Genomes to Life Contractor-Grantee Workshop III
February 6-9, 2005, Washington, D.C.
Genomics:GTL Program Projects
Sandia National Laboratories
20
Proteomic Analysis of the Synechococcus WH8102 CCM with Varying CO2 Concentrations
Arlene Gonzales, Yooli K. Light, Zhaoduo Zhang, Michael D. Leavell, Rajat Sapra, Tahera Iqbal, Todd W. Lane, and Anthony Martino* (martino@sandia.gov)
Sandia National Laboratories, Livermore, CA
The genera Synechococcus and Prochlorococcus are oxygenic photoautotroph cyanobacteria. They are the most abundant picophytoplankton in the world’s oceans where they form the foundation of the marine food web and are likely the largest contributors to primary production. Whole genome sequences are now available for a number of cyanobacteria including Synechococcus WH8102, Prochlorococcus MED4, and Prochlorococcus MIT9313. The sequences make it possible to use comparative analysis and high-throughput functional genomics and proteomics experiments to help better understand global diversity involved in carbon fixation.
Synechococcus WH8102’s 2.4 Mb genome has yielded a number of interesting results regarding the carbon concentrating mechanism (CCM) in this organism. The carboxysome encoding operon in 8102 resembles that of β-proteobacteria rather than cyanobacteria. The operon most likely was acquired through horizontal gene transfer from phage. Carbonic anhydrase (CA) activity in the carboxysome shell protein csoS3 has been determined experimentally. Genome analysis indicates a putative β-CA and a ferripyochelin binding protein CA may also exist. Finally, transport of inorganic carbon in 8102 may occur through the low affinity CO2 uptake genes ndhD4, ndhF4, and chpX. In Prochlorococcus, uptake genes have not been observed. Perhaps a unique transport mechanism exists in oceanic cyanobacteria.
We will present a high-throughput proteomic approach using mass spectrometry (MS), 2-hybrid analysis, and phage display to deconstruct components of the CCM and determine the effect of changing CO2 levels in Synechococcus 8102. Protein expression levels of CCM components and protein-protein interactions within the carboxysome will be presented. Protein fractions were separated in to particulate and soluble fractions, and western blots of the fractions indicated rbcL and carboxysome shell proteins partitioned exclusively with the particulate fractions. Developing and fully mature carboxysomes were observed in the particulate fractions using electron microscopy. The two putative CAs partitioned separately in the particulate and soluble fractions. Changes in expression of specific proteins in cultures bubbled under different CO2 levels were determined using 2D electrophoresis/MALDI-TOF MS. A synergistic whole proteome approach using capillary LC-MS/MS continues. Protein-protein interactions within the carboxysome have been determined using bacterial 2-hybrid techniques, and a number of pair wise interactions with be presented. Finally, rbcS-peptide interactions are being studied using phage display techniques.
* Presenting author
