A novel restriction-modification system, designated LlaJI, was identified on pNP40, a naturally occurring 65-kb plasmid from Lactococcus lactis. The system comprises four adjacent similarly oriented genes that are predicted to encode two m 5 C methylases and two restriction endonucleases. The LlaJI system, when cloned into a low-copy-number vector, was shown to confer resistance against representatives of the three most common lactococcal phage species. This phage resistance phenotype was found to be strongly temperature dependent, being most effective at 19°C. A functional analysis confirmed that the predicted methylase-encoding genes, llaJIM1 and llaJIM2, were both required to mediate complete methylation, while the assumed restriction enzymes, specified by llaJIR1 and llaJIR2, were both necessary for the complete restriction phenotype. A Northern blot analysis revealed that the four LlaJI genes are part of a 6-kb operon and that the relative abundance of the LlaJI-specific mRNA in the cells does not appear to contribute to the observed temperaturesensitive profile. This was substantiated by use of a LlaJI promoter-lacZ fusion, which further revealed that the LlaJI operon appears to be subject to transcriptional regulation by an as yet unidentified element(s) encoded by pNP40.Restriction-modification (R/M) systems are the most abundant bacteriophage resistance mechanism found in bacteria thus far. These systems are classified into four groups (designated type I, II, III, or IV) on the basis of their cofactor requirements, subunit composition, recognition sequence structure, and cleavage site relative to the recognition sequence (51). Members of type I are composed of the products of three distinct structural genes, which encode the subunits of a hetero-oligomeric enzyme complex that is required for restriction (R), modification (M), and specificity (S). Type I enzymes are present in two different oligomeric forms in vivo. The M 2 S oligomeric enzyme is capable of methylation in the presence of S-adenosylmethionine (AdoMet) and Mg 2ϩ , while the R 2 M 2 S oligomer is responsible for the restriction of unmethylated DNA in the presence of ATP, AdoMet, and Mg 2ϩ . Type I system representatives cleave target DNAs at sites that are distant from an asymmetrical recognition sequence. A model for cleavage by type I endonucleases has been proposed by Bourniquel and Bickle (4), who suggested that the endonuclease binds to its unmethylated recognition site, forming a dimer of two R 2 M 2 S oligomers. In the presence of ATP, all four HsdR subunits of this dimer complex promote the independent translocation of DNA directed toward the bound complex, and cleavage occurs when a physical barrier forces the translocation process to stop.A typical R/M system belonging to type II, which is the simplest group with respect to genetic structure and cofactor requirements, is composed of two distinct gene products, one of which acts as a Mg 2ϩ -dependent sequence-specific endonuclease (REase) while the other functions as a cognate AdoM...

The conjugative lactococcal plasmid pNP40, identified in Lactococcus lactis subsp. diacetylactis DRC3, possesses a potent complement of bacteriophage resistance systems, which has stimulated its application as a fitness-improving, food-grade genetic element for industrial starter cultures. The complete sequence of this plasmid allowed the mapping of previously known functions including replication, conjugation, bacteriocin resistance, heavy metal tolerance, and bacteriophage resistance. In addition, functions for cold shock adaptation and DNA damage repair were identified, further confirming pNP40's contribution to environmental stress protection. A plasmid cointegration event appears to have been part of the evolution of pNP40, resulting in a "stockpiling" of bacteriophage resistance systems.Lactococcus lactis, a gram-positive lactic acid bacterium, has been extensively exploited for the production of a variety of fermented dairy products. L. lactis strains exhibit biotechnologically important activities, which contribute to the character of the final food product, e.g., lactose utilization and protease production, and in addition encode properties that specifically provide a selective advantage to the bacterium itself, e.g., heavy metal resistance, bacteriocin production and/or immunity, and bacteriophage resistance (47). Many of these industrially significant traits have been found to be encoded by plasmids, which are omnipresent among this species, with most isolates containing multiple plasmids ranging in size from 2 to 80 kb (11).In recent decades, extensive research has established the molecular mechanisms governing many of these activities, in particular with respect to bacteriophage resistance (47). Lactococcal strains used for many food fermentations are known to be persistently challenged by phages and probably as a consequence have evolved numerous bacteriophage resistance strategies (48, 67).Presently, there are 30 completely sequenced lactococcal plasmids, the largest being pSK11P, a 75.8-kb plasmid isolated from L. lactis subsp. cremoris SK11 (47, 62). This plasmid encodes a variety of functions, including copper resistance, proteolytic activity, cold shock proteins, and cation transport activities, and displays clear "markings" of multiple recombination events that may have contributed to its evolution (62).Previous studies of a similarly sized plasmid, pNP40, originally identified in L. lactis subsp. diacetylactis DRC3 (45), revealed that this molecule, besides its encoded nisin and cadmium resistance determinants, is responsible for an impressive bacteriophage resistance profile (16,19,20,50,65). Two such systems, AbiE and AbiF, were found to provide significant resistance that correlates to an abortive infection phenotype (19).In addition, on the basis of phenotypic evidence, the presence of a third mechanism active at the stage of phage DNA injection was proposed (20). Most recently, a fourth resistance system, the LlaJI restriction-modification system, was identified (50).In the present stud...