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

Abstract

ABSTRACT As a result of a frameshift mutation, the hsdS locus of the NgoAV type IC restriction and modification (RM) system comprises two genes, hsdS NgoAV1 and hsdS NgoAV2 . The specificity subunit, HsdS NgoAV , the product of the hsdS NgoAV1 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 hsdS NgoAV1 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 hsdS NgoAV1 gene restored the fusion between the hsdS NgoAV1 and hsdS NgoAV2 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 hsdS NgoAV1 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(N 8 )TGC-3′ and methylates both strands. NgoAVΔ recognizes DNA sequences 5′-GCA(N 7 )GTCA-3′ and 5′-GCA(N 7 )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.