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 alpha- and beta-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 degrees 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.