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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

Genetic maps of drg or dam loci in the genomes of Neisseriaceae. Analysis included only the wholly-sequenced genomes of Neisseriaceae. leuS–gene encoding leucyl-tRNA synthase, drg—dam replacing gene, dam—gene encoding Dam methylase. For each type of locus organization, (A–F)—species and serogroup names of Neisseriaceae are given in the text.
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Figure 1: Genetic maps of drg or dam loci in the genomes of Neisseriaceae. Analysis included only the wholly-sequenced genomes of Neisseriaceae. leuS–gene encoding leucyl-tRNA synthase, drg—dam replacing gene, dam—gene encoding Dam methylase. For each type of locus organization, (A–F)—species and serogroup names of Neisseriaceae are given in the text.

Mentions: The drg gene was previously identified in the genomes of N. meningitidis and Neisseria lactamica (Cantalupo et al., 2001; Jolley et al., 2004). In N. gonorrhoeae FA1090, the Drg endonuclease seems to be encoded by the ngo0007 gene. By blasting the nucleotide sequence of the drg locus against the NCBI database 27 hits were obtained. Among them, 23 ORFs were drg. Within Neisseriaceae, only N. gonorrhoeae, N. meningitidis, and N. lactamica were determined to contain the drg locus. Only 21 hits were found in wholly-sequenced neisserial genomes. For these genomes, we have analyzed the genetic context of the drg and dam loci. In all cases, the drg/dam locus neighbored the leuS locus (leucyl-tRNA synthetase) (Figure 1). In three cases (N. gonorrhoeae FA1090, MS11, and TCDC-NG08107), the drg gene was followed by a type II DPNIIB—like enzyme (Figure 1A). Such organization is reminiscent of minimal mobile elements, where an acquired cassette-like is located between highly conserved flanking genes (Saunders and Snyder, 2002). The majority of these cassettes encode restriction and modification systems (Snyder et al., 2007). However, no small repeated elements with some transposon-like properties were found in proximity of the ngo0007 locus (Correia et al., 1988). The same organization was present in N. meningitidis NCCP11945, M01-240149, M01-240196, H44/76, NZ-05/33, 510612, WUE2594, and 8013 strains, but the drg gene was followed by different hypothetical genes (Figure 1B). In case of N. lactamica 020-06 and N. meningitidis alpha 522, the drg gene with other genes was flanked by leuS on both sides (Figure 1C). For N. meningitidis MC58, 053442, Z2491, and alpha710, the drg gene was inserted within the dam locus (Figures 1D,E), resulting in dam truncation. In the genomes of N. meningitidis strains MC58, 053442, and alpha710, the truncated dam gene was followed by a truncated leuS gene, suggesting that dam is also an acquired locus in these bacteria. For four strains, N. meningitidis G2136, FAM18, alpha14 and M01-240355, the drg locus is absent and leuS is followed by an intact dam gene (Figure 1F).


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)

Genetic maps of drg or dam loci in the genomes of Neisseriaceae. Analysis included only the wholly-sequenced genomes of Neisseriaceae. leuS–gene encoding leucyl-tRNA synthase, drg—dam replacing gene, dam—gene encoding Dam methylase. For each type of locus organization, (A–F)—species and serogroup names of Neisseriaceae are given in the text.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Genetic maps of drg or dam loci in the genomes of Neisseriaceae. Analysis included only the wholly-sequenced genomes of Neisseriaceae. leuS–gene encoding leucyl-tRNA synthase, drg—dam replacing gene, dam—gene encoding Dam methylase. For each type of locus organization, (A–F)—species and serogroup names of Neisseriaceae are given in the text.
Mentions: The drg gene was previously identified in the genomes of N. meningitidis and Neisseria lactamica (Cantalupo et al., 2001; Jolley et al., 2004). In N. gonorrhoeae FA1090, the Drg endonuclease seems to be encoded by the ngo0007 gene. By blasting the nucleotide sequence of the drg locus against the NCBI database 27 hits were obtained. Among them, 23 ORFs were drg. Within Neisseriaceae, only N. gonorrhoeae, N. meningitidis, and N. lactamica were determined to contain the drg locus. Only 21 hits were found in wholly-sequenced neisserial genomes. For these genomes, we have analyzed the genetic context of the drg and dam loci. In all cases, the drg/dam locus neighbored the leuS locus (leucyl-tRNA synthetase) (Figure 1). In three cases (N. gonorrhoeae FA1090, MS11, and TCDC-NG08107), the drg gene was followed by a type II DPNIIB—like enzyme (Figure 1A). Such organization is reminiscent of minimal mobile elements, where an acquired cassette-like is located between highly conserved flanking genes (Saunders and Snyder, 2002). The majority of these cassettes encode restriction and modification systems (Snyder et al., 2007). However, no small repeated elements with some transposon-like properties were found in proximity of the ngo0007 locus (Correia et al., 1988). The same organization was present in N. meningitidis NCCP11945, M01-240149, M01-240196, H44/76, NZ-05/33, 510612, WUE2594, and 8013 strains, but the drg gene was followed by different hypothetical genes (Figure 1B). In case of N. lactamica 020-06 and N. meningitidis alpha 522, the drg gene with other genes was flanked by leuS on both sides (Figure 1C). For N. meningitidis MC58, 053442, Z2491, and alpha710, the drg gene was inserted within the dam locus (Figures 1D,E), resulting in dam truncation. In the genomes of N. meningitidis strains MC58, 053442, and alpha710, the truncated dam gene was followed by a truncated leuS gene, suggesting that dam is also an acquired locus in these bacteria. For four strains, N. meningitidis G2136, FAM18, alpha14 and M01-240355, the drg locus is absent and leuS is followed by an intact dam gene (Figure 1F).

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