4 Discussion
In the environment, the cells undergo constant shifts in redox state
[12]. It has been revealed that the redox balance influences the
community structural development in biofilms [10,21]. Glucose is not
only a carbon source but also a reductant for microorganisms. Here, we
found that glucose significantly increased the biomass but reduced the
surfactin production in B. amyloliquefaciens WH1 and its
mutant strains like Δspx and ΔperR , consistent with
previous report [22]. If glucose is enough, the metabolism of
glucose to produce NADH is vigorous to keep the cytoplasm in a reductive
state [23,24]. In this study, although glucose was negatively
correlated with surfactin production, it was positively correlated with
biofilm formation by a dose-effect manner for WH1, Δspx and
ΔperR , suggesting that glucose can affect biofilm formation by a
surfactin-independent pathway in B. amyloliquefaciens .
This could also be verified by the result that ΔsrfA was also
able to form a robuster float pellicle in the presence of glucose
although it was unable to produce surfactin. In the presence of enough
glucose, the cells grew vigorously, and a substantial increase in
biomass robustness was correlated with an increase of genes expression
for producing matrix [2,9,25,26]. As a result, the biofilms
exhibited a strikingly wrinkled appearance, which is thought to maximize
access to oxygen by increasing the surface to volume ratio [12,27].
H2O2 is an oxidant containing reactive
oxygen, which can derepress the perR regulon includingsrfA to promote surfactin production [19,28]. However,
H2O2 led to a significant decrease of
surfactin accompanying with a decrease of genes transcription includingsrfAB , sfp , spx and perR in WH1.
Consistently, the biofilm formation was delayed and weakened in the
presence of H2O2 [1].
Spx controls the genes transcription responded to oxidative stress,
playing a key role in maintaining the cellular redox homeostasis exposed
to disulfide stress [14-16]. Also, Spx has been reported to repress
the transcription of srfA in B. subtilis [4,15]. Here,
knockout of spx resulted in a significant decrease of surfactin
accompanying with a decrease of srfAB transcription. This result
suggested that spx plays a positive role in biosynthesis of
surfactin, different from previous report [15]. Δspx showed a
similar response to reductants, but displayed a very different response
to oxidants from WH1, implying that Spx mainly responses to oxidative
rather than reductive stress [16]. Δspx was more resistant to
H2O2 stress than WH1. Perhaps, Spx
repressed some anti-oxidation genes for antagonizing
H2O2 [4,15], so deletion ofspx was favorable for expression of the anti-oxidation enzymes inB. amyloliquefaciens . Moreover,
H2O2 led to a significant decrease of
surfactin accompanying with down-regulation of the transcription ofsrfAB , sfp , spx and perR in WH1, but
resulted in a significant increase of surfactin accompanying with
up-regulation of the transcription of sfp and perR in
Δspx . This could be explained that the principal regulator for
biosynthesis of surfactin is ComA, and Spx plays a role in
fine-tuning[3,4,28]. For this reason, although deletion ofspx reduced the srfA transcription,
H2O2 could improve ComA or other
regulators to increase the surfactin production in Δspx.
Spx has been reported to inhibit biofilm formation [30], and
inactivation of spx can enhance the biofilm formation in B.
subtilis [31]. However, Δspx showed a colony morphology and
biofilm with less wrinkles but could be partially restored by
compensation of spx , indicating that spx plays a positive
role in the biofilm formation here. Unexpectedly,
H2O2 significantly increased surfactin
production but inhibited biofilm formation in Δspx .
H2O2 can be catalyzed to
O2, which suppresses production of extracellular matrix
[1]. Thereby, the biofilm formation in Δspx was dependent on
oxygen rather than surfactin. This was also supported by the result that
glucose could enhance biofilm formation although it inhibited surfactin
production in Δspx . The key factor was O2, which
was rich in the presence of enough H2O2and was poor with enough glucose.
PerR senses H2O2 by Fe-mediated
histidine oxidation resulting in an inactive style [29,32]. Here,
ΔperR showed a different colony morphology, defective floating
pellicle without wrinkles, and weaker growth compared to WH1, consistent
with previous report [19]. Knockout of perR led to Spx
accumulation [15], resulting in a failure to produce surfactin inB. subtilis [16]. Here, surfactin was decreased in
ΔperR , but could be partially restored by compensation ofperR . The results confirmed that perR was positive for
surfactin production in B. amyloliquefaciens [28].
Interestingly, compensation of perR could not restore colony
morphology and float biofilm.
ΔperR s howed a similar response to reductants, but displayed a
very different response to oxidants from WH1 and Δspx , suggesting
that PerR mainly responded to oxidative rather than reductive stress
[16]. H2O2 inhibited the growth of
ΔperR , non-consistent with WH1 and Δspx . WH1 was only
sensitive to 4 mM H2O2, while
Δspx was not sensitive to H2O2 at
all concentrations used here. Thereby, Δspx was more resistant,
while ΔperR was more sensitive to
H2O2 stress. Possibly, deletion ofspx was favorable for expression of the anti-oxidation genes likekatA , aphC , sodA , etc, but deletion of perRwas unfavourable for these genes transcription. However, it has been
reported that the perR null strain of B. subtilis is
resistant to H2O2 [17-19], different
from our results. Also, H2O2 improved
surfactin production in Δspx , while reduced it in ΔperR .
The float biofilm in ΔperR was impaired so it could not respond
to H2O2 via a surfactin-dependent
manner. All of the above results suggested that the biofilm formation in
ΔperR was not dependent on surfactin.
Rex senses variation of NAD+/NADH to balance the
intracellular redox reactions [33]. Here, Δrex grew
significantly weaker, and showed a significant decrease of surfactin
production than WH1. Compensation of rex could form a robuster
biofilm, but could not efficiently restore the surfactin production
compared to WH1. This result also suggested that B.amyloliquefaciens could form biofilm by a surfactin-independent
way. The spx transcription is positively regulated by SigB
[15,34]. Here, knockout of sigB also led to a significant
decrease of spx transcription and surfactin production inB. amyloliquefaciens .
In B. amyloliquefaciens , the reductant glucose could
reduce surfactin and enhance biofilm formation by a
surfactin-independent way. The oxidant
H2O2 led to a decrease of surfactin
accompanying with weakened biofilm formation.
H2O2 improved surfactin production but
inhibited biofilm formation by a surfactin-independent manner in
Δspx . Moreover, Δspx was more tolerant to
H2O2 stress than WH1. PerR was essential
for surfactin production and biofilm formation, and knockout ofperR led to a significant decrease of surfactin and very
defective biofilm thus it could not respond to
H2O2 via a surfactin-dependent manner.
Contrary to Δspx , H2O2 reduced
surfactin production, and the ability against
H2O2 stress was weakened in
ΔperR . Collectively, PerR is favorable for resisting oxidative
stress, while Spx plays a negative role in this action. Surfactin is not
a unique signal to trigger biofilm formation, and the cellular redox
state can influence biofilm formation by a surfactin - dependent or -
independent way in B. amyloliquefaciens .