A novel bleb-dependent polysaccharide export system in nitrogen-fixing Azotobacter vinelandii subjected to low nitrogen gas levels

The alginate biofilm-producing bacterium Azotobacter vinelandii aerobically fixes nitrogen by oxygensensitivenitrogenases. Here we investigated the bacterial response to nitrogen/oxygen gas mixtures. A. vinelandii cellswere cultured in nitrogen-free minimal media containing gas mixtures differing in their ratios of nitrogen and oxygen.The bacteria did not grow at oxygen concentrations >75 % but grew well in the presence of 5 % nitrogen/25 % oxygen.Growth of wild-type and alginate-deficient strains when cultured with 50 % oxygen did not differ substantially, indicatingthat alginate is not required for the protection of nitrogenases from oxygen damage. In response to decreasing nitrogenlevels, A. vinelandii produced greater amounts of alginate, accompanied by the formation of blebs on the cell surface. Theencystment of vegetative cells occurred in tandem with the release of blebs and the development of a multilayered exine.Immunoelectron microscopy using anti alginate-antibody revealed that the blebs contained alginate molecules. Bycontrast, alginate-deficient mutants could not form blebs. Taken together, our data provide evidence for a novel blebdependentpolysaccharide export system in A. vinelandii that is activated in response to low nitrogen gas levels