Positive and Negative Selection of Mutant Forms of Prokaryotic (Cyanobacterial) Ribulose-1,5-bisphosphate Carboxylase/Oxygenase

A system for biological selection of randomly mutagenized ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) genes from the cyanobacterium Synechococcus PCC6301 was designed in which a Rubisco deletion mutant of the photosynthetic bacterium Rhodobacter capsulatus served as a host. Trans-complementation with the Synechococcus PCC6301 rbcLS genes enabled anaerobic photoautotrophic growth of the R. capsulatus deletion strain with 5% CO2, but not with 1.5% CO2 in the atmosphere, and this strain could not grow under aerobic chemoautotrophic conditions. Phenotypic differences between the R. capsulatus host strain complemented with the wild-type rbcLS genes and transconjugates carrying mutated genes were used to identify mutants that were able to complement to photoautotrophic growth with 1.5% CO2. These “positive” mutant proteins were unaffected for any measured kinetic properties, with a single exception. A mutant with a valine substitution at phenylalanine 342 had an increased affinity for ribulose-1,5-bisphosphate. Mutants with changes in the affinity for CO2 were isolated through negative selection, in which mutants incapable of complementing R. capsulatus to photoautotrophic growth with 5% CO2 were identified. Mutations at aspartate 103 resulted in enzymes that were greatly affected for different kinetic parameters, including an increased Km for CO2. This study demonstrated that random mutagenesis and bioselection procedures could be used to identify mutations that influence important properties of bacterial Rubisco; these residues would not have been identified by other methods.