Genetic Complementation and Kinetic Analyses ofRhodobacter capsulatus ORF1696 Mutants Indicate that the ORF1696 Protein Enhances Assembly of the Light-Harvesting I Complex

摘要

Rhodobacter capsulatus ORF1696 mutant strains were created by insertion of antibiotic resistance cartridges at different sites within the ORF1696 gene in a strain that lacks the light-harvesting II (LHII) complex. Steady-state absorption spectroscopy profiles and the kinetics of the light-harvesting I (LHI) complex assembly and decay were used to evaluate the function of the ORF1696 protein in various strains. All of the mutant strains were found to be deficient in the LHI complex, including one (ΔNae) with a disruption located 13 codons before the 3′ end of the gene. A 5′-proximal disruption after the 31st codon of ORF1696resulted in a mutant strain (ΔMun) with a novel absorption spectrum. The two strains with more 3′ disruptions (ΔStu and ΔNae) were restored nearly to the parental strain phenotype when transcomplemented with a plasmid expressing the ORF1696 gene, but ΔMun was not. The absorption spectrum of ΔMun resembled that of a strain which had a polar mutation in ORF1696. We suggest that a rho-dependent transcription termination site exists between the MunI and proximal StuI sites ofORF1696. A comparison of LHI complex assembly kinetics showed that assembly occurred 2.6-fold faster in the parental strain than in strain ΔStu. In contrast, LHI complex decay occurred 1.7-fold faster in the ORF1696 parental strain than in ΔStu. These results indicate that the ORF1696 protein has a major effect on LHI complex assembly, and models of ORF1696 function are proposed.