Analysis of type I restriction modification systems in the Neisseriaceae: genetic organization and properties of the gene products

Abstract

SummaryThe hsd locus (host specificity of DNA) was identified in the Neisseria gonorrhoeae genome. The DNA fragment encoding this locus produced an active restriction and modification (R/M) system when cloned into Escherichia coli. This R/M system was designated NgoAV. The cloned genomic fragment (7800 bp) has the potential to encode seven open reading frames (ORFs). Several of these ORFs had significant homology with other proteins found in the databases: ORF1, the hsdM, a methylase subunit (HsdM); ORF2, a homologue of dinD; ORF3, a homologue of hsdS; ORF4, a homologue of hsdS; and ORF5, an endonuclease subunit hsdR. The endonuclease and methylase subunits possessed strongest protein sequence homology to the Eco R124II R/M system, indicating that NgoAV belongs to the type IC R/M family. Deletion analysis showed that only ORF3 imparted the sequence specificity of the RM.NgoAV system, which recognizes an interrupted palindrome sequence (GCAN 8-TGC). The genetic structure of ORF3 (208 amino acids) is almost identical to the structure of the 5 0 truncated hsdS genes of Eco DXXI or Eco R124II R/M systems obtained by in vitro manipulation. Genomic sequence analysis allowed us to identify hsd loci with a very high homology to RM.NgoAV in two strains of Neisseria meningitidis. However, significant differences in the organization and structure of the hsdS genes in both these systems suggests that, if functional, they would possess recognition sites that differ from the gonococcus and from themselves.

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

“…All routine DNA manipulations were carried out according to standard protocols (39). Plasmid pMS2, carrying hsdM NgoAV , dinD, hsdS NgoAV1 , and hsdS NgoAV2 , was used as the template for mutagenesis (34). Plasmids pMS2⌬T and pMS2⌬40 were constructed by long PCR using appropriate primers (containing the site-directed mutations) annealing either side of the deleted sequences (inside-out).…”

“…1, panels 2a and 2b). The presence or absence of the hsdS NgoAV2 gene does not affect NgoAV specificity (34). The polypeptide encoded by hsdS NgoAV2 is nonfunctional, probably because it lacks a central CD and has a truncated TRD (Fig.…”

“…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).…”

“…Previously, we described the first active, naturally truncated form of the HsdS subunit, which forms part of the NgoAV RM system of N. gonorrhoeae (34). This type IC RM system is encoded by four genes: hsdM NgoAV , hsdR NgoAV , hsdS NgoAV1 , and hsdS NgoAV2 (Fig.…”

Comparative proteogenomic analysis of the Leptospira interrogans virulence-attenuated strain IPAV against the pathogenic strain 56601

“…This defense mechanism system consists of HsdM, HsdS, HsdR and sometimes a tRNA Lys -specific anticodon nuclease (ACNase), which, in combination, carries out DNA methylation and restriction functions [22]. In the IPAV strain, we identified the intact Type I R/M gene cluster with an anticodon nuclease gene incorporated (LIF_A2572-LIF_A2575).…”