacterium pseudotuberculosis in Escherichia coli. 1990

J G Songer, and S J Libby, and J J Iandolo, and W A Cuevas

Department of Veterinary Science, University of Arizona, Tucson 85721.

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.

UI	MeSH Term	Description	Entries
D010739	Phospholipase D	An enzyme found mostly in plant tissue. It hydrolyzes glycerophosphatidates with the formation of a phosphatidic acid and a nitrogenous base such as choline. This enzyme also catalyzes transphosphatidylation reactions. EC 3.1.4.4.	Lecithinase D,Phosphatidylcholine Phosphohydrolase
D010740	Phospholipases	A class of enzymes that catalyze the hydrolysis of phosphoglycerides or glycerophosphatidates. EC 3.1.-.	Lecithinases,Lecithinase,Phospholipase
D003001	Cloning, Molecular	The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells.	Molecular Cloning
D003352	Corynebacterium	A genus of asporogenous bacteria that is widely distributed in nature. Its organisms appear as straight to slightly curved rods and are known to be human and animal parasites and pathogens.
D004269	DNA, Bacterial	Deoxyribonucleic acid that makes up the genetic material of bacteria.	Bacterial DNA
D004926	Escherichia coli	A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc.	Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D005798	Genes, Bacterial	The functional hereditary units of BACTERIA.	Bacterial Gene,Bacterial Genes,Gene, Bacterial
D015139	Blotting, Southern	A method (first developed by E.M. Southern) for detection of DNA that has been electrophoretically separated and immobilized by blotting on nitrocellulose or other type of paper or nylon membrane followed by hybridization with labeled NUCLEIC ACID PROBES.	Southern Blotting,Blot, Southern,Southern Blot
D015153	Blotting, Western	Identification of proteins or peptides that have been electrophoretically separated by blot transferring from the electrophoresis gel to strips of nitrocellulose paper, followed by labeling with antibody probes.	Immunoblotting, Western,Western Blotting,Western Immunoblotting,Blot, Western,Immunoblot, Western,Western Blot,Western Immunoblot,Blots, Western,Blottings, Western,Immunoblots, Western,Immunoblottings, Western,Western Blots,Western Blottings,Western Immunoblots,Western Immunoblottings
D015183	Restriction Mapping	Use of restriction endonucleases to analyze and generate a physical map of genomes, genes, or other segments of DNA.	Endonuclease Mapping, Restriction,Enzyme Mapping, Restriction,Site Mapping, Restriction,Analysis, Restriction Enzyme,Enzyme Analysis, Restriction,Restriction Enzyme Analysis,Analyses, Restriction Enzyme,Endonuclease Mappings, Restriction,Enzyme Analyses, Restriction,Enzyme Mappings, Restriction,Mapping, Restriction,Mapping, Restriction Endonuclease,Mapping, Restriction Enzyme,Mapping, Restriction Site,Mappings, Restriction,Mappings, Restriction Endonuclease,Mappings, Restriction Enzyme,Mappings, Restriction Site,Restriction Endonuclease Mapping,Restriction Endonuclease Mappings,Restriction Enzyme Analyses,Restriction Enzyme Mapping,Restriction Enzyme Mappings,Restriction Mappings,Restriction Site Mapping,Restriction Site Mappings,Site Mappings, Restriction