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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

COGs classification of proteins encoded by up- and down-regulated genes in N. gonorrhoeae with inserted dam gene vs. wild-type strain. Letters represent COGs categories; numbers—the number of proteins in each category; black bars–proteins encoded by up-regulated genes; white bars–proteins encoded by down-regulated genes.
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Figure 4: COGs classification of proteins encoded by up- and down-regulated genes in N. gonorrhoeae with inserted dam gene vs. wild-type strain. Letters represent COGs categories; numbers—the number of proteins in each category; black bars–proteins encoded by up-regulated genes; white bars–proteins encoded by down-regulated genes.

Mentions: Expression microarray analysis was also performed for the N. gonorrhoeae drg::dam mutant. Obtained results showed that expression of 240 genes (11% of total genes) was deregulated by at least 1.5 fold (Supplementary Material, Table 3). Expression of 117 genes (48.75%) was increased and expression of 123 genes (51.25%) was decreased in comparison to the wt N. gonorrhoeae strain. We have identified 173 functional COGs (Supplementary Material, Table 3 and Figure 4). Most of the proteins which genes were deregulated (9 up- and 11 down-regulated) belonged to the “Amino acid transport and metabolism” (E category). The second most represented category was “Translation, ribosomal structure and biogenesis” (J category) within the “Information storage and processing” group (15 proteins). 21 proteins which genes were deregulated were classified to COGs involved in “Replication, recombination and repair” (L category) and “Transcription” (K category). 14 deregulated genes encoded proteins that matched COGs engaged in “Energy production and conversion”(C category). All identified categories are represented on Figure 4 and in Supplementary Material, Table 3. Many proteins, encoded by deregulated genes, did not exhibit any conserved domain (73 proteins) or their conserved domains did not have a characterized function (38 proteins in R and S category).


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)

COGs classification of proteins encoded by up- and down-regulated genes in N. gonorrhoeae with inserted dam gene vs. wild-type strain. Letters represent COGs categories; numbers—the number of proteins in each category; black bars–proteins encoded by up-regulated genes; white bars–proteins encoded by down-regulated genes.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: COGs classification of proteins encoded by up- and down-regulated genes in N. gonorrhoeae with inserted dam gene vs. wild-type strain. Letters represent COGs categories; numbers—the number of proteins in each category; black bars–proteins encoded by up-regulated genes; white bars–proteins encoded by down-regulated genes.
Mentions: Expression microarray analysis was also performed for the N. gonorrhoeae drg::dam mutant. Obtained results showed that expression of 240 genes (11% of total genes) was deregulated by at least 1.5 fold (Supplementary Material, Table 3). Expression of 117 genes (48.75%) was increased and expression of 123 genes (51.25%) was decreased in comparison to the wt N. gonorrhoeae strain. We have identified 173 functional COGs (Supplementary Material, Table 3 and Figure 4). Most of the proteins which genes were deregulated (9 up- and 11 down-regulated) belonged to the “Amino acid transport and metabolism” (E category). The second most represented category was “Translation, ribosomal structure and biogenesis” (J category) within the “Information storage and processing” group (15 proteins). 21 proteins which genes were deregulated were classified to COGs involved in “Replication, recombination and repair” (L category) and “Transcription” (K category). 14 deregulated genes encoded proteins that matched COGs engaged in “Energy production and conversion”(C category). All identified categories are represented on Figure 4 and in Supplementary Material, Table 3. Many proteins, encoded by deregulated genes, did not exhibit any conserved domain (73 proteins) or their conserved domains did not have a characterized function (38 proteins in R and S category).

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