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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

Biofilm formed by N. gonorrhoeae on cover glass after 24 h visualized by Field Emission Scanning Electron Microscopy. (A) Biofilm produced by wild-type FA1090 strain; (B) Biofilm produced by N. gonorrhoeae drg::cm mutant and (C) Biofilm produced by N. gonorrhoeae drg::dam mutant. Experiments were triplicated and representative images are shown.
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Figure 5: Biofilm formed by N. gonorrhoeae on cover glass after 24 h visualized by Field Emission Scanning Electron Microscopy. (A) Biofilm produced by wild-type FA1090 strain; (B) Biofilm produced by N. gonorrhoeae drg::cm mutant and (C) Biofilm produced by N. gonorrhoeae drg::dam mutant. Experiments were triplicated and representative images are shown.

Mentions: To evaluate the ability of N. gonorrhoeae FA1090 wt, drg::cm and drg::dam mutants to form biofilm, we used FE SEM and SCLM. For FE SEM, gonococci were grown on a cover slip glass for 24 h in standard growth conditions. The wt strain formed a homogenous biofilm covering almost the whole glass with cells associated in layers (Figure 5A). Biofilm presented a structural roughness, yet, cells seems to be attached to each other in a relaxed, dispersed way. Gonococcal cells exhibited on their surface characteristic, discrete vesicles (blebbings) and had proper coffee-bean shape. In contrast, the biofilm formed by the N. gonorrhoeae drg-deficient mutant was leakier. Many single cells could be observed with numerous empty spaces between them (Figure 5B). However, some structural clumps were also noted. Such formations were well-structured like in the biofilm produced by wt strain. Cells were covered with many small tabs, and their surface was wrinkled. Biofilm created by the gonococcal drg::dam mutant seemed not to adhere to the glass surface as well as the wt strain (Figure 5C). Biofilm formations were “stressed” and seemed to be condensed. Cells were embedded in an extracellular structure that gave a cemented appearance. Single cells were crumpled.


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)

Biofilm formed by N. gonorrhoeae on cover glass after 24 h visualized by Field Emission Scanning Electron Microscopy. (A) Biofilm produced by wild-type FA1090 strain; (B) Biofilm produced by N. gonorrhoeae drg::cm mutant and (C) Biofilm produced by N. gonorrhoeae 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 5: Biofilm formed by N. gonorrhoeae on cover glass after 24 h visualized by Field Emission Scanning Electron Microscopy. (A) Biofilm produced by wild-type FA1090 strain; (B) Biofilm produced by N. gonorrhoeae drg::cm mutant and (C) Biofilm produced by N. gonorrhoeae drg::dam mutant. Experiments were triplicated and representative images are shown.
Mentions: To evaluate the ability of N. gonorrhoeae FA1090 wt, drg::cm and drg::dam mutants to form biofilm, we used FE SEM and SCLM. For FE SEM, gonococci were grown on a cover slip glass for 24 h in standard growth conditions. The wt strain formed a homogenous biofilm covering almost the whole glass with cells associated in layers (Figure 5A). Biofilm presented a structural roughness, yet, cells seems to be attached to each other in a relaxed, dispersed way. Gonococcal cells exhibited on their surface characteristic, discrete vesicles (blebbings) and had proper coffee-bean shape. In contrast, the biofilm formed by the N. gonorrhoeae drg-deficient mutant was leakier. Many single cells could be observed with numerous empty spaces between them (Figure 5B). However, some structural clumps were also noted. Such formations were well-structured like in the biofilm produced by wt strain. Cells were covered with many small tabs, and their surface was wrinkled. Biofilm created by the gonococcal drg::dam mutant seemed not to adhere to the glass surface as well as the wt strain (Figure 5C). Biofilm formations were “stressed” and seemed to be condensed. Cells were embedded in an extracellular structure that gave a cemented appearance. Single cells were crumpled.

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