, Volume 11, Issue 2, pp 283-293
Date: 24 Nov 2006

Cloning and expression of heterologous genes in Rhodothermus marinus

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Abstract

The construction of a shuttle cloning system for Rhodothermus marinus and Escherichia coli is described. It is based on the shuttle vector pRM3000, which contains a multiple cloning site as well as the shuttle marker trpB and TrpB recipients of both species. The vector is stable and in 25 ± 2 and 91 ± 3 copies in R. marinus SB-1 and E. coli SDH-1, respectively. Three different R. marinus regulatory sequences of the dnaJ, dnaK and recA genes were integrated into pRM3000 to make the expression vectors pRM5100, pRM5200 and pRM5300, respectively. Genes encoding α- and β-galactosidase (agaT and bgaT) from Thermus brockianus were cloned in R. marinus. Expression of both recombinant genes in R. marinus was demonstrated. The agaT gene was used as a reporter to study transcription from the expression vectors. Induced expression by ten- and twentyfold was observed from the dnaK and dnaJ regulatory sequences, respectively, after a temperature shift from 63 to 77°C. This is the first report of cloning and expression of heterologous genes in R. marinus and the first study of promoter activity in the species.

Communicated by J. N. Reeve.