Figure 1.
Ultrastructural carboxysome phenotypes in BMC shell mutants of S. elongatus PCC 7942.
Representative transmission electron micrographs of each mutant generated here are shown, as well as the carboxysomeless ΔccmM mutant for reference [16], [37]. A, Wild-type PCC 7942. B, PCC 7942 ΔccmM. C, HIND mutant ccmK2::CmR [20]. D, ΔccmK2. E, ΔccmK3. F, ΔccmK4. G, ΔccmK3-4. H, ΔccmO. Scale bars are 500 nm and HIND and ΔccmK4 were embedded in Epon-araldite rather than LR-white resin.
Table 1.
Ultrastructural, physiological and growth parameters for BMC mutants.
Figure 2.
Photosynthetic oxygen evolution in response to external Ci by BMC shell mutants.
Shown are a representative set of mass-spectrometric measurements of Ci-dependent O2 evolution by wild type and mutant strains of S. elongatus PCC 7942 over a range of Ci concentrations. Ci is the sum of CO2 and HCO3− in solution (pH 7.9).
Figure 3.
Carboxysomal proteins in wild-type S. elongatus PCC 7942 and BMC mutants.
Western immunoblots show the presence or absence of carboxysomal proteins in carboxysomes purified to the Triton X-100-Percoll stage of the Epps-EDTA method for β-carboxysome purification [43], [48]. Polyclonal antibodies against CcaA, RbcLS, CcmM-35, CcmK2, and CcmO were used [15]. The CcmM-35 antiserum detects CcmM-58 and CcmM-35, as well as a non-specific band (ns) corresponding to RbcL that is routinely visible in CcmM western immunoblots [15]. The CcmK2 antibody detects all three CcmK homologues, but not CcmO, whereas the CcmO antibody detects CcmO specifically.
Figure 4.
Protein:protein interactions between CcmO and CcmK2.
Western immunoblots showing carboxysome components IMAC-purified with H6-Ub-CcmO from TP pellets in the presence and absence of 1.0 m urea (+/−urea). UB, IMAC unbound fraction. B, IMAC bound fraction. The presence of a small amount of CcmK in the ΔccmO mutant is attributed to the very high loading of these SDS-PAGE gels in comparison to those presented in Figure 3. The specificities of the different antibodies are explained in the caption to Fig. 3.
Figure 5.
Two models for the outer shell facets of β-carboxysomes in S. elongatus PCC 7942.
A, a β-carboxysome facet model where the entire edge structure is formed from CcmO. B, a β-carboxysome facet where CcmO only occupies the EutS-like role. Two facets are shown with the top facet extending into the plane of the drawing at 138° from the bottom facet. Structural roles are: edge (CcmO, red), facet (CcmK2, grey), vertex (CcmL, brown) and niche (CcmK3, K4 and CcmP, Purple). The number of individual protein oligomers forming a single facet is listed below. The carboxysome edge length (90.2 nm) was calculated as the number of CcmK2 hexamers (CcmK2 hexamer edge length = 35 Å [9]) sufficient to account for the facet-edge length (92.0 nm) calculated from the maximum cross-sectional diameter reported here (175±37 nm). The maximum cross-sectional diameter was assumed to represent the radius of a sphere circumscribing the icosahedral carboxysome.
Table 2.
Protein components of the outer shell of idealised models of the β-carboxysome presented in Figure 5.