Two restriction-modification (R-M) systems KpnAI and KpnBl, found in Klebsiella pneumoniae strains M5a1 and GM236, respectively, have been studied and confirmed to he different from other R-M systems reported in K. pneumoniae. Mutant studies that the KpnAl and KpnBI systems may belong to either a type I or type III systems since approximately equal numbers of r-m+ and r-m- mutants were obtained. However, a DNA hybridization study using representative type I and type III probes from E. coli and S. typhimurium failed to show homologies to either KpnAI or KpnBI. The restriction endonuclease KpnBI was found to be temperature-sensitive with maximum restriction activity at 30°c and no restriction activity at 42°C. Further, the activity of endonuclease KpnBI was found to be reduced to almost zero level by growing the bacteria in the presence of 10% glycerol. Although the mechanism is not known, this is the first time such a phenomenon has been observed in any of the reported R-M systems. These studies also compared the efficiency of transformation in K. pneumoniae of three plasmid transformation methods; CaCl2 heat-shock; freezing and thawing in the presence of CaCl2; and electroporation. Electroporation was shown to be the most efficient method. Transformation efficiency in both the r+ kpnAL and r+ KpnBL strains was 20- to 100-fold less than the transformation efficiency of the r- strains, depending on plasmid size. Four different approaches have been used to clone the hsd genes of the KpnBI system. Two clones were obtained; these were named pKpnBl and pKpnB2. The pKpnBl and pKpnB2 clones were found to complement the restriction activity of a r-kpnBLm+ kpaBL K. pneumoniae mutant and were also found to complement both the restriction and modification activities of a r-kpnBLm+ kpaBL K. pneumoniae mutant. A quick subcloning method which involves making subclones from a plasmid clone in a single step was also developed. A preliminary analysis, based on complementation studies, of the gene structure suggested that the KpnBI system may consist of three structural genes, a characteristic of the type I R-M system.
Leonard R. Bullas
David A. Hessinger
Barry L. Taylor
Anthony J. Zuccarelli
Doctor of Philosophy (PhD)
Year Degree Awarded
Date (Title Page)
Library of Congress/MESH Subject Headings
DNA Restriction-Modification Enzymes -- analysis; Klebsiella Pneumoniae -- isolation & purification; Transformation, Bacterial
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This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.
Valinluck, Boontar, "Characterization of Restriction-Modification Systems in KLEBSIELLA PNEUMONIAE" (1992). Loma Linda University Electronic Theses, Dissertations & Projects. 1911.
Loma Linda University Electronic Theses and Dissertations
Loma Linda University. Del E. Webb Memorial Library. University Archives