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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

N. gonorrhoeae adhesion to human Hec-1-B cells. Human epithelial cells were infected for 3.5 h with the gonococcal wild-type strain, drg::cm or drg::dam mutant. The adhesion index was calculated as a ratio between the number of bacteria that adhered to human cells to the total cell number used for infection. The asterisk represents statistical difference in comparison to the wild-type strain (P < 0.05).
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Figure 8: N. gonorrhoeae adhesion to human Hec-1-B cells. Human epithelial cells were infected for 3.5 h with the gonococcal wild-type strain, drg::cm or drg::dam mutant. The adhesion index was calculated as a ratio between the number of bacteria that adhered to human cells to the total cell number used for infection. The asterisk represents statistical difference in comparison to the wild-type strain (P < 0.05).

Mentions: After demonstrating that gonococcal drg-deficient and dam-expressing mutant strains differ from the parental strain in adherence to different surfaces (glass and polystyrene), we investigated their ability to attach to human epithelial cells. Hec-1-B cells were cultured to monolayer and infected with N. gonorrhoeae for 3.5 h. After incubation, the bacteria that were attached to the human cell membrane or have penetrated into the human cells were liberated. In parallel, we counted all bacterial cells added to the eukaryotic cells (total CFU). Under our experimental conditions, the adhesion index of the drg::cm mutant was 0.26 (±0.13) and for the wt strain the adhesion index was 0.47 (±0.30) (Figure 8). However, this difference was not statistically significant. The gonococcal drg::dam mutant presented almost a 5-time lower adhesion compared to the wt strain (Figure 8). The adhesion index determined for drg::dam strain was 0.102 (±0.09; P < 0.05).


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)

N. gonorrhoeae adhesion to human Hec-1-B cells. Human epithelial cells were infected for 3.5 h with the gonococcal wild-type strain, drg::cm or drg::dam mutant. The adhesion index was calculated as a ratio between the number of bacteria that adhered to human cells to the total cell number used for infection. The asterisk represents statistical difference in comparison to the wild-type strain (P < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: N. gonorrhoeae adhesion to human Hec-1-B cells. Human epithelial cells were infected for 3.5 h with the gonococcal wild-type strain, drg::cm or drg::dam mutant. The adhesion index was calculated as a ratio between the number of bacteria that adhered to human cells to the total cell number used for infection. The asterisk represents statistical difference in comparison to the wild-type strain (P < 0.05).
Mentions: After demonstrating that gonococcal drg-deficient and dam-expressing mutant strains differ from the parental strain in adherence to different surfaces (glass and polystyrene), we investigated their ability to attach to human epithelial cells. Hec-1-B cells were cultured to monolayer and infected with N. gonorrhoeae for 3.5 h. After incubation, the bacteria that were attached to the human cell membrane or have penetrated into the human cells were liberated. In parallel, we counted all bacterial cells added to the eukaryotic cells (total CFU). Under our experimental conditions, the adhesion index of the drg::cm mutant was 0.26 (±0.13) and for the wt strain the adhesion index was 0.47 (±0.30) (Figure 8). However, this difference was not statistically significant. The gonococcal drg::dam mutant presented almost a 5-time lower adhesion compared to the wt strain (Figure 8). The adhesion index determined for drg::dam strain was 0.102 (±0.09; P < 0.05).

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