Limits...
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 the drg-deficient N. gonorrhoeae 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
getmorefigures.php?uid=PMC4269198&req=5

Figure 3: COGs classification of proteins encoded by up- and down-regulated genes in the drg-deficient N. gonorrhoeae 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: The gonococcal gene expression was determined by expression microarray analysis. For the N. gonorrhoeae drg::cm mutant, a total of 195 genes exhibited an altered expression by at least 1.5 fold compared to the wt strain (Supplementary Material, Table 2). This group of genes represented 8.94% of the total gene pool. Expression of 136 genes (69.74%) was up-regulated and 59 genes (30.26%) were down-regulated. We have determined the cluster of orthologous gene, COG, (Natale et al., 2000; Tatusov et al., 2000) category for each gene (with deregulated expression), which encode proteins of known function (Supplementary Material, Table 2 and Figure 3). We found 102 COGs (70 among up-regulated genes and 32 among down-regulated), while 97 proteins did not exhibit any known conserved domain. As seen in Figure 3, genes which expression was enhanced in the gonococcal drg::cm mutant encoded proteins that mostly belong to the “Information storage and processing” or “Metabolism” category. In the first group, 25 proteins belonged to COGs involved in “Replication, recombination and repair” (L category) and “Translation, ribosomal structure and biogenesis” (J category). A total of 27 proteins was grouped in the “Metabolism” category. Among them, we found COGs classified to “Energy production and conversion” (C category), “Amino Acid metabolism and transport” (E category) and “Inorganic ion transport and metabolism” (P category). A predicted general function (R category) was determined for five proteins and eight were classified to the S category (Function Unknown). Detailed information can be found in Supplementary Material, Table 2.


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 the drg-deficient N. gonorrhoeae 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 3: COGs classification of proteins encoded by up- and down-regulated genes in the drg-deficient N. gonorrhoeae 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: The gonococcal gene expression was determined by expression microarray analysis. For the N. gonorrhoeae drg::cm mutant, a total of 195 genes exhibited an altered expression by at least 1.5 fold compared to the wt strain (Supplementary Material, Table 2). This group of genes represented 8.94% of the total gene pool. Expression of 136 genes (69.74%) was up-regulated and 59 genes (30.26%) were down-regulated. We have determined the cluster of orthologous gene, COG, (Natale et al., 2000; Tatusov et al., 2000) category for each gene (with deregulated expression), which encode proteins of known function (Supplementary Material, Table 2 and Figure 3). We found 102 COGs (70 among up-regulated genes and 32 among down-regulated), while 97 proteins did not exhibit any known conserved domain. As seen in Figure 3, genes which expression was enhanced in the gonococcal drg::cm mutant encoded proteins that mostly belong to the “Information storage and processing” or “Metabolism” category. In the first group, 25 proteins belonged to COGs involved in “Replication, recombination and repair” (L category) and “Translation, ribosomal structure and biogenesis” (J category). A total of 27 proteins was grouped in the “Metabolism” category. Among them, we found COGs classified to “Energy production and conversion” (C category), “Amino Acid metabolism and transport” (E category) and “Inorganic ion transport and metabolism” (P category). A predicted general function (R category) was determined for five proteins and eight were classified to the S category (Function Unknown). Detailed information can be found in Supplementary Material, Table 2.

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