BackgroundBioinformatic analysis of the genome sequence of Neisseria gonorrhoeae revealed the presence of nine probable prophage islands. The distribution, conservation and function of many of these sequences, and their ability to produce bacteriophage particles are unknown.ResultsOur analysis of the genomic sequence of FA1090 identified five genomic regions (NgoΦ1 – 5) that are related to dsDNA lysogenic phage. The genetic content of the dsDNA prophage sequences were examined in detail and found to contain blocks of genes encoding for proteins homologous to proteins responsible for phage DNA replication, structural proteins and proteins responsible for phage assembly. The DNA sequences from NgoΦ1, NgoΦ2 and NgoΦ3 contain some significant regions of identity. A unique region of NgoΦ2 showed very high similarity with the Pseudomonas aeruginosa generalized transducing phage F116. Comparative analysis at the nucleotide and protein levels suggests that the sequences of NgoΦ1 and NgoΦ2 encode functionally active phages, while NgoΦ3, NgoΦ4 and NgoΦ5 encode incomplete genomes. Expression of the NgoΦ1 and NgoΦ2 repressors in Escherichia coli inhibit the growth of E. coli and the propagation of phage λ. The NgoΦ2 repressor was able to inhibit transcription of N. gonorrhoeae genes and Haemophilus influenzae HP1 phage promoters. The holin gene of NgoΦ1 (identical to that encoded by NgoΦ2), when expressed in E. coli, could serve as substitute for the phage λ s gene. We were able to detect the presence of the DNA derived from NgoΦ1 in the cultures of N. gonorrhoeae. Electron microscopy analysis of culture supernatants revealed the presence of multiple forms of bacteriophage particles.ConclusionThese data suggest that the genes similar to dsDNA lysogenic phage present in the gonococcus are generally conserved in this pathogen and that they are able to regulate the expression of other neisserial genes. Since phage particles were only present in culture supernatants after induction with mitomycin C, it indicates that the gonococcus also regulates the expression of bacteriophage genes.

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

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 ngoAXmod 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 twofold deregulation of a total of 121 genes in the NgoAX knock-out mutant compared to the wild-type (wt) strain under standard grow conditions. Genes with changed expression levels encoded mostly proteins involved in cell metabolism, DNA replication and repair or regulating cellular processes and signaling (such as cell wall/envelop biogenesis). 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. Live biofilm observations showed that the biofilm formed by the gonococcal ngoAXmod gene mutant is more relaxed, dispersed and thicker than the one formed by the wt strain. This more relaxed feature of the biofilm, in respect to adhesion and bacterial interactions, can be involved in pathogenesis. Moreover, 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 knock-out 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.

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Many Neisseriaceae 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 cells embedded in an extracellular matrix. This strain has also a five 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.