Molecular characterization of the Lactococcus lactis LlaKR2I restriction-modification system and effect of an IS982 element positioned between the restriction and modification genes.

Restriction and modification (R-M) systems are widespread among prokaryotes, where they have a role in counteracting bacteriophage proliferation in a manner akin to a primitive immune system (reviewed in reference 20). Their presence in lactococci in conjunction with other natural phage defense mechanisms (i.e., adsorption blocking, injection blocking, and abortive infection [Abi] mechanisms) is of particular economic importance to the dairy fermentation industry, since phage infection of commercial lactococcal starter cultures persists as one of the primary sources of failed or suboptimal fermentations. The persistent challenge posed by phage infection has required the continued search for strains with superior phage-resistant traits to meet the demands of the dairy fermentation industry. To date, at least eight complete lactococcal R-M systems have been sequenced. Two of these have been classified as type I systems (46, 47), and five have been classified as type II systems; the latter include LlaDCHI (32), LlaBI (34), LlaCI (26), LlaDII (27), and ScrFI (7, 54, 55). The other R-M system described, LlaI, has characteristics reminiscent of both type IIS and type I R-M systems (17, 35). In general, type II systems are composed of two structural genes, an endonuclease and a methyltransferase. However, both the LlaDCHI and ScrFI R-M systems encode two methyltransferases. Comparison of LlaDCHI with the well-characterized DpnII system indicated that in addition to the conventional double-stranded DNA methyltransferase, a single-stranded DNA methyltransferase was encoded, a property which potentially facilitates the uptake of intact DNA via conjugation in Lactococcus lactis (32). Two 5-methylcytosine methyltransferases were identified in the ScrFI R-M system; while the exact role of each has not been established, the second methyltransferase may have evolved to counteract the degeneracy of the ScrFI endonuclease (55). A prerequisite to fully developing and enhancing phage resistance systems from a biotechnological standpoint is gaining an understanding of how these defense mechanisms are expressed and regulated in vivo. A number of factors have been shown to have an influence on the phenotypic expression of a number of native lactococcal phage resistance determinants. These factors include heat, whereby elevated temperatures similar to those used during the Cheddar cheese cooking process (40°C) render such mechanisms as the LlaI R-M system and the AbiA abortive infection mechanism (originally designated Hsp due to its heat-sensitive phage resistance) less effective in counteracting phage. In addition, insertion sequence (IS) elements have been demonstrated to play a role in the expression of some Abi determinants. A promoter sequence within an iso-ISS1 element located upstream of the abiB gene from L. lactis subsp. lactis IL416 was required for transcription of this gene (6), whereas insertion of IS981 into a fragment originating from the native lactococcal plasmid, pKR223, accounted for the loss of the Abi phenotype (37). Furthermore, IS-mediated rearrangements have contributed to altered resistance phenotypes in the conjugal plasmid pTR2030 (43). The native lactococcal phage resistance plasmid pKR223, first identified in L. lactis subsp. lactis biovar diacetylactis KR2, was shown to mediate two distinct mechanisms of resistance to bacteriophage infection: an R-M system active against the small isometric-headed phage φsk1, and an Abi mechanism which retards the proliferation of the prolate-headed phage φc2 (25, 30). Other phages, such as φstl1 and φeb1, also exhibited a reduced plaque size on hosts harboring pKR223, an observation predicted to be linked to the presence of the Abi mechanism. By using a streptococcal cloning vector, both resistance mechanisms of pKR223 were subcloned on a 19-kb HpaII fragment, generating a construct designated pGBK17 (25). A combination of deletion derivatives and a natural mutant arising from the fortuitous insertion of a novel lactococcal insertion sequence permitted the general location of both defense mechanisms to be established (30, 37). In this study, we determined the nucleotide sequence and established the precise location of the R-M determinants which contained an IS982 element. We subcloned this region and investigated the effect of the IS element on expression of the R-M phenotype.