3.2 Effects of reductants on surfactin production and biofilm formation in WH1
Glucose at high concentrations (e.g. 10 or 20 g/L) significantly increased the biomass in 24 h, while it significantly delayed and reduced the surfactin production in a concentration-dependent effect (Fig. 1A). We detected the genes transcription related with surfactin production in different strains using qRT-PCR. With glucose , the transcription of genes including srfAB , sfp that encodes 4’-phosphopantetheinyl transferase, spx and perR, were all significantly down-regulated (Fig. 1B), consistent with the result that these reductants led to a significant decrease of surfactin production in WH1.
The perR gene was amplified from WH1, then ligated into pET28a for expression. After induction with IPTG, the recombinant PerR inE. coli was purified by Ni-NTA column (Fig. S2 in supplementary materials). The purified rPerR was used for immunization of mice for producing antibodies, then the antisera were collected for Western blotting assay. The cells were collected after being cultured for 24 h, lyzed by ultrasonication, then the total cell proteins were adjusted to the same concentration for Western blotting assay. The result showed the content of PerR was decreased in the group added with 10 g/L glucose compared to WH1, consistent with the perR transcription described above (Fig. 1B). rPerR was used as control, which showed a higher molecular weight because it includes an extra sequence containing His-tag from the vector.
Glucose was capable of enhancing biofilm formation in a concentration-dependent manner (Fig. 1C). Interestingly, glucose could also restore and enhance biofilm formation in the strain ΔsrfA(Fig. 1D), which had no surfactin production at all as described above.