Characterization of the Selective Permeability of Carboxysome Shell Proteins and Implications for CO2 Fixation Efficiency

• Applicant: Dr. Manuel Sommer
• Subject Area: Plant Physiology ; Plant Biochemistry and Biophysics
• Term: from 2015 to 2017
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 289026613

Photosynthesis powers life on earth by using light energy to produce sugars from gaseous CO2. Photoautotrophic prokaryotes named cyanobacteria contribute a major portion to this way of sugar production, as they account for 60% of marine photosynthesis. A key enzyme for photosynthetic CO2 assimilation is RuBisCO. The carboxylating activity of this enzyme is enhanced by CO2 and inhibited in the presence of O2 and thus photoautotrophs evolved carbon concentrating mechanisms (CCMs) to improve photosynthesis in oxygenic environments. The cyanobacterial CCM restrains the majority of the cells RuBisCO in proteinaceous microcompartments called carboxysomes, where CO2 is enriched and O2 is excluded. The carboxysome shell forms an icosahedron that is confined by layers of hexagonal protein oligomers. But despite fair knowledge about carboxysome structure, diffusion routes that metabolites take across the carboxysome shell have not been demonstrated. Structural analysis revealed that the carboxysome shell proteins CcmK3, CcmK4 and CcmP form oligomers that contain a central pore. The pore diameter is 4 Å in CcmK oligomers and 13 Å in CcmP oligomers, which is sufficient for the substrates and products of the RuBisCO reaction. The aim of this project is to analyze mutants of CcmK3, CcmK4 and CcmP regarding their effect on carboxysome permeability and carbon fixation efficiency in order to characterize their function in the cyanobacterium Synechococcus elongatus PCC 7942. To reach this aim, I will grow wildtype, ccmK3, ccmK3/4 and ccmP mutants of S. elongatus PCC 7942 in moderate light and shift the cultures to a) high light and b) low CO2. I will compare alterations of the metabolome of wildtype and mutants during the shift, since ccm mutation will most likely affect the pool size of carbon metabolism intermediates. In order to show implications on carbon fixation and other metabolic processes of CcmK3, CcmK4 or CcmP mutation, I will analyze the response to the shift on the gene expression level using transcriptomics and proteomics. Preliminary results show a growth phenotype of both ccmK3 ccmK4 and the ccmP mutant, which depends on the light intensity. This indicates that the function of these genes is involved in assimilatory processes. I hypothesize that CcmK3, CcmK4 and CcmP are involved in metabolite transport across the carboxysome shell and that their loss-of-function mutants display a change of the shells permeability towards RuBisCO substrates and/or products. Metabolomics of mutants shifted to high light or low CO2 will reveal putative bottlenecks in carboxysome transport. Transcriptomics and proteomics will help to determine the dynamics of carboxysome adaptation to high light environment. Furthermore, they will show how the response of gene expression to environmental changes is affected by the new properties of the carboxysome in the mutants.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Professorin Cheryl Kerfeld, Ph.D.