Low Activation State of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Carboxysome-Defective Synechococcus Mutants

Abstract

The high-C0,-requiring mutant of Synechococcus sp. PCC 7942, EK6, was obtained after extension of the C terminus of the small subunit of ribulose-I ,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco). The carboxysomes in EK6 were much larger than in the wild type, but the cellular distribution of the large and small subunits of Rubisco was not affected. The kinetic parameters of in vitro-activated Rubisco were similar in EK6 and in the wild type. On the other hand, Rubisco appeared to be in a low state of activation in situ in EK6 cells pretreated with an air leve1 of CO,. This was deduced from the appearance of a lag phase when carboxylation was followed with time in cells permeabilized by detergent and subsequently supplied with saturating CO, and RuBP. Pretreatment of the cells with high CO, virtually abolished the lag. After low-CO, treatment, the internal RuBP pool was much higher in mutant cells than in the wild-type cells; pretreatment with high CO, reduced the pool in mutant cells. We suggest that the high-C0,-requiring phenotype in mutants that possess aberrant carboxysomes arises from the inactivated state of Rubisco when the cells are exposed to low co,.Cyanobacteria possess polyhedral bodies that contain most of the cellular Rubisco (see Codd, 1988;McKay et al., 1992) and CA activities . Quantitative models (Reinhold et al., 1989) assigned a critica1 role to the structural organization of the carboxysomes in the operation of the CCM. They postulate that CO, is generated within the carboxysomes at CA sites in close proximity to Rubisco. The CCM enable the cells to overcome the 20-fold difference between the concentration of dissolved CO, at equilibrium with air and the K, (CO,) of their Rubisco (see Aizawa and Miyachi, 1986;Badger, 1987;Pierce and Omata, 1988;Kaplan et al., 1990Kaplan et al., , 1991Kaplan et al., , 1994Miller et al., 1990;Coleman, 1991;Badger and Price, 1992, for recent reviews and literature citations).Aberrant carboxysomes were observed in severa1 Syneckococcus sp. PCC 7942 mutants, which demand high CO, for growth. These mutants were isolated after modifications of ORF78, ccmL, ccmM, ccmN, and ccmO, which were located upstream of rbc (Friedberg et al., 1989;

How Do Algae Concentrate CO2 to Increase the Efficiency of Photosynthetic Carbon Fixation?1

“…One example of this is the loss of carboxysomes through loss of the carboxysomal shell proteins (Orú s et al, 1995), in which case Rubisco is distributed in the cytoplasm. A similar situation occurs in the mutant EK6, which contains a 30-amino acid extension of the small subunit of Rubisco and has empty carboxysomes (Schwarz et al, 1995). Even though the kinetics of this Rubisco appear normal, this strain requires high concentrations of CO 2 for normal growth.…”

“…Immunolocalization studies using antibodies raised against Rubisco indicate that the carboxysome is the primary location in cyanobacteria (McKay et al, 1993). A mutation that causes a 30-amino acid extension of the Rubisco small subunit leads to a Rubisco that does not pack into the carboxysome, which leaves the carboxysome empty (Schwarz et al, 1995). Mutations in any of the genes affecting the assembly, functioning, or shape of the carboxysome result in cells that cannot grow on air levels of CO 2 .…”

The photorespiratory glycolate metabolism is essential for cyanobacteria and might have been conveyed endosymbiontically to plants

“…In earlier studies, HCR phenotypes were observed in CCM mutants of cyanobacteria defective in the ability to accumulate C i internally (22,27) or in the structural organization of the carboxysomes (16,28,29). In both cases, the phenotype emerged from the very low apparent photosynthetic affinity for external C i .…”

Analysis of Carboxysomes from Synechococcus PCC7942 Reveals Multiple Rubisco Complexes with Carboxysomal Proteins CcmM and CcaA

“…Also noteworthy, however, is the potential for introduction of CcmM forms with a large fusion tag to disrupt the usual packing of carboxysomal proteins, resulting in oversized carboxysomes. Schwarz et al (49), for example, reported larger than usual, nonfunctional carboxysomes in the EK6 mutant of Synechococcus PCC7942, which has a C-terminal extension on RbcS. A further, more detailed analysis of carboxysome sizes from WT, ⌬ccmM expressing untagged CcmM, and chimeric CcmM mutants is required in order to determine first if there is a statistical variation in carboxysome size and, second, if the Nor C-terminal extension is responsible for any carboxysome size variations.…”