Bacterial cells contain various restriction-modification (R-M) systems to protect themselves from incoming foreign DNA. Most bacteria have at least one such system. In this study, the restriction genes and the location of the genes on the chromosome were examined in three enteric bacteria. Salmonella typhimurium LT2, Klebsiella pneumoniae GM 236, and Klebsiella pneumoniae M5al. The 98 minute region of the Salmonella typhimurium LT2 chromosome comprises at least two R-M systems. From genetic data this region was previously thought to be only one minute in length but was found to be larger than two minutes using a physical mapping technique, pulsed field gel electrophoresis (PFGE).
The KpnAI and KpnBI R-M systems were recognized in K. pneumoniae strain M5al and GM236, respectively. A macro-restriction map of Klebsiella pneumoniae GM236 was constructed in this study using PFGE and Southern hybridization. The genome was digested with three rare cutting restriction enzymes (BlnI, I-Cuel and XBal). The estimated size of the GM236 genome was 4,582 (±80) kb, in accordance with the range of other enteric bacteria. The partial digest of I-Cuel allowed a tentative ordering of the eight fragments on a circular map. The map was remarkably similar to the map of Escherichia coli and S. typhimurium with the exception of two fragments. I-Cuel cuts the GM236 chromosome in eight fragments whereas it cuts E. coli and Salmonella genomes This suggests a possible in seven. duplication of the ribosomal cluster in the GM236 strain. The constructed map, although tentative, has permitted the mapping of hsdR KpnBI’ a restriction gene located on a 322 kpmBI+ fragment and also on a 40 kb Xbal fragment.
In this study, hsdRKpnA1, another restriction gene recognized in K. pneumoniae M5al, was subcloned and sequenced. The DNA sequence of 4.5 kb was determined and only one open reading frame (ORF) of 3, 305 base pair (bp) was considered as the coding region for the HsdRKpnAI polypeptide. The nucleotide sequence of the HsdRKpnA gene showed no significant similarity to any other sequences in the GenBank database. However, the deduced amino acid sequence showed a moderate degree of homology (26%) with EcoR\2AH (former £cK. pneumoniae.
Ronald L. Carter
David L. Cowels
E. Clifford Herrmann
Doctor of Philosophy (PhD)
Year Degree Awarded
Date (Title Page)
Library of Congress/MESH Subject Headings
Bacteria; Genome, Bacterial; DNA, Bacterial; DNA Restriction Enzymes; Salmonella typhimurium; Klebsiella pneumoniae; Escherichia coli; Bacteriophages
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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.
Rutebuka, Obed B., "Molecular Studies of Restriction Genes in Enteric Bacteria" (1996). Loma Linda University Electronic Theses, Dissertations & Projects. 757.
Loma Linda University Electronic Theses and Dissertations
Loma Linda University. Del E. Webb Memorial Library. University Archives