CLONING AND DNA-SEQUENCE ANALYSIS OF 2 ABORTIVE INFECTION PHAGE RESISTANCE DETERMINANTS FROM THE LACTOCOCCAL PLASMID PNP40

The lactococcal plasmid pNP40, from Lactococcus lactis subsp, lactis b iovar diacetylactis DRC3, confers complete resistance to the prolate-h eaded phage phi c2 and the small isometric-headed phage phi 712 in L. lactis subsp, lactis MG1614. A 6.0-kb NcoI fragment of pNP40 cloned in the lactococcal Escherichia coil shuttle vector pAM401 was found to c onfer partial resistance to phi 712. Subcloning and deletion analysis of the recombinant plasmid pPG01 defined a 2.5-kb ScaIHpaI fragment as conferring phage insensitivity, Sequence analysis of this region conf irmed the presence of two overlapping open reading frames (ORFs), Furt her subcloning of pNP40 to characterize the resistance determinant act ive against phi c2 identified a 5.6-kb EcoRV fragment of pNP40 which, when cloned in pAM401, conferred partial resistance to both phi c2 and phi 712. Subcloning and deletion analysis of the recombinant plasmid pCG1 defined a 3.7-kb EcoRV-XbaI fragment as encoding phage insensitiv ity. DNA sequence analysis of this region revealed the presence of a s ingle complete ORF, The introduction of a frameshift mutation at the u nique Bg/II site within this ORF disrupted the phage resistance phenot ype, confirming that this ORF is responsible for the observed phage in sensitivity, The mechanisms encoded by pPG01 and pCG1 in L. lactis sub sp. lactis MG1614 conformed to the criteria defining abortive infectio n and were designated AbiE and AbiF, respectively, Analysis of the pha ge DNA content of phi 712-infected hosts containing AbiF demonstrated that it inhibited the rate of phage DNA replication, while AbiE had li ttle effect on phage DNA replication, suggesting a later target of inh ibition, The predicted protein product of abiF shows significant homol ogy to the products of two other lactococcal abortive infection genes, abiD and abiD1.