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 K-m 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. (C) 2003 Elsevier Ltd. All rights reserved.