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

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 NgoAV⌬ RM system were able to restrict 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. NgoAV⌬ 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.Restriction and modification (RM) systems are composed of two enzymatic activities that recognize the same specific DNA sequence. The restriction endonuclease cleaves the DNA, unless the recognized sequence is methylated by the modification methylase. RM systems are found in a wide variety of bacteria and archaea, and their main role is to protect cells from bacteriophage infections or the uptake of undesirable foreign DNA (7, 33). The importance of RM systems in host protection is difficult to estimate in the case of bacteria for which no phages or no phage detection methods are known and that encode several RM systems, such as Neisseria gonorrhoeae (43,34). Recently, a new role for RM systems as factors regulating other gene expression was proposed for type III RM systems (16,42).Type I DNA RM systems are multimeric enzyme complexes that recognize and methylate an adenine residue within a specific DNA sequence. The cleavage of double-stranded DNA takes place at an undefined region, several hundreds to thousands of base pairs away from the recognized sequence. The archetypal type I methyltransferases are encoded by two genes, hsdS and hsdM, and are composed of one HsdS subunit and two HsdM subunits. The HsdS subunit is responsible for recognition and binding of DNA by the enzymatic complex and for the interaction of the other subunits of the enzyme. The

DNA sequence repeats identify numerous Type I restriction‐modification systems that are potential epigenetic regulators controlling phase‐variable regulons; phasevarions

“…We predict that variation in length of SSRs located in hsdM genes likely leads to ON-OFF switching, akin to that seen with Type III mod genes 9,17 ( Figure 5). SSR variation has been demonstrated to result in a switch between specificities due to expression of a full-length HsdS or truncated HsdS has been demonstrated in N. gonorrhoeae, 31 and full and extended versions of HsdS proteins are expressed by M. haemolytica commensurate with SSR tract variation (this work). For example, SSR tracts in hsdS genes invariably occur between two TRDs, which leads to expression of multiple HsdS variants, be they full length two TRD proteins, truncated HsdS subunits which then dimerise to form a functional HsdS protein, or an extended three TRD containing HsdS protein (Figure 1 and Figure 3).…”

“…Type I R-M systems that phase vary via changes in the length of SSRs are less well studied. The full-length and truncated HsdS proteins have differing methyltransferase specificities, 31 with two of these truncated HsdS subunits combining to participate in sequence recognition 32 (see Figure 1A). An hsdM gene containing a pentanucleotide SSR tract was identified in H. influenza, 30 and we previously noted changes in the length of this tract in multiple nontypeable H. influenzae (NTHi) isolates from paired samples taken from the human nasopharynx and middle ear during cases of otitis media.…”

The Complete Genome and Phenome of a Community-Acquired Acinetobacter baumannii

“…Analysis indicated the gene encoding the specificity subunit ( hsdS ) of the restriction-modification system contained a frameshift mutation. However, a truncated hsdS can still potentially translate and dimerize into a functional HsdS subunit, albeit with an altered DNA specificity [66]-[68]. Nevertheless, the presence of a DNA restriction-modification system in this genomic island may act as a ‘selfish’ genetic element to ensure its dissemination [69] and/or may function in bacteriophage defence and hindrance of lateral gene transfer [70].…”

The role of variable DNA tandem repeats in bacterial adaptation

“…Interestingly, a recent study uncovered a variable poly-G tract within the 3′ end of hsdS NgoAV1 in N. gonorrhoeae, and loss of a guanine in that tract restores the fusion of the hsdS NgoAV1 and hsdS NgoAV2 genes, resulting in the generation of a new HsdS NgoAVD protein, responsible for a novel NgoAV R-M system, termed 'NgoAVD'. The NgoAVD system has a modified DNA recognition specificity, thereby conferring an altered susceptibility to various phages (Adamczyk-Poplawska et al, 2011).…”