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The dam replacing gene product enhances Neisseria gonorrhoeae FA1090 viability and biofilm formation.

Kwiatek A, Bacal P, Wasiluk A, Trybunko A, Adamczyk-Poplawska M - Front Microbiol (2014)

Bottom Line: 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.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).This strain has also a five times reduced ability for adhesion to human epithelial cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Virology, Institute of Microbiology, Faculty of Biology, University of Warsaw Warsaw, Poland.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Three-dimensional structures of biofilms formed by N. gonorrhoeae after 24 h visualized by Scanning Confocal Laser Microscopy. (A–C) Three-dimensional biofilm structures and density; (D–F) Biofilm thickness. (A,D) Biofilm produced by the wild-type strain; (B,E) Biofilm produced by the gonococcal drg::cm mutant; (C,F) Biofilm produced by the gonococcal drg::dam mutant. Experiments were triplicated and representative images are shown.
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Figure 6: Three-dimensional structures of biofilms formed by N. gonorrhoeae after 24 h visualized by Scanning Confocal Laser Microscopy. (A–C) Three-dimensional biofilm structures and density; (D–F) Biofilm thickness. (A,D) Biofilm produced by the wild-type strain; (B,E) Biofilm produced by the gonococcal drg::cm mutant; (C,F) Biofilm produced by the gonococcal drg::dam mutant. Experiments were triplicated and representative images are shown.

Mentions: To observe live gonococcal biofilm, we used SCLM. For this purpose, N. gonorrhoeae was cultivated on glass plates and after a 24-h growth, cells were stained by acridine orange. As shown on Figure 6, the biofilm formed by the N. gonorrhoeae drg::cm mutant was seriously confined (Figure 6B) in comparison to that formed by the wt strain (Figure 6A). The biofilm produced by the wt strain was dense and uniformly distributed on the glass surface. In contrast, the biofilm produced by the gonococcal drg::cm strain was represented by scattered clusters. The cross section (Figures 6D,E) indicated that only a small number of cells was attached to the glass. Finally, the biofilm produced by the N. gonorrhoeae drg::dam mutant (Figure 6C) resembled the one formed by the wt strain, however, it was less dense and unoccupied spaces between clumps could be noticed. Moreover, the biofilm seemed to be slightly thicker in comparison to the wt biofilm, indicating more complex structures.


The dam replacing gene product enhances Neisseria gonorrhoeae FA1090 viability and biofilm formation.

Kwiatek A, Bacal P, Wasiluk A, Trybunko A, Adamczyk-Poplawska M - Front Microbiol (2014)

Three-dimensional structures of biofilms formed by N. gonorrhoeae after 24 h visualized by Scanning Confocal Laser Microscopy. (A–C) Three-dimensional biofilm structures and density; (D–F) Biofilm thickness. (A,D) Biofilm produced by the wild-type strain; (B,E) Biofilm produced by the gonococcal drg::cm mutant; (C,F) Biofilm produced by the gonococcal drg::dam mutant. Experiments were triplicated and representative images are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4269198&req=5

Figure 6: Three-dimensional structures of biofilms formed by N. gonorrhoeae after 24 h visualized by Scanning Confocal Laser Microscopy. (A–C) Three-dimensional biofilm structures and density; (D–F) Biofilm thickness. (A,D) Biofilm produced by the wild-type strain; (B,E) Biofilm produced by the gonococcal drg::cm mutant; (C,F) Biofilm produced by the gonococcal drg::dam mutant. Experiments were triplicated and representative images are shown.
Mentions: To observe live gonococcal biofilm, we used SCLM. For this purpose, N. gonorrhoeae was cultivated on glass plates and after a 24-h growth, cells were stained by acridine orange. As shown on Figure 6, the biofilm formed by the N. gonorrhoeae drg::cm mutant was seriously confined (Figure 6B) in comparison to that formed by the wt strain (Figure 6A). The biofilm produced by the wt strain was dense and uniformly distributed on the glass surface. In contrast, the biofilm produced by the gonococcal drg::cm strain was represented by scattered clusters. The cross section (Figures 6D,E) indicated that only a small number of cells was attached to the glass. Finally, the biofilm produced by the N. gonorrhoeae drg::dam mutant (Figure 6C) resembled the one formed by the wt strain, however, it was less dense and unoccupied spaces between clumps could be noticed. Moreover, the biofilm seemed to be slightly thicker in comparison to the wt biofilm, indicating more complex structures.

Bottom Line: 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.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).This strain has also a five times reduced ability for adhesion to human epithelial cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Virology, Institute of Microbiology, Faculty of Biology, University of Warsaw Warsaw, Poland.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus