Molecular characterization of a genomic region in a Lactococcus bacteriophage that is involved in its sensitivity to the phage defense mechanism AbiA

A spontaneous mutant of the lactococcal phage phi 31 that is insensitive to the phage defense mechanism AbiA was characterized in an effort to identify the phage factor(s) involved in sensitivity of phi 31 to AbiA, A point mutation was localized in the genome of the AbiA-insensitive phage (phi 31A) by heteroduplex analysis of a 9-kb region. The mutation (G to T) was within a 738-bp open reading frame (ORF245) and resulted in an arginine-to-leucine change in the predicted amino acid sequence of the protein, The mutant phi 31A-ORF245 reduced the sensitivity of phi 31 to AbiA when present in trans, indicating that the mutation in ORF245 is responsible for the AbiA insensitivity of phi 31A. Transcription of ORF245 occurs early in the phage infection cycles of phi 31 and phi 31A and is unaffected by AbiA, Expansion of the phi 31 sequence revealed ORF169 (immediately upstream of ORF245) and ORF71 (which ends 84 bp upstream of ORF169). Two inverted repeats lie within the 84-bp region between ORF71 and ORF169, Sequence analysis of an independently isolated AbiA-insensitive phage, phi 31B, identified a mutation (G to A) in one of the inverted repeats, A 118-bp fragment from phi 31, encompassing the 84-bp region between ORF71 and ORF169, eliminates AbiA activity against phi 31 when present in trans, establishing a relationship between AbiA and this fragment, The study of this region of phage phi 31 has identified an open reading frame (ORF245) and a 118-bp DNA fragment that interact with AbiA and are likely to be involved in the sensitivity of this phage to AbiA.