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Title:Plasmid studies in Clostridium perfringens: Plasmid isolation; bacterial conjugation; interstrain mobilization of nonconjugative plasmids
Author(s):Hampson, Brian Clark
Doctoral Committee Chair(s):Witter, Lloyd D.
Department / Program:Food Science and Human Nutrition
Discipline:Food Science and Human Nutrition
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Agriculture, Food Science and Technology
Biology, Genetics
Biology, Microbiology
Abstract:A procedure for the isolation of both high and low molecular weight DNA from Clostridium perfringens was developed. Cells were washed, subjected to an extended lysozyme treatment and lysed under alkaline conditions (pH 12.4) with a 4% SDS solution. Following neutralization, salt precipitation and protein extractions with organic solvents, the DNA was ammonium acetate/ethanol precipitated up to three times. The procedure may be scaled up or used for mini-prep isolations.
Another procedure, called the rapid boiling method, was also developed. It is a quick, alternative method for plasmid isolation. Cell lysis is achieved by boiling the lysozyme-treated cell suspension for 2 minutes in a 2% SDS solution followed by ultracentrifugation (45,000 rpm for 30 minutes at 4$\sp\circ$C) of the lysate to remove cellular debris. Protein extractions using organic solvents, ammonium acetate and ethanol precipitations complete the procedure.
A method for improving the frequency of conjugation between strains of C. perfringens is described. The interstrain plasmid conjugation frequency following filter-mating between a naladixic acid and spectinimycin-resistant C. perfringens 3626B donor strain containing plasmids pIP406 (encoding tetracycline resistance) and pHB101 (caseinase activity) and a rifampicin and streptomycin-resistant, plasmid-free 3624A recipient was increased seven orders of magnitude when compared to the frequency obtained using standard agar plate mating. Adjusting the donor to recipient cell ratio to 1:4 resulted in a conjugation frequency of 1.47 $\times$ 10$\sp{-2}$ tetracycline resistant 3624A-based transconjugants per viable post-conjugative donor cell. This improved frequency made possible the mobilization of the 3.2 kb caseinase enzyme activity (Lambda toxin) encoding nonconjugative plasmid designated pHB101. Greater than 39% mobilization of the Lambda toxin phenotype was observed. Southern blotting and hybridization using a radio-labelled pHB101 probe indicated that the transconjugants contained pHB101 in its autonomous form as well as co-integrated with other high molecular weight plasmids. The high frequency of mobilization and the variability in size of plasmids in the transconjugants suggested that the mechanism of mobilization is of the Class II type, as proposed by Kilbane and Malamy (1980). Class II plasmid mobilization involves the recA system, concomitant transfer of the mobilizing plasmid (pIP406), and altered plasmids are yielded at high frequencies.
Issue Date:1989
Rights Information:Copyright 1989 Hampson, Brian Clark
Date Available in IDEALS:2011-05-07
Identifier in Online Catalog:AAI8916258
OCLC Identifier:(UMI)AAI8916258

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