Osmoregulation in Azospirillum brasilense: glycine betaine transport enhances growth and nitrogen fixation under salt stress

Abstract

Addition of glycine betaine (1 mM) stimulated aerobic growth of Azospirillum brasilense Sp 7 in the presence of 0.3 M-NaC1. The nitrogenase activity of whole cells was particularly sensitive to salt stress, being almost totally inhibited in the presence of the same concentration of salt. Added glycine betaine strongly enhanced nitrogen fixation activity under salt stress. Under such conditions, maximal nitrogenase activity was obtained at a p(02),value (1 kPa) that inhibits nitrogen fixation activity in the absence of salt. We demonstrated the presence of a high affinity transport system for glycine betaine, with an apparent K(m), of 10 μM. The osmolarity of the medium regulated the activity of the transport system. The maximal transport rates were 4 and 20 nmol min(-l) (mg protein)(-l) in cells grown in low-salt and high-salt medium, respectively. A high intracellular concentration of glycine betaine (480 mM) was observed only at a high osmolarity (0.3 M-NaC1). Glycine betaine uptake was significantly reduced in osmotically shocked cells and a glycine betaine binding activity was detected in the crude periplasmic shock fluid. This suggests a transport mechanism involving a periplasmic glycine betaine binding protein. A. brasilense was unable to use the transported glycine betaine as a carbon- or nitrogen-source, in low- or high-salt medium. Intracellular glycine betaine was not catabolized.