Adaptive responses of Azotobacter vinelandii biofilm to oxidative stress: functional role of the rhodanese-like protein RhdA

In both managed and natural soil system bacteria experience environmental stressors such as xenobiotics, temperature shock, changes in pH and osmolarity, water and oxygen limitation, as well as competition for nutrients. Plant-associated bacteria also encounter oxidative stress in the rhizosphere. Plant roots produce reactive oxygen species (ROS) in response to many stimuli and several studies implicate ROS in root development and in interactions between roots and microorganisms (Colburn-Clifford et al., 2010). The nitrogen-fixing plant growth-promoting rhizobacterium (PGPR) Azotobacter vinelandii plays a key role in supporting and increasing plant health and growth. Thus, A. vinelandii is of interest for application in agriculture either as biofertiliser as well as for phytoremediation applications (Kennedy et al., 2004). Rhizosphere colonization is a prerequisite for a successful plant-bacteria interaction and the evolutionary prospective on life in biofilm enhances the ability of rhizobacteria to adapt to the selective pressures of this complex and competitive environment. A. vinelandii has long served as a model for biochemical and functional studies of the rhodanese RhdA that in vitro catalyzes the transfer of a sulfur atom from thiosulfate to cyanide with concurrent formation of thiocyanate, and belongs to the family of rhodanese-like proteins. Considering the fairly tight connection between biofilm formation and cellular response to various stresses including oxidative damage (Landini, 2009), the functionality of RhdA as a redox switch in planktonic cells (Remelli et al., 2010), the redundancy and ubiquitary distribution of rhodanese-like proteins, a possible involvement of RhdA in biofilm genesis was postulated. Consistent with this hypothesis, the present study investigated the adaptive response of A. vinelandii biofilm to oxidative stress by using a mutant strain, in which the rhdA gene was disrupted by deletion. The MV474 biofilm growth curve revealed that the lack of the antioxidant protein RhdA enhanced the ability of A. vinelandii to develop biofilm while the intracellular level of ROS decreased over time reaching the lower values in mature and late-stage biofilm. Particularly noteworthy was the observation that MV474 sustained a surface-associated movement resulting in a faster and efficient colonization of the polycarbonate membrane. Indeed, cryosectioning combined with microscopy revealed that MV474 biofilm was significantly thinner than the film formed by UW136 and the mutant strain of A. vinelandii synthesized a polysaccharide-rich extracellular polymer matrix. Developed biofilms of the wild-type and the mutant strains of A. vinelandii showed different protein profiles. Finally, we demonstrated that MV474 biofilm was less susceptible to biocide activities than UW136 biofilm. Likely, the deletion of the rhdA gene exposes A. vinelandii mutant strain to stress condition, which triggers a more efficient bacterial oxidative stress response and the activation of alternative defensive mechanisms like the social behavior in the sessile lifestyle. Taken together, our results indicate an involvement of bacterial rhodanese-like proteins in physiological processes related to biofilm formation.