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 lacZ fusions in association with wheat. In situ staining of the bacteria with 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-Gal) as a chromogenic substrate and examination by light microscopy revealed bacteria localized at the surface of the root and also within the cortex. Determination of ß-galactosidase activity allowed a comparison of the expression of nif promoters in the plant-associated bacteria. Plasmid-borne nifA-, nifB-, and nifH-lacZ fusions carried by the wild-type A. brasilense strain Sp7 were significantly expressed in association with the plants, compared to the basal level observed with bacteria lacking a lacZ fusion or carrying a nifH-lacZ fusion in nif A- and glnB-mutated backgrounds. A chromosomally borne nifH-lacZ fusion was also expressed. Colonization efficiency was examined with a partially constitutive nifA-lacZ fusion or with a lacZ fusion to a kan promoter. The colonization efficiency of a nifA-Tn5 mutant strain was identical to that of the wild type, while glutamine and tryptophan auxotrophs showed less colonization. As the protocol is applicable to a range of A. brasilense wild-type and mutant strains, it can be used to compare gene expression in free-living bacteria and those in association with plants and to screen for hyperperforming strains in association with plants.