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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

In vitro characterization of the N. gonorrhoeae FA1090 Drg endonuclease. (A) Pre-purified Drg protein. The enzyme was separated on a 12% SDS PAGE gel and stained with Coomassie Brilliant Blue R250. M, PageRuler™ Prestained Protein Ladder (10, 15, 25, 35, 40, 55, 70, 100, 130, and 170 kDa) was used as the protein molecular mass marker. The arrow indicates the pre-purified Drg protein. (B) Determination of the activity and specificity of the Drg endonuclease. Lanes 1–4 methylated pUC19 DNA with GmeATC specificity. Lanes 6–9 unmethylated pUC19 DNA. 1 and 6, undigested DNA; 2 and 7, plasmids digested with DpnI (cuts only at GmeATC); 3 and 8, plasmids digested by MboI (cuts only at GATC); 4 and 9, plasmids digested by the pre-purified Drg endonuclease from N. gonorrhoeae FA1090; 5, GeneRuler™ DNA Ladder Mix: 10,000, 8000, 6000, 5000, 4000, 3500, 3000, 2500, 2000, 1500, 1200, 1000, 900, 800, 700, 600, 500, 400, 300, 200, and 100 bp.
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Figure 9: In vitro characterization of the N. gonorrhoeae FA1090 Drg endonuclease. (A) Pre-purified Drg protein. The enzyme was separated on a 12% SDS PAGE gel and stained with Coomassie Brilliant Blue R250. M, PageRuler™ Prestained Protein Ladder (10, 15, 25, 35, 40, 55, 70, 100, 130, and 170 kDa) was used as the protein molecular mass marker. The arrow indicates the pre-purified Drg protein. (B) Determination of the activity and specificity of the Drg endonuclease. Lanes 1–4 methylated pUC19 DNA with GmeATC specificity. Lanes 6–9 unmethylated pUC19 DNA. 1 and 6, undigested DNA; 2 and 7, plasmids digested with DpnI (cuts only at GmeATC); 3 and 8, plasmids digested by MboI (cuts only at GATC); 4 and 9, plasmids digested by the pre-purified Drg endonuclease from N. gonorrhoeae FA1090; 5, GeneRuler™ DNA Ladder Mix: 10,000, 8000, 6000, 5000, 4000, 3500, 3000, 2500, 2000, 1500, 1200, 1000, 900, 800, 700, 600, 500, 400, 300, 200, and 100 bp.

Mentions: To characterize the gonococcal Drg endonuclease, the ngo0007 gene was cloned into the pET28a vector and protein encoded by the gene was pre-purified from GM2163 (T7) (pET28a::drg) cells using metal affinity chromatography and a Ni-NTA Agarose column. The molecular mass of Drg was M(r) = 38000 ± 1000 Da as estimated from SDS-PAGE (Figure 9A) and was consistent with that predicted on the basis of the amino acid sequence.


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)

In vitro characterization of the N. gonorrhoeae FA1090 Drg endonuclease. (A) Pre-purified Drg protein. The enzyme was separated on a 12% SDS PAGE gel and stained with Coomassie Brilliant Blue R250. M, PageRuler™ Prestained Protein Ladder (10, 15, 25, 35, 40, 55, 70, 100, 130, and 170 kDa) was used as the protein molecular mass marker. The arrow indicates the pre-purified Drg protein. (B) Determination of the activity and specificity of the Drg endonuclease. Lanes 1–4 methylated pUC19 DNA with GmeATC specificity. Lanes 6–9 unmethylated pUC19 DNA. 1 and 6, undigested DNA; 2 and 7, plasmids digested with DpnI (cuts only at GmeATC); 3 and 8, plasmids digested by MboI (cuts only at GATC); 4 and 9, plasmids digested by the pre-purified Drg endonuclease from N. gonorrhoeae FA1090; 5, GeneRuler™ DNA Ladder Mix: 10,000, 8000, 6000, 5000, 4000, 3500, 3000, 2500, 2000, 1500, 1200, 1000, 900, 800, 700, 600, 500, 400, 300, 200, and 100 bp.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 9: In vitro characterization of the N. gonorrhoeae FA1090 Drg endonuclease. (A) Pre-purified Drg protein. The enzyme was separated on a 12% SDS PAGE gel and stained with Coomassie Brilliant Blue R250. M, PageRuler™ Prestained Protein Ladder (10, 15, 25, 35, 40, 55, 70, 100, 130, and 170 kDa) was used as the protein molecular mass marker. The arrow indicates the pre-purified Drg protein. (B) Determination of the activity and specificity of the Drg endonuclease. Lanes 1–4 methylated pUC19 DNA with GmeATC specificity. Lanes 6–9 unmethylated pUC19 DNA. 1 and 6, undigested DNA; 2 and 7, plasmids digested with DpnI (cuts only at GmeATC); 3 and 8, plasmids digested by MboI (cuts only at GATC); 4 and 9, plasmids digested by the pre-purified Drg endonuclease from N. gonorrhoeae FA1090; 5, GeneRuler™ DNA Ladder Mix: 10,000, 8000, 6000, 5000, 4000, 3500, 3000, 2500, 2000, 1500, 1200, 1000, 900, 800, 700, 600, 500, 400, 300, 200, and 100 bp.
Mentions: To characterize the gonococcal Drg endonuclease, the ngo0007 gene was cloned into the pET28a vector and protein encoded by the gene was pre-purified from GM2163 (T7) (pET28a::drg) cells using metal affinity chromatography and a Ni-NTA Agarose column. The molecular mass of Drg was M(r) = 38000 ± 1000 Da as estimated from SDS-PAGE (Figure 9A) and was consistent with that predicted on the basis of the amino acid sequence.

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