Use of lacZ Fusions to Study the Expression of nif Genes of Azospirillum brasilense in Association with Plants. Florence Arsene. Unite de Physiologie Cellulase, Departement des Biotechnologies, URA 1300 CNRS, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France. Sunietha Katupitiya (2), Ivan R. Kennedy (2), and Claudine Elmerich (1) (1) Unite de Physiologie Cellulase, Departement des Biotechnologies, URA 1300 CNRS, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France, and (2) Department of Agricultural Chemistry and Soil Science, University of Sydney, Sydney 2006 Australia. MPMI 7:748-757. Accepted 12 July 1994. Copyright 1994 The American Phytopathological Society.

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.