Proteomic assessment of an established technique for carboxysome enrichment from Synechococcus PCC7942

Carboxysomes are protein-bound, polyhedral microbodies within cyanobacteria, containing the key enzyme for photosynthetic CO2 fixation, ribulose-1,5-bisphosphate carboxylase–oxygenase (Rubisco). Sequencing of cyanobacterial genomes has revealed that cyanobacteria possess one or other of two types of carboxysomes. Cyanobacteria containing form 1A Rubisco possess α-carboxysomes, while those with form 1B Rubisco possess β-carboxysomes. Given the central importance of carboxysomes in the CO2-concentrating mechanism of cyanobacteria, understanding the nature and composition of these structures is of considerable importance. In an effort to develop techniques for the characterization of the structure of β-carboxysomes, particularly the outer protein shell, we have undertaken a proteomic assessment of the Percoll–Mg2+ carboxysome enrichment technique using the freshwater cyanobacterium Synechococcus sp. PCC7942. Both matrix-assisted laser desorption–ionization – time of flight mass spectrometry (MALDI-TOF MS) and multidimensional protein identification technology (MuDPIT) methods were used to determine the protein content of a novel carboxysome-rich fraction. A total of 17 proteins were identified using MALDI-TOF MS from enriched carboxysome preparations, while 122 proteins were identified using MuDPIT analysis on the same material. The carboxysomal protein CcmM was identified by MALDI-TOF MS as two distinct proteins of 38 and 58 kDa. The only other carboxysomal proteins identified were the large and small subunits of Rubisco (RbcL and RbcS). Reasons for the lack of evidence for the expected full complement of carboxysomal proteins and future directions are discussed.Key words: CO2-concentrating mechanism, cyanobacteria, carboxysomes, proteomics.

Regulation of cyanobacterial CO2-concentrating mechanisms through transcriptional induction of high-affinity Ci-transport systems

Approximately 50% of global CO2-based productivity is now attributed to the activity of phytoplankton, including ocean-dwelling cyanobacteria. In response to inherent restrictions on the rate of CO2 supply in the aquatic environment, cyanobacteria have evolved a very efficient means of capturing inorganic carbon (Ci), as either CO2 or HCO3–. for photosynthetic carbon fixation. This capturing mechanism, known as a CO2-concentrating mechanism (CCM), involves the operation of active CO2 and HCO3– transporters and results in the concentration of CO2 around RuBisCO, in a unique microcompartment called the carboxysome. The CCM exhibits two basic physiological states: a constitutive, low-affinity state; and a high-affinity state, which is induced in response to Ci limitation. Many of the genetic components of the CCM, including genes encoding Ci transporters, have been identified. It is apparent that the expression of genes encoding the inducible, high-affinity Ci transporters is particularly sensitive to Ci availability, and we are now interested in defining how cyanobacterial cells sense and respond to Ci limitation at the transcriptional level. Current theories include direct sensing of external Ci; sensing of internal Ci-pool fluctuations; and detection of changes in photorespiratory intermediates, carbon metabolites, or redox potential. At present, there is no consensual view. We have investigated the physiological and transcriptional responses of CCM mutants and wildtype strains to pharmacological treatments and various light, O2, and Ci regimes. Our data suggest that perception of Ci limitation by a cyanobacterial cell is either directly or indirectly related to the size of the internal Ci pool within the cell, in an oxygen-dependent manner.Key words: CO2-concentrating mechanisms, CO2 sensing, Ci transporters, Synechococcus PCC7942.