Molecular Cloning and Analysis of the Modification Genes of the KpnBl Restriction- Modification System of Klebsiella pneumoniae Strain GM236

The KpnBl restriction-modification (R-M) system has been recognized for some time in Klebsiella pneumoniae strain GM236. Previously the restriction subunit (hsdRkpmBI) was cloned and sequenced, based on the sequence infonnation the KpnBl RM system was suspected to be either a type I or type III R-M system In this project the modification genes of KpnBl were cloned into a pMECA plasmid by using a modification expression method. The modification genes were identified on an 8.2 kb EcoRl fragment from a chromosomal library of the GM236 strain. The complete 8.2 kb fragment was sequenced and two large open reading frames (ORE) were identified. The first ORE was 2,631 bp in length and concluded to be the hsdRkpmBI gene based on sequence homologies. Similarily, the second ORE which was 1,344 bp in length was concluded to be the hsdRkpmBI gene. The presence of both hsdM and hsdS gene is a unique characteristic of type I systems. Therefore, it was concluded on the basis of the experimental evidence provided by this study that the KpnBI system is a type I system. Both phage and plasmid modification tests as well as complementation tests with the hsdRkpmBIsubunit indicate that the modification genes were expressed in both Kebsiella strains and E. coli. The deduced amino acid sequences of all three subunit proteins of the KpnBl system (HsdRKpnBi, HsdMitpnBi, and HsdSicpnBi) were compared to the corresponding proteins of all known type I systems. Because the Kpn&l system does not show sufficient homology when compared to any of the four existing type I families (IA, IB, IC, and ID) I concluded that KpnB\ represents a prototype of a previously undescribed type I family and proposed as type IE. Close analysis of the inter- and intrafamily protein sequence comparisons among currently known type I systems has allowed us to develop criteria for grouping type I systems based on protein sequence homologies. Using this method all the type I systems including numerous type I systems deduced from bacterial genome sequence projects were grouped into 24 new type I families in addition to the 4 existing families. In this project the DNA sequence recognized by the Kpn&l R-M enzyme has also been deduced using a new analytical method currently being developed in our laboratory. This method is based upon the comparative transfer efficiency of various plasmids with known DNA sequence into restriction (R+) competent and restriction impaired (R\u27) bacterial strains. It was assumed that plasmids restricted by R+ cells contained the recognition sites. The plasmid sequences were compared using a computer program which is designed to find the DNA sequences unique to restricted plasmids. This analysis showed that the KpnBl recognition site is most likely the 14 mer TGA (7N) TTTG. In this project a chromosomal library of K. pneumoniae GM236 was also constructed to facilitate completion of a GM236 chromosomal restriction map. A partial map was constructed in our lab by O. Rutebuka (1996). The library constructed here contains 100 HindlW clones and covers an estimated 455 kb segement, which represents approximately 10% of the total chromosome size. The use of these clones in future Southern hybridization tests will help to complete the restriction map and allow the accurate mapping of the KpnBl R-M genes on the chromosomal map of GM236