AUTHOR=Kwiatek Agnieszka , Bacal Pawel , Wasiluk Adrian , Trybunko Anastasiya , Adamczyk-Poplawska Monika 

TITLE=The dam replacing gene product enhances Neisseria gonorrhoeae FA1090 viability and biofilm formation

JOURNAL=Frontiers in Microbiology

VOLUME=5

YEAR=2014

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2014.00712

DOI=10.3389/fmicb.2014.00712

ISSN=1664-302X

ABSTRACT=<p>Many <italic>Neisseriaceae</italic> do not exhibit Dam methyltransferase activity and, instead of the <italic>dam</italic> gene, possess <italic>drg</italic> (<italic>dam replacing gene</italic>) inserted in the <italic>leuS/dam</italic> locus. The <italic>drg</italic> locus in <italic>Neisseria gonorrhoeae</italic> FA1090 has a lower GC-pairs content (40.5%) compared to the whole genome of <italic>N. gonorrhoeae</italic> FA1090 (52%). The gonococcal <italic>drg</italic> gene encodes a DNA endonuclease Drg, with GmeATC specificity. Disruption of <italic>drg</italic> or insertion of the <italic>dam</italic> gene in gonococcal genome changes the level of expression of genes as shown by transcriptome analysis. For the <italic>drg</italic>-deficient <italic>N. gonorrhoeae</italic> 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 <italic>dam</italic>-expressing <italic>N. gonorrhoeae</italic> 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. <italic>In vivo</italic>, the inactivation of <italic>drg</italic> gene causes the decrease of the number of live neisserial cells and long lag phase of growth. The insertion of <italic>dam</italic> gene instead of <italic>drg</italic> locus restores cell viability. We have also shown that presence of the <italic>drg</italic> gene product is important for <italic>N. gonorrhoeae</italic> FA1090 in adhesion, including human epithelial cells, and biofilm formation. Biofilm produced by <italic>drg</italic>-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 (OD<sub>570</sub> = 0.242 ± 0.038) for <italic>drg</italic>-deficient strain, compared to wild-type strain (OD<sub>570</sub> = 0.378 ± 0.057). <italic>Dam</italic>-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 <italic>N. gonorrhoeae</italic> biology than Dam presence.</p>