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. 2009 Oct 21;4(10):e7521.
doi: 10.1371/journal.pone.0007521.

VSports app下载 - The pentameric vertex proteins are necessary for the icosahedral carboxysome shell to function as a CO2 leakage barrier

Fei Cai  1 , Balaraj B Menon, Gordon C Cannon, Kenneth J Curry, Jessup M Shively, Sabine Heinhorst
Affiliations

The pentameric vertex proteins are necessary for the icosahedral carboxysome shell to function as a CO2 leakage barrier

Fei Cai et al. PLoS One. .

Abstract

Background: Carboxysomes are polyhedral protein microcompartments found in many autotrophic bacteria; they encapsulate the CO(2) fixing enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) within a thin protein shell and provide an environment that enhances the catalytic capabilities of the enzyme. Two types of shell protein constituents are common to carboxysomes and related microcompartments of heterotrophic bacteria, and the genes for these proteins are found in a large variety of bacteria. VSports手机版.

Methodology/principal findings: We have created a Halothiobacillus neapolitanus knockout mutant that does not produce the two paralogous CsoS4 proteins thought to occupy the vertices of the icosahedral carboxysomes and related microcompartments. Biochemical and ultrastructural analyses indicated that the mutant predominantly forms carboxysomes of normal appearance, in addition to some elongated microcompartments. Despite their normal shape, purified mutant carboxysomes are functionally impaired, although the activities of the encapsulated enzymes are not negatively affected V体育安卓版. .

Conclusions/significance: In the absence of the CsoS4 proteins the carboxysome shell loses its limited permeability to CO(2) and is no longer able to provide the catalytic advantage RubisCO derives from microcompartmentalization. This study presents direct evidence that the diffusion barrier property of the carboxysome shell contributes significantly to the biological function of the carboxysome. V体育ios版.

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"VSports app下载" Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Two-dimensional separation of carboxysome shell proteins.
Purified wild type carboxysomes were broken and a shell-enriched fraction was recovered after high-speed centrifugation . (A) Proteins (90 µg) separated by two-dimensional SDS-PAGE and stained with Coomassie Blue. (B) A blot of the gel probed with anti-CsoS4B antiserum. The two immunoreactive spots representing CsoS4A (right) and CsoS4B (left) are indicated by white triangles.
Figure 2
Figure 2. Genotype of HncsoS4AB::Km.
The csoS4A and csoS4B genes of the H. neapolitanus cso operon were replaced with a KanR cassette by homologous recombination in vivo as described previously , . The mutant genotype was verified by genomic DNA sequencing. The wild type cso operon from the start codon of cbbL to the stop codon of csoS1B is 7686 nucleotides long.
Figure 3
Figure 3. Growth curve of wild type and mutant H. neapolitanus cultures.
Batch cultures were grown in air (wild type: open circles; mutant: open triangles) or in air supplemented with 5% CO2 (wild type: filled circles; mutant: filled triangles). The HncsoS4AB::Km mutant reaches lower cell densities than the wild type.
Figure 4
Figure 4. Transmission electron micrographs of HncsoS4AB::Km cells.
The sectioned cells shown in (A) – (D) contain elongated carboxysomes (triangles) and carboxysomes of apparently icosahedral shape (arrows). Panels (C) and (D) show dividing cells with greatly elongated carboxysomes that extend across the constriction sites. Scale bar  = 100 nm.
Figure 5
Figure 5. Transmission electron micrographs of purified HncsoS4AB::Km carboxysomes.
The images in (A) – (C) show sucrose gradient-purified wild type (A) and mutant (B, C) carboxysomes that were negatively stained with ammonium molybdate. Scale bar  = 100 nm.
Figure 6
Figure 6. The HncsoS4AB::Km mutant does not produce CsoS4 protein.
(A) Crude cell extract (50 µg) and (B) purified carboxysome (10 µg) proteins were separated by SDS-PAGE (lanes 1, 2). Blots were probed with anti-CsoS4B (lanes 3–5) and anti-CsoS1B (lanes 6, 7) antiserum. Wild type: lanes 1, 3, 6; mutant: lanes 2, 4, 7; lane 5: 10 ng rCsoS4 protein (1∶1 mixture of CsoS4A and CsoS4B). The reduced migration rate of the recombinant CsoS4 proteins is due to their C-terminal hexa-histidine tag. The mutant does not produce CsoS4A and CsoS4B.
Figure 7
Figure 7. Catalytic activity of the carboxysomal carbonic anhydrase CsoSCA.
The increase in pH upon dehydration of bicarbonate to CO2 by CsoSCA was followed using a colorimetric stopped-flow assay . In the representative plot shown, the open circle trace shows the apparently faster kinetics (steeper initial slope) of CsoSCA activity in HncsoS4AB::Km mutant carboxysomes; the filled circle trace indicates wild type.
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"V体育官网入口" References

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