Identification and cloning of the promoter for the Thiobacillus ferrooxidans ribulose 1,5-bisphosphate carboxylase/oxygenase.

摘要

The ribulose 1,5-bisphosphate carboxylase/oxygenase {dollar}rm (Lsb8Ssb8,{dollar} form I; rbcL-rbcS) structural gene(s) from the iron-oxidizing, obligately acidophilic chemolithotroph Thiobacillus ferrooxidans (ATCC 19859) has been identified and subcloned into the Escherichia coli plasmid pUC18. The recombinant plasmid produced was screened by colony level hybridization and a 5.5 kbp HpaI fragment, including the entire coding region for rbcL-rbcS form I Rubisco genes, was isolated and subcloned into plasmid pRK404 and transferred into a rubisco deletion mutant, Rhodobacter sphaeroides strain 16 (rbcL-rbcS) {dollar}sp-,{dollar} (rbpL) {dollar}sp-.{dollar} This strain requires complementation by the genes rbcL, rbcS or rbpL in order to grow anaerobically and photoautotrophically in an H{dollar}sb2{dollar}-CO{dollar}sb2{dollar} atmosphere. A restriction map of the isolated DNA fragment was obtained and the presence of the putative Rubisco promoter was confirmed by subcloning the 5.5 kbp HpaI fragment into the commercial promoter vector pRL124.