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Neisseria gonorrhoeae FA1090 carries genes encoding two classes of Vsr endonucleases.

ABSTRACT: A very short patch repair system prevents mutations resulting from deamination of 5-methylcytosine to thymine. The Vsr endonuclease is the key enzyme of this system, providing sequence specificity. We identified two genes encoding Vsr endonucleases V.NgoAXIII and V.NgoAXIV from Neisseria gonorrhoeae FA1090 based on DNA sequence similarity to genes encoding Vsr endonucleases from other bacteria. After expression of the gonococcal genes in Escherichia coli, the proteins were biochemically characterized and the endonucleolytic activities and specificities of V.NgoAXIII and V.NgoAXIV were determined. V.NgoAXIII was found to be multispecific and to recognize T:G mismatches in every nucleotide context tested, whereas V.NgoAXIV recognized T:G mismatches in the following sequences: GTGG, CTGG, GTGC, ATGC, and CTGC. Alanine mutagenesis of conserved residues showed that Asp50 and His68 of V.NgoAXIII and Asp51 and His69 of V.NgoAXIV are essential for hydrolytic activity. Glu25, His64, and Asp97 of V.NgoAXIV and Glu24, Asp63, and Asp97 of V.NgoAXIII are important but not crucial for the activity of V.NgoAXIII and V.NgoAXIV. However, Glu24 and Asp63 are also important for the specificity of V.NgoAXIII. On the basis of our results concerning features of Vsr endonucleases expressed by N. gonorrhoeae FA1090, we postulate that at least two types of Vsr endonucleases can be distinguished.

SUBMITTER: Kwiatek A

PROVIDER: S-EPMC2916375 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Neisseria gonorrhoeae FA1090 carries genes encoding two classes of Vsr endonucleases.

Kwiatek Agnieszka A Luczkiewicz Maciej M Bandyra Katarzyna K Stein Daniel C DC Piekarowicz Andrzej A

Journal of bacteriology 20100528 15

A very short patch repair system prevents mutations resulting from deamination of 5-methylcytosine to thymine. The Vsr endonuclease is the key enzyme of this system, providing sequence specificity. We identified two genes encoding Vsr endonucleases V.NgoAXIII and V.NgoAXIV from Neisseria gonorrhoeae FA1090 based on DNA sequence similarity to genes encoding Vsr endonucleases from other bacteria. After expression of the gonococcal genes in Escherichia coli, the proteins were biochemically characte ...[more]

PMID: 20511499

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Project description:BackgroundThe functioning of DNA repair systems is based on correct interactions between proteins involved in DNA repair. Very Short Patch (VSP) repair is a DNA repair system that corrects mismatches resulting from the deamination of 5-methylcytosine. The key enzyme in the VSP system is Vsr endonuclease, which can cleave mismatched DNA independently of accessory proteins. Until now, in vivo activity has only been shown for V.EcoKDcm - the only Vsr endonuclease in Escherichia coli. Additionally, the VSP system of E. coli is the only one for which interactions between proteins of the system have been demonstrated. Neisseria gonorrhoeae FA1090 is the first bacterium that we previously demonstrated to encode two active in vitro Vsr endonucleases: V.NgoAXIII and V.NgoAXIV.ResultsWe elucidate the mutator phenotype of N. gonorrhoeae mutants with disrupted genes encoding V.NgoAXIII or V.NgoAXIV endonuclease. Furthermore, we investigate the interactions between gonococcal Vsr endonucleases and MutL and MutS proteins. The Vsr endonucleases physically interact with gonococcal MutL protein but not with MutS protein. In the presence of the MutL protein, the efficiency of DNA cleavage by both V.NgoAXIII and V.NgoAXIV endonucleases increases, resulting in a decrease in the amount of Vsr enzyme required to complete digestion of mismatched DNA. Both Vsr endonucleases are also stimulated in vitro by the MutL protein of E. coli. In turn, the gonococcal MutS protein hinders DNA cleavage by the Vsr endonucleases. However, this effect is overridden in the presence of MutL, and furthermore, the simultaneous presence of MutL and MutS causes an increase in the efficiency of DNA cleavage by the Vsr endonucleases compared to the reaction catalyzed by V.NgoAXIII or V.NgoAXIV alone.ConclusionsFor the first time, interactions between proteins of the DNA repair system encoded by N. gonorrhoeae that are responsible for the correction of mismatches resulting from the 5-methylcytosine deamination were identified. The increase in activity of Vsr endonucleases in the presence of MutL protein could allow for reduced synthesis of the Vsr endonucleases in cells, and the susceptibility of gonococcal Vsr endonucleases on MutL protein of E. coli implies a universal mechanism of Vsr stimulation by MutL protein.

Project description: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.

Project description: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) × 10(5) for mutant and 4.67 (±3.09) × 10(4) 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.

Dataset Information

Neisseria gonorrhoeae FA1090 carries genes encoding two classes of Vsr endonucleases.

Publications

Neisseria gonorrhoeae FA1090 carries genes encoding two classes of Vsr endonucleases.

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