Axenic Biofilm Formation and Aggregation bySynechocystisPCC 6803 is Induced by Changes in Nutrient Concentration, and Requires Cell Surface Structures

Abstract

AbstractPhototrophic biofilms are key to nutrient cycling in natural environments and bioremediation technologies, but few studies describe biofilm formation by pure (axenic) cultures of a phototrophic microbe. The cyanobacteriumSynechocystissp. PCC 6803 (hereafterSynechocystis) is a model micro-organism for the study of oxygenic photosynthesis and biofuel production. We report here that wild-type (WT)Synechocystiscaused extensive biofilm formation in a 2000 liter outdoor non-axenic photobioreactor under conditions attributed to nutrient limitation. We developed a biofilm assay and found that axenicSynechocystisforms biofilms of cells and extracellular material, but only when induced by an environmental signal, such as by reducing the concentration of growth medium BG11. Mutants lacking cell surface structures, namely type IV pili and the S-layer, do not form biofilms.To further characterize the molecular mechanisms of cell-cell binding bySynechocystis, we also developed a rapid (8 hour) axenic aggregation assay. Mutants lacking Type IV pili were unable to aggregate, but mutants lacking a homolog to Wza, a protein required for Type 1 exopolysaccharide export inEscherichia coli, had a super-binding phenotype. In WT cultures, 1.2x BG11 induced aggregation to the same degree as 0.8x BG11. Overall, our data support that Wza-dependant exopolysaccharide is essential to maintain stable, uniform suspensions of WTSynechocystiscells in unmodified growth medium, and this mechanism is counter-acted in a pili-dependent manner under altered BG11 concentrations.ImportanceMicrobes can exist as suspensions of individual cells in liquids, and also commonly form multicellular communities attached to surfaces. Surface-attached communities, called biofilms, can confer antibiotic resistance to pathogenic bacteria during infections, and establish food webs for global nutrient cycling in the environment. Phototrophic biofilm formation is one of the earliest phenotypes visible in the fossil record, dating back over 3 billion years. Despite the importance and ubiquity of phototrophic biofilms, most of what we know about the molecular mechanisms, genetic regulation, and environmental signals of biofilm formation comes from studies of heterotrophic bacteria. We aim to help bridge this knowledge gap by developing new assays forSynechocystis, a phototrophic cyanobacterium used to study oxygenic phototsynthesis and biofuel production. With the aid of these new assays, we contribute to the development ofSynechocystisas a model organism for the study of axenic phototrophic biofilm formation.