Azotobacter vinelandii biofilm genesis in response to endogenously induced oxidative stress

In both natural and engineered ecosystems bacteria experience environmental stress factors known to be sources for oxidative injuries. Little is known about the mechanisms by which bacteria adapt to stress encountered in the environment, but it is now widely accepted that cells exist, at least for a portion of their lives, in a biofilm state, enabling bacteria to develop coordinated survival strategies. Studying the role of exogenously as well as endogenously induced oxidative stress in biofilm genesis may shed light on the determinants required by bacteria to successfully colonize hostile habitats, improving the exploitation of microorganisms from stressed or contaminated environments. The nitrogen-fixing plant growth-promoting rhizobacterium A. vinelandii is of interest for application in agriculture as biofertiliser as well as for phytoremediation treatments. The picture emerged from recent studies indicates that rhodaneses RhdA (thiosulfate:cyanide sulfurtransferase, E.C. 2.8.1.1, catalyzing in vitro the transfer of a sulfane sulfur atom from thiosulfate to cyanide) play a key role in maintaining the cellular redox balance in planktonic cells. Given the relationship between biofilm genesis and stress conditions, we determined whether the deletion of rhdA might impact the social behaviour in the sessile lifestyle. In particular, 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. Biofilm experiments revealed that the lack of the protein RhdA enhanced the ability of A. vinelandii to develop biofilm while the level of endogenously generated reactive oxygen species decreased over time reaching lower values in mature biofilm. Biofilm cryosectioning revealed that ΔrhdA biofilm was significantly thicker than the biofilm formed by the parental strain and showed a polysaccharide-richer extracellular polymer matrix. The mutant strain sustained a surface-associated movement resulting in a faster and efficient colonization of the surface. Developed biofilms of the wild-type and the mutant strains of A. vinelandii showed different protein profiles. Finally, we demonstrated that ΔrhdA biofilm was less susceptible to the oxidative stressor hydrogen peroxide than the wild-type biofilm. In conclusion, during the sessile growth A. vinelandii lacking the protein RhdA experiences intracellular oxidative stress conditions to which the bacterium responds efficiently by improving its ability to develop biofilm, a protective action from unfavourable conditions. Thus, the stress condition generated endogenously by the absence of RhdA acts as environmental cue that evokes the social behaviour in the sessile lifestyle.