A plasmid-encoded type IIB restriction-modification system in cyanobacteria blocks conjugative gene transfer

Abstract

Anabaena (aka Trichormus) variabilis ATCC 29413 is a filamentous, heterocyst-forming cyanobacterium with a 6.36 Mb chromosome, four circular plasmids, A (366 kb), B (35.8 kb), C (301 kb), and D (27 kb), and a 37-kb excision element. The hsdRMS genes on plasmid D may encode a type IIB restriction-modification system that protects the cells from invasion by foreign DNA. A variant of A. variabilis, strain FD, lacking plasmid D, grew with the same generation time as A. variabilis ATCC 29413, which stably maintained plasmid D. Nostoc sp. PCC 7120 and Nostoc sp. M131, which lack plasmid D and hsdRMS, grew similarly with a synthetic replicating plasmid, with or without added hsdRMS genes. Although the natural plasmid D was very stable in A. variabilis ATCC 29413, the synthetic plasmid was easily lost in Nostoc sp. PCC 7120 and Nostoc sp. M131, with or without hsdRMS. Strain FD, lacking plasmid D, and an A. variabilis hsdRM deletion mutant were much better hosts for conjugation of a non-replicative, integrative plasmid than wild-type A. variabilis. The conjugative nonreplicating plasmid formed circular molecules in strain FD and in the A. variabilis hsdRM deletion mutant, allowing for a high percentage of single-recombinant exconjugants. In contrast, the relatively few exconjugants in A. variabilis were all double recombinants, suggesting that the restriction-modification system encoded by the genes on plasmid D resulted in only linear molecules that recombined by double crossovers. Expression of the hsdRMS genes in strains Nostoc sp. PCC 7120 and Nostoc sp. M131 drastically reduced conjugation frequency and recombination of a non-replicative plasmid. Together, these data indicate that HsdRMS acts as a defense against the successful transfer of foreign DNA into these cyanobacteria, likely by double-stranded DNA cleavage. Many cyanobacterial strains that are not amenable to gene transfer might have similar restriction-modification systems that lead to cleavage and subsequent degradation of foreign DNA. Although such systems inhibit gene transfer, they may yield a high proportion, although a low number, of gene replacement recombinants.