Isolation of novel strains of Agrobacterium radiobacter with altered capacities for lactose metabolism and succinoglucan production

Abstract

Agrobacterium radiobacter NCIB 11883 was grown in continuous culture at low dilution rate under lactose limitation. Washed cells rapidly transported lactose [and its non-metabolizable analogue MTG (methyl β d-thiogalactoside)] via a lactose-binding protein (LBP)-dependent uptake system, and subsequently hydrolysed the lactose using a highly active β-galactosidase composed of two identical subunits of M r approximately 86000. Growth under these conditions for <40 generations led to the selection of a novel strain (AR50) which overexpressed both LBP and β-galactosidase, and exhibited lactose (MTG) uptake and β-galactosidase activities that were two to three times those of the wild-type organism. Both enzymes were expressed constitutively in strain AR50, in contrast to the wild-type organism, but remained subject to catabolite repression (particularly by galactose). Southern blotting of restricted DNA from the two organisms using an oligonucleotide probe for the structural gene for LBP showed a 2·7-fold amplification in strain AR50, together with a deletion of at least 1·7 kb which may be part of a regulatory gene. The wild-type organism and strain AR50 exhibited similar lactose (MTG) uptake rates during growth under ammonia limitation, and also synthesized an exocellular succinoglucan polysaccharide at only marginally different rates [q p 0·21--0·24 g h−1 (g cells)−1]; both organisms exhibited a flux control coefficient for lactose uptake on succinoglucan production of < 0·45, indicating that lactose uptake is a major kinetic control point for polysaccharide production. Growth of strain AR50 under ammonia limitation for >; 40 generations led to the selection of another novel strain (AR60) which exhibited a decreased q p [0·16 g h−1 (g cells)−1]. Washed cells of strain AR60 exhibited significantly lower rates of lactose (MTG) uptake than strain AR50, an observation which was commensurate with the rate of polysaccharide production being predominantly controlled by the rate of lactose uptake, but β-galactosidase activity was substantially higher. Both the lactose uptake system and β-galactosidase were expressed constitutively in strain AR60, but catabolite repression of β-galactosidase was much weaker than in the wild-type organism or strain AR50.