Characterization of additional host restriction-modification systems in the unicellular cyanobacterium Cyanothece sp

In order to develop a gene transfer system for the unicellular diazotrophic cyanobacterium Cyanothece sp. strain BH68K, this organism has been further investigated for the presence of additional host restriction-modification enzymes other than Csp68KI, previously reported for Cyanothece sp. Analysis of cell extracts by phosphocellulose and Mono Q fast protein liquid chromatography (FPLC) has led to the identification of three new restriction endonucleases. These enzymes have been designated Csp68KII, Csp68KIII, and Csp68KVI. Csp68KII is an isoschizomer of AsuII and restricts DNA at the recognition sequence 5'-TT/CGAA-3'. Cleavage occurred between thymine and cytosine producing 2 bp 5' overhang ends. The third restriction endonuclease, Csp68KIII, is an isoschizomer of AvaIII and restricts DNA at the recognition sequence 5'-ATGCA/T-3'. Cleavage occurred between the 3' adenosine and thymine nucleotides producing 4 bp 3' overhang ends. The fourth enzyme identified, Csp68KVI, recognizes CGCG and cleaves this sequence between the internal guanine and cytosine nucleotides producing blunt ends.