CLONING OF AN AZORHIZOBIUM-CAULINODANS ENDOGLUCANASE GENE AND ANALYSIS OF ITS ROLE IN SYMBIOSIS

Azorhizobium caulinodans ORS571, a symbiont of the tropical leguminous plant Sesbania rostrata, showed low, constitutive levels of endogluca nase (Egl) activity. A clone carrying the gene responsible for this ph enotype was isolated via introduction of a genomic library into the wi ld-type strain and screening for transconjugants with enhanced Egl act ivity. By subcloning and expression in Escherichia coli, the Egl pheno type was allocated to a 3-kb EcoRI-BamHI fragment. However, sequence a nalysis showed the egl gene to be much larger consisting of an open re ading frame of 1,836 amino acids, Within the deduced polypeptide, thre e kinds of putative domains were identified: a catalytic domain, two c ellulose-binding domains, and an eightfold reiterated motif. The catal ytic domain belongs to the family A of cellulases. A C-terminal stretc h of 100 amino acids was similar to family II cellulose-binding domain s, A second copy of this domain occurred near the middle of the polype ptide, flanked by reiterated moths. ORS571 mutants carrying a Tn5 inse rtion in the egl gene had lost the Egl activity, These mutants as well as Egl-overproducing strains showed a normal nodulation behavior, ind istinguishable from wild-type nodulation on Sesbania rostrata under la boratory conditions.