USE OF LACZ FUSIONS TO STUDY THE EXPRESSION OF NIF GENES OF AZOSPIRILLUM-BRASILENSE IN ASSOCIATION WITH PLANTS

A simple protocol has been developed to detect Azospirillum carrying l acZ fusions in association with wheat. In situ staining of the bacteri a with -bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) as a chromogenic substrate and examination by light microscopy revealed ba cteria localized at the surface of the root and also within the cortex . Determination of P-galactosidase activity allowed a comparison of th e expression of nif promoters In the plant-associated bacteria. Plasmi d-borne nifA-, nifB-, and nifH-lacZ fusions carried by the wild-type A . brasilense strain Sp7 were significantly expressed in association wi th the plants, compared to the basal level observed with bacteria lack ing a lacZ fusion or carrying a nifH-lacZ fusion in nifA- and glnB-mut ated backgrounds. A chromosomally borne nifH-lacZ fusion was also expr essed. Colonization efficiency was examined with a partially constitut ive nifA-lacZ fusion or with a lacZ fusion to a kan promoter. The colo nization efficiency of a nifA-Tn5 mutant strain was identical to that of the wild type, while glutamine and tryptophan auxotrophs showed les s colonization. As the protocol is applicable to a range of A. brasile nse wild-type and mutant strains, it can be used to compare gene expre ssion in free-living bacteria and those in association with plants and to screen for hyperperforming strains in association with plants.