Cloning and expression of the D-ribulose-1,5-bisphosphate carboxylase/oxygenase form II gene from Thiobacillus intermedius in Escherichia coli.

Both form I and II ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) genes were detected in Thiobacillus intermedius by heterologous hybridization using specific probes from Anacystis nidulans and Rhodobacter sphaeroides, respectively. However, only the previously reported form I enzyme could be demonstrated in cells grown under a number of different conditions. The reason(s) why the form II gene is not expressed in T. intermedius is/are not clear at this time. The form II gene was isolated from a lambda library by screening with the Rb. sphaeroides probe. A SalI fragment from this clone was ligated into pUC8 and transformed into Escherichia coli DH5 alpha. Subclones pTi20IIA and pTi20IIB representing both orientations relative to the lac promoter were isolated. Low levels of RuBisCO activity were detected in both induced and non-induced pTi20IIA indicating the probable expression from a T. intermedius promoter. Induced pTi20IIB produced much higher levels of enzyme activity. Analysis of cell-free extracts using sucrose density gradients confirmed the expression of a form II RuBisCO similar in size to that found in Rhodobacter capsulatus. Other Calvin cycle genes were not clustered with either the form I or form II genes.