Abstract

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.

LLU Discipline

Microbiology

Department

Microbiology

School

Graduate School

First Advisor

Junichi Ryu

Second Advisor

Leonard R. Bullas

Third Advisor

David A. Hessinger

Fourth Advisor

Barry L. Taylor

Fifth Advisor

Anthony J. Zuccarelli

Degree Name

Doctor of Philosophy (PhD)

Degree Level

Ph.D.

Year Degree Awarded

1992

Date (Title Page)

7-1992

Language

English

Library of Congress/MESH Subject Headings

DNA Restriction-Modification Enzymes -- analysis; Klebsiella Pneumoniae -- isolation & purification; Transformation, Bacterial

Type

Dissertation

Page Count

xi; 162

Digital Format

PDF

Digital Publisher

Loma Linda University Libraries

Usage Rights

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.

Collection

Loma Linda University Electronic Theses and Dissertations

Collection Website

http://scholarsrepository.llu.edu/etd/

Repository

Loma Linda University. Del E. Webb Memorial Library. University Archives

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