Neisseria gonorrhoeae is the etiological factor of the sexually transmitted gonorrhea disease that may lead, under specific conditions, to systemic infections. The gonococcal genome encodes many Restriction Modification (RM) systems, which main biological role is to defend the pathogen from potentially harmful foreign DNA. However, RM systems seem also to be involved in several other functions. In this study, we examined the effect of inactivation the N. gonorrhoeae FA1090 ngo0545 gene encoding M.NgoAX methyltransferase on the global gene expression, biofilm formation, interactions with human epithelial host cells and overall bacterial growth. Expression microarrays showed at least a two-fold deregulation of a total of 121 genes in the NgoAX knock-out mutant compared to the wt strain under standard grow conditions. As determined by the assay with crystal violet, the NgoAX knock-out strain formed a slightly larger biofilm biomass per cell than the wt strain (OD570/600 = 13.8  2.24 and 9.35  2.06, respectively). SCLM observations showed that the biofilm formed by the gonococcal ngo0545 gene mutant is more relaxed and dispersed than the one formed by the wt strain. Thickness of the biofilm formed by both strains was 48.3 (14.9) µm for the mutant and 28.6 (4.0) µm for the wt. This more relaxed feature of the biofilm in respect to adhesion and bacterial interactions seems advantageous for pathogenesis of the NgoAX-deficient gonococci at the stage of human epithelial cell invasion. Indeed, the overall adhesion of mutant bacterial cells to human cells was lower than adhesion of the wt gonococci (adhesion index = 0.672 ( 0.2) and 2.15 ( 1.53), respectively); yet, a higher number of mutant than wt bacteria were found inside the Hec-1-B epithelial cells (invasion index = 3.38 ( 0.93)  105 for mutant and 4.67 ( 3.09)  104 for the wt strain). These results indicate that NgoAX-deficient cells have lower ability to attach to human cells, but more easily penetrate inside the host cells. All these data suggest that the NgoAX methyltransferase, may be implicated in N. gonorrhoeae pathogenicity, involving regulation of biofilm formation, adhesion to host cells and epithelial cell invasion.

Numerous bacterial pathogens express an ortholog of the enzyme TlyA, which is an rRNA 2′-O-methyltransferase associated with resistance to cyclic peptide antibiotics such as capreomycin. Several other virulence traits have also been attributed to TlyA, and these appear to be unrelated to its methyltransferase activity. The bacterial pathogen Campylobacter jejuni possesses the TlyA homolog Cj0588, which has been shown to contribute to virulence. Here, we investigate the mechanism of Cj0588 action and demonstrate that it is a type I homolog of TlyA that 2′-O-methylates 23S rRNA nucleotide C1920. This same specific function is retained by Cj0588 both in vitro and also when expressed in Escherichia coli. Deletion of the cj0588 gene in C. jejuni or substitution with alanine of K⁸⁰, D¹⁶², or K¹⁸⁸ in the catalytic center of the enzyme cause complete loss of 2′-O-methylation activity. Cofactor interactions remain unchanged and binding affinity to the ribosomal substrate is only slightly reduced, indicating that the inactivated proteins are folded correctly. The substitution mutations thus dissociate the 2′-O-methylation function of Cj0588/TlyA from any other putative roles that the protein might play. C. jejuni strains expressing catalytically inactive versions of Cj0588 have the same phenotype as cj0588-null mutants, and show altered tolerance to capreomycin due to perturbed ribosomal subunit association, reduced motility and impaired ability to form biofilms. These functions are reestablished when methyltransferase activity is restored and we conclude that the contribution of Cj0588 to virulence in C. jejuni is a consequence of the enzyme's ability to methylate its rRNA.

Neisseriacae do not exhibit Dam methyltransferase activity and, instead of the dam gene, possess drg (dam replacing gene) inserted in the leuS/dam locus. The drg locus in Neisseria gonorrhoeae FA1090 has a lower GC-pairs content (40.5%) compared to the whole genome of N. gonorrhoeae FA1090 (52%). The gonococcal drg gene encodes a DNA endonuclease Drg, with GmeATC specificity. Disruption of drg or insertion of the dam gene in gonococcal genome changes the level of expression of genes as shown by transcriptome analysis. For the drg-deficient N. gonorrhoeae mutant, a total of 195 (8.94% of the total gene pool) genes exhibited an altered expression compared to the wt strain by at least 1.5 fold. In Dam-expressing N. gonorrhoeae mutant, the expression of 240 genes (11% of total genes) was deregulated. Most of these deregulated genes were involved in translation, DNA repair, membrane biogenesis and energy production as shown by cluster of orthologous group analysis. In vivo, the inactivation of drg gene causes the decrease of the number of live neisserial cells and long lag phase of growth. The insertion of dam gene instead of drg locus restores cell viability. We have also shown that presence of the drg gene product is important for N. gonorrhoeae FA1090 in adhesion, including human epithelial cells, and biofilm formation. Biofilm produced by drg-deficient strain is formed by more dispersed cells, compared to this one formed by parental strain as shown by scanning electron and confocal microscopy. Also adherence assays show a significantly smaller biomass of formed biofilm (OD570= 0.242 +/- 0.038) for drg-deficient strain, compared to wild type strain (OD570= 0.378 +/- 0.057). Dam-expressing gonococcal cells produce slightly weaker biofilm with embedded in extracellular matrix cells. This strain has also a 5 times reduced ability for adhesion to human epithelial cells. In this context, the presence of Drg is more advantageous for N. gonorrhoeae biology than Dam presence.