019 and p = 0.032, respectively). Figure 7 Damage of biofilms of S. mutans wildtype and knock-out mutants for comC , comD and comE https://www.selleckchem.com/products/blz945.html by carolacton. Biofilms were grown under anaerobic conditions
for 24 h and stained with the LIVE/DEAD BacLight Bacterial Viability staining kit. Green and red fluorescence was determined in triplicate samples, and biofilm damage was calculated as check details reduction of the fluorescence ratio green/red compared to untreated controls. Standard deviations were calculated from 3 – 5 independent experiments. Thus, the comD knockout mutant was slightly less sensitive to carolacton than the wildtype. This could indicate that carolacton interferes with the membrane bound histidine kinase ComD. However, since the comC and comE mutants were
just as sensitive for carolacton as the wildtype, and since there was still considerable activity of carolacton against the comD mutant, other mechanisms must be more important. Influence of carolacton on a pcomX luciferase reporter strain ComX, an alternative sigma-factor, plays a key role in the quorum sensing system of S. mutans which controls not only genetic competence, but also stress tolerance and biofilm formation, leading to the suggestion to call it the “”X-state”" rather than competence see more . ComX is positively induced by CSP through the response regulator ComE, but also by another two component system, CiaRH, and environmental stress . ComX controls the late competence genes, including the machinery for DNA-uptake and processing, but also many other density dependent traits [36, 40–42]. Altogether 240 genes are directly or indirectly controlled by ComX . To investigate the effect of carolacton on the promoter activity of comX a pcomX-luciferase reporter strain was
constructed. For the experiment a concentration of CSP (200 nM) was chosen that induced competence without causing substantial growth inhibition . Figure 8A shows that a severe reduction of CSP-induced comX expression Thiamet G was caused by addition of carolacton to biofilms grown anaerobically. Furthermore carolacton led to a decrease of growth-dependent, basal comX-reporter activity. Maximum inhibition was seen 60 min post induction at the peak of comX expression. In planktonic culture (Figure 8B) similar results were obtained, but both the CSP induced expression of comX and its inhibition through carolacton occurred over a longer time, e.g. from 45 to 180 min post induction, possibly reflecting the lower cell density in the planktonic culture. Furthermore we found that carolacton reduced the growth-dependent comX-promoter activity of this reporter strain also in the absence of externally added CSP, both in biofilms and in planktonic culture. Figure 8 Effect of carolacton on the comX -promoter activity of S. mutans.