IDENTIFICATION OF A NUCLEASE AND HOST RESTRICTION-MODIFICATION IN THEUNICELLULAR, AEROBIC NITROGEN-FIXING CYANOBACTERIUM CYANOTHECE SP

In the process of developing a gene transfer system for the marine, un icellular, nitrogen-fixing cyanobacterium Cyanothece sp. strain BH68K, two major restriction barriers have been identified. A tell wall-asso ciated nuclease exhibited non-site-specific degradation of covalently closed circular and linear double-stranded DNA molecules, including Cy anothece sp. strain BH68K chromosomal DNA. The nuclease is easily rele ased from intact cells by using water or buffer containing Triton X-10 0. Nuclease activity was undetectable in cell extracts prepared from w ater-washed cells. Comparison of the restriction endonuclease suscepti bility of Cyanothece sp. strain BH68K DNA to that of Anabaena sp. stra in PCC 7120 revealed that these organisms have a nearly identical patt ern of restriction and therefore may contain similar systems for DNA m ethylation. Restriction by DpnI, MboI, and Sau3AI indicated the presen ce of adenine methylation. Cyanothece sp. strain BH68K cell extracts c ontain a type II restriction endonuclease, Csp68KI. The activity of Cs p68KI aas easily detected in cell extracts without extensive purificat ion. Csp68KI is an isoschizomer of AvaII and recognizes the nucleotide sequence 5'-GG(A/T)CC-3'. Cleavage occurs between the guanosine nucle otides producing 3-bp 5' overhang ends.