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).