Olafur H. Fridjonsson

Thermoadaptation of α-Galactosidase AgaB1 in Thermus thermophilus

The evolutionary potential of a thermostable α-galactosidase, with regard to improved catalytic activity at high temperatures, was investigated by employing an in vivo selection system based on thermophilic bacteria. For this purpose, hybrid α-galactosidase genes of agaA and agaB from Bacillus stearothermophilus KVE39, designated agaA1 and agaB1, were cloned into an autonomously replicating Thermus vector and introduced into Thermus thermophilus OF1053GD (ΔagaT) by transformation. This selector strain is unable to metabolize melibiose (α-galactoside) without recombinant α-galactosidases, because the native α-galactosidase gene, agaT, has been deleted. Growth conditions were established under which the strain was able to utilize melibiose as a single carbohydrate source when harboring a plasmid-encoded agaA1 gene but unable when harboring a plasmid-encoded agaB1 gene. With incubation of the agaB1 plasmid-harboring strain under selective pressure at a restrictive temperature (67°C) in a minimal melibiose medium, spontaneous mutants as well as N-methyl-N′-nitro-N-nitrosoguanidine-induced mutants able to grow on the selective medium were isolated. The mutant α-galactosidase genes were amplified by PCR, cloned in Escherichia coli, and sequenced. A single-base substitution that replaces glutamic acid residue 355 with glycine or valine was found in the mutant agaB1 genes. The mutant enzymes displayed the optimum hydrolyzing activity at higher temperatures together with improved catalytic capacity compared to the wild-type enzyme and furthermore showed an enhanced thermal stability. To our knowledge, this is the first report of an in vivo evolution of glycoside-hydrolyzing enzyme and selection within a thermophilic host cell.

Microbial speciation in the geothermal ecosystem

Geothermal areas are unique in many aspects as microbial habitats. They are rare on a global scale and geographically confined. They can be regarded as islands, ecologically separated by large distances and physicochemical dispersal barriers. In a sense the global geothermal ecosystem can be considered to be a world of widely dispersed, often very different “archipelagos” with no mainland. These and other features make geothermal sites an attractive and perhaps ideal model system for studies of microbial divergence and speciation. Microbial speciation may even be more easily observable in geothermal habitats than in other ecosystems.

Divergence of Species in the Geothermal Environment

Geothermal areas are unique in many aspects as microbial habitats. They are rare on a global scale and geographically confined. They can be regarded as islands, ecologically separated by large distances and physicochemical dispersal barriers. In a sense the global geothermal ecosystem can be considered to be a world of widely dispersed, often very different ‘archipelagos’ with no mainland. These and other features make geothermal sites an attractive and perhaps ideal model system for studies of microbial divergence and speciation. Microbial speciation may even be more easily observable in geothermal habitats than in other ecosystems.