CLONING, EXPRESSION IN ESCHERICHIA-COLI, AND CHARACTERIZATION OF CELLULOLYTIC ENZYMES OF AZOARCUS SP, A ROOT-INVADING DIAZOTROPH

We screened members of a new genus of grass-associated diazotrophs (Az oarcus spp.) for the presence of cellulolytic enzymes. Out of five Azo arcus strains representing different species, only in the endorhizosph ere isolate BH72, which is also capable of invading grass roots, was s ignificant endoglucanase activity, in addition to beta-glucosidase and cellobiohydrolase activity, present. Reducing sugars were readily rel eased from medium-viscosity carboxymethylcellulose (CMC), but neither CMC, cellulose filter strips, Avicel, cellobiose, nor D-glucose served as the sole carbon source for growth of Azoarcus spp. Clones from a p lasmid library of strain BH72 expressed all three enzymes in Escherich ia coli, apparently not from their own promoter. According to restrict ion endonuclease mapping and subclone analysis, beta-glucosidase and c ellobiohydrolase activities were localized on a single 2.6-kb fragment not physically linked to a 1.45-kb fragment from which endoglucanase (egl) was expressed. Two isoenzymes of endoglucanase probably resultin g from proteolytic cleavage had pI values of 6.4 and 6.1 and an appare nt molecular mass of approximately 36 kDa. Cellobiohydrolase and beta- glucosidase activity were conferred by one enzyme 41 kDa in size with a pl of 5.4, which we classified as an unspecific exoglycanase (exg) a ccording to substrate utilization and specificity mapping; hydrolysis of various oligomeric substrates differentiated it from endoglucanase, which degraded substituted soluble cellulose derivatives but not micr ocrystalline cellulose. Both enzymes were not excreted but were associ ated with the surface of Azoarcus cells. Both activities were only sli ghtly influenced by the presence of CMC or D-glucose in the growth med ium but were enhanced by ethanol. egl was located on a large transcrip t approximately 15 kb in size, which was detectable only in cells grow n under microaerobic conditions on N2. Surface-bound exo- and endogluc anases with some unusual regulatory features, detected in this study i n a strain which is unable to metabolize cellulose or sugars, might as sist Azoarcus sp. strain BH72 in infection of grass roots.