Deletion of One Nucleotide within the Homonucleotide Tract Present in the hsdS Gene Alters the DNA Sequence Specificity of Type I Restriction-Modification System NgoAV

As a result of a frameshift mutation, the hsdS locus of the NgoAV type IC restriction and modification (RM) system comprises two genes, hsdSNgoAV1 and hsdSNgoAV2. The specificity subunit, HsdSNgoAV, the product of the hsdSNgoAV1 gene, is a naturally truncated form of an archetypal specificity subunit (208 N-terminal amino acids instead of 410). The presence of a homonucleotide tract of seven guanines (poly[G]) at the 3' end of the hsdSNgoAV1 gene makes the NgoAV system a strong candidate for phase variation, i.e., stochastic addition or reduction in the guanine number. We have constructed mutants with 6 guanines instead of 7 and demonstrated that the deletion of a single nucleotide within the 3' end of the hsdSNgoAV1 gene restored the fusion between the hsdSNgoAV1 and hsdSNgoAV2 genes. We have demonstrated that such a contraction of the homonucleotide tract may occur in vivo: in a Neisseria gonorrhoeae population, a minor subpopulation of cells appeared to have only 6 guanines at the 3' end of the hsdSNgoAV1 gene. Escherichia coli cells carrying the fused gene and expressing the NgoAVΔ RM system were able to restrict λ phage at a level comparable to that for the wild-type NgoAV system. NgoAV recognizes the quasipalindromic interrupted sequence 5'-GCA(N8)TGC-3' and methylates both strands. NgoAVΔ recognizes DNA sequences 5'-GCA(N7)GTCA-3' and 5'-GCA(N7)CTCA-3', although the latter sequence is methylated only on the complementary strand within the 5'-CTCA-3' region of the second recognition target sequence.