Molecular Studies of KpnAI and KpnBI R-M Systems : in KLEBSIELLA PNEUMONIAE M5a1 and GM236

<p>Two possible type I restriction-modification (R-M) systems, <em>Kpn</em>AI and <em>Kpn</em>Bl, were recognized in <em>Klebsiella pneumoniae</em> strains M5al and GM236, respectively. In this project, four <em>hsdR</em>_KpnAI clones (pNLA1 to pNLA4) were obtained by using an endonuclease expression-selection method. The <em>hsdR</em>_KpnAI gene was identified on a 3.6 kb <em>Sau3</em>Al fragment. To map the gene, the <em>hsdR</em>_KpnAI clone was hybridized to a membrane which contained pulsed-field gel electrophoresed fragments of the chromosome of the strain M5al. The <em>hsdR</em>_KpnAIclone was hybridized to a 19 kb <em>Xba</em>l fragment and a 420 kb <em>Bln</em>l fragment, located at about 16 to 20 min on the recently constructed physical map of the M5al chromosome.</p> <p>Similarly, the <em>hsdR</em>_KpnBI clone was hybridized to a 40 kb <em>Xba</em>l fragment and a 330 kb <em>Bln</em>l fragment of the GM236 chromosome. Among five subclones (pNLB1 to pNLB5), the 3.5 kb <em>Bam</em>HI-<em>Eco</em>Rl fragment of pNLB2 was the smallest DNA fragment to retain restriction activity through complementation testing. The DNA sequence of the 3.5 kb fragment was determined. An open reading frame (ORF) of 3,039 bp was concluded to be the coding region for the HsdR_kpnBIpolypeptide. The 1,013 amino acid product (111,430 daltons) deduced from the nucleotide sequence is in excellent agreement with the 116 kD size estimated from the electrophoretic mobility of the putative HsdR_kpnBIprotein in SDS polyacrylamide gels. The nucleotide sequence of the hsdR_kpnBIgene showed no significant similarity to any other sequence in the GenBank database. However, a homology search of the amino acid sequence scored highly with <em>Eco</em>R124/3I, a type IC restriction enzyme. After alignment of the two proteins, two ATP binding domains, a DNA binding domain and seven helicase motifs were found. Eight small ORFs were identified within a few kb on either side of the <em>hsdR</em>_KpnBI gene. When subcloned, none of these ORFs showed any modification activity. However, complementation with r^-_KpnBI m^-_KpnBImutant suggests that one downstream ORF is a positive effector for expression of the KpnBI modification activity.</p>

Name thesis:degree_name

Doctor of Philosophy (Medical Science)

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Microbiology

Year

1994

Author dc:creator

• Lee, Nan Sook

Contributors dc:contributor

• Junichi Ryu
• Leonard R. Bullas
• Kelvin A. W. Hill
• John J. Rossi
• Anthony J. Zuccarelli

• Microbiology
• Klebsiella pneumoniae -- genetics; Restriction Mapping Cloning; Molecular

Statement dc: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.

Language dc:language

English

Repository record dc:identifier

https://scholarsrepository.llu.edu/etd/1404

OAI identifier oai:identifier

oai:scholarsrepository.llu.edu:etd-2175