Combinatorial mutagenesis and pseudorevertant analysis to characterize regions in loop E of the CP47 protein in...

Deletion of the I265-F268 and T271-K277 regions in the large lumenally exposed loop of the CP47 protein are known to lead to a loss of photoautotrophic growth. Here, these regions have been investigated by combinatorial mutagenesis and pseudorevertant mapping. No single amino-acid residue in the I265-F268 region was found to be critical for function, but a large hydrophobic residue at position 267 and preferentially an aromatic residue at position 268 appeared to be required for photoautotrophic growth. Starting from an obligate photoheterotrophic mutant lacking the T271-K277 region, photoautotrophic pseudorevertants were generated with short in-frame tandem repeats near the site of the original deletion, partially or fully restoring the length of the original protein. These pseudorevertants were sensitive to oxygen indicating that the T271-K277 region may provide PS II stability and/or protection against oxygen-dependent photoinactivation. Pseudorevertants with much improved photoautotrophic growth were also generated for one of the combinatorial mutants in the I265-F268 region. Surprisingly, the secondary mutations in these pseudorevertants mapped to the ferrochelatase gene. We speculate that the secondary mutation in ferrochelatase gene resulted in altered ferrochelatase activity. Decreased heme (phycobilin) biosynthesis and/or increased chlorophyll biosynthesis could then lead to improved PS II performance of the combinatorial CP47 mutant.