3.3 Effects of oxidants on surfactin production and biofilm formation in WH1
H2O2 at high concentrations had negative influences on the cell growth. Consistently, H2O2 led to a decrease of surfactin production in a dose-dependent manner (Fig. 2A). Accompanying with reduced surfactin production, biofilm formation was also delayed and weakened. At 24 h, float biofilm was unobserved in all groups added with H2O2. At 36 h, the group added with 0.2 or 0.5 mM H2O2 was observed with growing biofilm. At 48 h, float pellicle was still unobserved in the group added with 2.0 or 4.0 mM H2O2 (Fig. 2B). However, H2O2 had no obvious influence on biofilm formation in ΔsrfA because this strain lost the ability to produce biofilm in a surfactin-dependent manner (Fig. 2C).
With H2O2, the transcription ofsrfAB , sfp , spx and perR were all significantly down-regulated (Fig. 2D), consistent with the result that H2O2 led to a significant decrease of surfactin production in WH1. Western blot analysis showed that the content of PerR was decreased in the group added with 2 mM H2O2 (Fig. 2E), also consistent with the result of perR transcription level described above.
3.4 Effects of reductants on surfactin production and biofilm formation in Δspx
The spx knockout strain (Δspx ) was constructed (Fig. S3 in supplementary materials). Δspx showed a colony morphology with less wrinkles, and displayed a weaker floating pellicle with less wrinkles than WH1 (Fig. 3A). Δspx showed a slightly weaker growth but a significant decrease of surfactin production compared to WH1 (Fig. 3B). The weakened ability to form biofilm in Δspx could be partially restored by compensation of spx , and the reduced surfactin production was also restored in the complementary strain (C-Δspx ) (Fig. 3A&B). The transcription of srfAB was significantly decreased in Δspx compared to WH1. The PerR protein was slightly decreased in Δspx compared to WH1, consistent with the transcription level of perR in Δspx (Fig. 3C).
Just like that in WH1, glucose at high concentrations (e.g. 10 or 20 g/L) significantly improved the cell growth but significantly inhibited the surfactin production in Δspx (Fig. 3D). qRT-PCR showed that glucose resulted in a significant down-regulation of genes transcription like srfAB , sfp and perR . Western blot analysis showed that glucose could slightly increase the content of PerR in Δspx (Fig. 3G).
Like that in WH1, glucose was favorable for forming a robuster floating pellicle with more wrinkles in Δspx (Fig. 3F). Thereby, glucose affected the biofilm formation by a surfactin-independent manner.
3.5 Effects of oxidants on surfactin production and biofilm formation in Δspx
H2O2 had no significant influence on the cell growth, but significantly improved the surfactin production in Δspx (Fig. 4A), very different from that in WH1. qRT-PCR showed that H2O2 could up-regulate the transcription of sfp and perR. C onsistent with the result that these two oxidants were able to improve the surfactin production in Δspx . Western blot assay also showed that H2O2 could slightly increase the PerR protein in Δspx (Fig. 4B&D).
The biofilm formation in Δspx was inhibited by H2O2 in a dose-dependent manner, just like that in WH1 (Fig. 4C).
3.6 Effects of reductants on surfactin production and biofilm formation in ΔperR
The perR gene knockout strain (ΔperR ) was constructed (Fig. S3 in supplementary materials). ΔperR displayed a flat colony morphology and defective floating pellicle without wrinkles (Fig. 5A). ΔperR showed a weaker growth than WH1, and surfactin production was significantly decreased in ΔperR but could be partially restored by compensation of perR (Fig. 5B). However, the colony morphology and float pellicle could not be well restored in the complementary strain (C-ΔperR ) (Fig. 5A). Consistently, qRT-PCR showed that knockout of perR led to a significant decrease of srfAB transcription (Fig. 5B).
Glucose had obvious influence on the cell growth in ΔperR . In 12 h, glucose promoted the cell growth at all concentrations used here. At 24 h, only 10 or 20 g/L glucose improved the cell growth. Similar to WH1, glucose reduced the surfactin production by a dose-dependent manner in ΔperR (Fig. 5C).Just like that in WH1 and Δspx . Unexpectedly, the transcription of srfAB was significantly up-regulated in the group added with glucose(Fig. 5D). Glucose was favorable for forming a robuster floating pellicle with more wrinkles in ΔperR (Fig. 5E), consistent with that in WH1 and Δspx .
3.7 Effects of oxidants on surfactin production and biofilm formation in ΔperR
H2O2 at 2 mM or 4 mM significantly inhibited the cell growth in ΔperR , non-consistent with that in WH1 or Δspx . Similar to WH1 but different from Δspx , H2O2 at 2 mM or 4 mM significantly reduced the surfactin production in ΔperR (Fig. 6A). H2O2 significantly up-regulated the transcription of srfAB , sfp and spx , while FeCl3 mainly increased the transcription of srfABin ΔperR (Fig. 6B). H2O2obviously inhibited the biofilm formation (Fig. 6C).