Figure 5 S. epidermidis agr system regulates biofilm formation and initial cell attachment through atlE . ( a-d) S. epidermidis 1457 wild type (wt, a

and d), agr mutant (△ agr, b and e) and agr/atlE double mutant (△ agr/atlE, c and f) were grown for 24 h in flow chambers irrigated with minimal medium, and were then stained with SYTO 9 and PI, upon which microscopic investigation was performed by CLSM. The 3-D images (d-f) were generated using the IMARIS, bars, 50 μm. (g) Biofilm biomass in microtitre plates was quantified using a crystal violet assay. (h) Initial GF120918 nmr attachment of S. epidermidis strains in static chambers was quantified as described in Methods. Error bars represent GDC-0449 in vivo the S.E.M. for three independent experiments. Agr regulates se release of extracellular DNA and autolysis through suppression of atlE Our previous study revealed that mutation of atlE in Se 1457 significantly reduced extracellular DNA release and impairs biofilm

formation [11]. Consistent with those results, qRT-PCR revealed that expression of atlE was significantly increased for 1457 △agr, but almost no atlE learn more transcripts were detected in 1457 △agr/atlE (Figure 6A). Our qRT-PCR also confirmed that no RNAIII transcripts were detected in Se 1457 △agr, when compared with its wt strain (Figure 6A). Furthermore, 1457 △agr exhibited increased extracellular DNA relative to 1457 wt using both microtitre plate assays and DDAO staining in the flow-chamber systems (Figure 6C-F), while 1457 △agr/atlE abolished most extracellular DNA (Figure 6B6G-H). In addition, 1457 △agr displayed higher cell autolysis abilities than its wt strain, when induced by Triton X-100, whereas poor cell autolysis was seen in 1457 △agr/atlE GNE-0877 (Additional file 4: Figure S3). Notably, expression of icaA transcripts was almost unchanged for 1457 △agr relative to its wt strain, however, icaA transcripts were partially reduced in 1457 △agr/atlE (Figure 6A). Figure 6 S. epidermidis agr system controls extracellular DNA

release through atlE . (a) Biofilm-associated gene transcripts were compared between 1457 wt, △ agr and △ agr/atlE by using qRT-PCR. (b) Extracellular DNA release from cultures in microtitre plates was quantified as described above. Error bars represent the S.E.M. for three independent experiments. (c-h) S. epidermidis 1457 wild type (wt, c-d) agr mutant (△ agr, e and f) and agr/atlE double mutant (△ agr/atlE, g and h) were grown for 24 h in flow chambers irrigated with minimal medium, and were then stained with DDAO for extracellular DNA in biofilms, upon which microscopic investigation was performed by CLSM. The 3-D images ( d/ f/ h) were generated using the IMARIS, bars, 50 μm. Chemical inhibition of agr increases biofilm formation, initial attachment and cell autolysis through upregulation of atlE A recent study has revealed that inhibition of S.