1 Introduction
Bacillus species like B. subtilis , B. amyloliquefaciens , B. licheniformis , are Gram-positive, spore-forming bacteria widely distributed in soil, water, air, etc. During times of environmental insult such as poor nutrient, the bacteria undergo developmental changes leading to quorum sensing responses, by which the cells form multicellular communities known as biofilm [1,2]. Now it is known that upon harsh environment, the cells produce a secondary metabolite called surfactin, which is a nonribosomal lipopeptide biosynthesized by a multienzyme system encoded by the gene cluster srfA [3-5], as a signal to trigger biofilm formation [6-8].
Generally, harsh environments lead to a change of cellular redox state, which is an important parameter to affect surfactin production and biofilm formation [9]. For example, B. subtilis can switch from an unicellular state to a multicellular state of biofilm via responding to a redox switch1. The NADH/NAD+ ratio, or another indicator of the cellular redox potential, is sensed by regulatory pathways to control features of colony wrinkling. Also, colony development can both respond to and affect redox homeostasis [9]. For instance, isocitrate dehydrogenase is capable of regulating biofilm formation by modulating intracellular redox homeostasis [10]. Also, the cells are capable of transferring glucose to acetoin as a response to enough carbon source and reducing power, which are essential for robust biofilm growth and conserving redox balance [11,12].
Despite importance of redox, we know little about how the shifts in redox state regulates biosynthesis of surfactin for triggering biofilm formation. Surfactin production can be regulated by the cellular redox status via some regulatory proteins such as Spx and PerR. Spx interacts with RNA polymerase to control transcription in response to oxidative stress [13,14], which plays a key role in maintaining the redox homeostasis exposed to disulfide stress, ensuring an immediate response to oxidative stress [15]. The redox state of cytoplasm is the major effector driving Spx activation, which activity is enhanced by reversible formation of a disulfide bond and thereby directly modulated by the intracellular redox status [16]. The transcription ofspx is negatively regulated by the repressor PerR and is positively regulated by a sigma factor of SigB [15]. SigB is a general stress response transcription factor [17], and PerR senses H2O2 to mediate adaptation to peroxide stress. PerR also represses oxidative stress resistance genes including the catalase katA , alkylhydroperoxide reductase ahpC , iron uptake repressor (fur ), and perR  itself [18,19]. After oxidation PerR is inactivated, so the oxidative response genes includingspx are capable of being successfully expressed. Thereby, PerR plays an auxiliary role in coordinating the disulfide stress responses [19].
Previously, we reported that mutation of srfA resulted in a very seriously defective growth and biofilm formation but could be restored by glucose in B. amyloliquefaciens WH1 [20]. Glucose an its intermediate metabolites such as NADH are reductive substances, we hypothesized that these reductants might influence biofilm formation via some regulators such as Spx and PerR to regulate surfactin production as a response to the change of ceullular redox homeostasis. In this study, we characterized the interplay among redox state, surfactin production and biofilm formation, and found the redox status influenced by oxidants and reductants could affect biofilm formation via regulation of surfactin production in B. amyloliquefaciens . Moreover, some reductants such as glucose could also influence biofilm formation by a surfactin-independent way in this bacterium.