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Advances in Understanding Carboxysome Assembly in Prochlorococcus and Synechococcus Implicate CsoS2 as a Critical Component.

Cai F, Dou Z, Bernstein SL, Leverenz R, Williams EB, Heinhorst S, Shively J, Cannon GC, Kerfeld CA - Life (Basel) (2015)

Bottom Line: Two types of carboxysome, α and β, encapsulating form IA and form IB d-ribulose-1,5-bisphosphate carboxylase/oxygenase, respectively, differ in gene organization and associated proteins.Based on our results from bioinformatic, biophysical, genetic and biochemical approaches, including peptide array scanning for protein-protein interactions, we propose a model for CsoS2 function and its spatial location in the α-carboxysome.Analogies between the pathway for β-carboxysome biogenesis and our model for α-carboxysome assembly are discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA. fcai@lbl.gov.

ABSTRACT
The marine Synechococcus and Prochlorococcus are the numerically dominant cyanobacteria in the ocean and important in global carbon fixation. They have evolved a CO2-concentrating-mechanism, of which the central component is the carboxysome, a self-assembling proteinaceous organelle. Two types of carboxysome, α and β, encapsulating form IA and form IB d-ribulose-1,5-bisphosphate carboxylase/oxygenase, respectively, differ in gene organization and associated proteins. In contrast to the β-carboxysome, the assembly process of the α-carboxysome is enigmatic. Moreover, an absolutely conserved α-carboxysome protein, CsoS2, is of unknown function and has proven recalcitrant to crystallization. Here, we present studies on the CsoS2 protein in three model organisms and show that CsoS2 is vital for α-carboxysome biogenesis. The primary structure of CsoS2 appears tripartite, composed of an N-terminal, middle (M)-, and C-terminal region. Repetitive motifs can be identified in the N- and M-regions. Multiple lines of evidence suggest CsoS2 is highly flexible, possibly an intrinsically disordered protein. Based on our results from bioinformatic, biophysical, genetic and biochemical approaches, including peptide array scanning for protein-protein interactions, we propose a model for CsoS2 function and its spatial location in the α-carboxysome. Analogies between the pathway for β-carboxysome biogenesis and our model for α-carboxysome assembly are discussed.

No MeSH data available.


Knockout of CsoS2 abolishes carboxysome formation in Hnea. When the csoS2 gene is interrupted by the insertion of a KmR cassette (a); no carboxysomes are apparent in mutant cells comparing to wildtype Hnea cells with carboxysomes (indicated by red arrows) under the same growth condition (b).
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life-05-01141-f003: Knockout of CsoS2 abolishes carboxysome formation in Hnea. When the csoS2 gene is interrupted by the insertion of a KmR cassette (a); no carboxysomes are apparent in mutant cells comparing to wildtype Hnea cells with carboxysomes (indicated by red arrows) under the same growth condition (b).

Mentions: A HneacsoS2 gene disruption mutant was generated by inserting a kanamycin resistance cassette (KmR) in the csoS2 coding region at an EcoRV site (Figure 3a). This mutant presents a high CO2 requiring (hcr) phenotype and does not grow in air (Figure S2). This is in contrast to the hcr phenotype observed in the Hnea csoS3 insertion mutant that was similarly constructed, which does grow but at a significantly slower rate than wildtype in air [11]. Thin sections of Hnea csoS2::KmR mutant cells completely lack carboxysomes (Figure 3b), which accounts for the observed hcr phenotype. This is distinctly different from the Hnea csoS3::KmR mutant, in which the elimination of the CsoSCA protein results in mutant carboxysomes that are indistinguishable in size and appearance from wildtype but functionally defective [11]. The fact that all other carboxysomal proteins are present at a similar level in the CsoSCA knockout mutant relative to wildtype suggests that insertion of a KmR cassette does not affect expression of downstream genes. These results indicate that the CsoS2 protein is important for the formation or stability of α-carboxysomes.


Advances in Understanding Carboxysome Assembly in Prochlorococcus and Synechococcus Implicate CsoS2 as a Critical Component.

Cai F, Dou Z, Bernstein SL, Leverenz R, Williams EB, Heinhorst S, Shively J, Cannon GC, Kerfeld CA - Life (Basel) (2015)

Knockout of CsoS2 abolishes carboxysome formation in Hnea. When the csoS2 gene is interrupted by the insertion of a KmR cassette (a); no carboxysomes are apparent in mutant cells comparing to wildtype Hnea cells with carboxysomes (indicated by red arrows) under the same growth condition (b).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4499774&req=5

life-05-01141-f003: Knockout of CsoS2 abolishes carboxysome formation in Hnea. When the csoS2 gene is interrupted by the insertion of a KmR cassette (a); no carboxysomes are apparent in mutant cells comparing to wildtype Hnea cells with carboxysomes (indicated by red arrows) under the same growth condition (b).
Mentions: A HneacsoS2 gene disruption mutant was generated by inserting a kanamycin resistance cassette (KmR) in the csoS2 coding region at an EcoRV site (Figure 3a). This mutant presents a high CO2 requiring (hcr) phenotype and does not grow in air (Figure S2). This is in contrast to the hcr phenotype observed in the Hnea csoS3 insertion mutant that was similarly constructed, which does grow but at a significantly slower rate than wildtype in air [11]. Thin sections of Hnea csoS2::KmR mutant cells completely lack carboxysomes (Figure 3b), which accounts for the observed hcr phenotype. This is distinctly different from the Hnea csoS3::KmR mutant, in which the elimination of the CsoSCA protein results in mutant carboxysomes that are indistinguishable in size and appearance from wildtype but functionally defective [11]. The fact that all other carboxysomal proteins are present at a similar level in the CsoSCA knockout mutant relative to wildtype suggests that insertion of a KmR cassette does not affect expression of downstream genes. These results indicate that the CsoS2 protein is important for the formation or stability of α-carboxysomes.

Bottom Line: Two types of carboxysome, α and β, encapsulating form IA and form IB d-ribulose-1,5-bisphosphate carboxylase/oxygenase, respectively, differ in gene organization and associated proteins.Based on our results from bioinformatic, biophysical, genetic and biochemical approaches, including peptide array scanning for protein-protein interactions, we propose a model for CsoS2 function and its spatial location in the α-carboxysome.Analogies between the pathway for β-carboxysome biogenesis and our model for α-carboxysome assembly are discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA. fcai@lbl.gov.

ABSTRACT
The marine Synechococcus and Prochlorococcus are the numerically dominant cyanobacteria in the ocean and important in global carbon fixation. They have evolved a CO2-concentrating-mechanism, of which the central component is the carboxysome, a self-assembling proteinaceous organelle. Two types of carboxysome, α and β, encapsulating form IA and form IB d-ribulose-1,5-bisphosphate carboxylase/oxygenase, respectively, differ in gene organization and associated proteins. In contrast to the β-carboxysome, the assembly process of the α-carboxysome is enigmatic. Moreover, an absolutely conserved α-carboxysome protein, CsoS2, is of unknown function and has proven recalcitrant to crystallization. Here, we present studies on the CsoS2 protein in three model organisms and show that CsoS2 is vital for α-carboxysome biogenesis. The primary structure of CsoS2 appears tripartite, composed of an N-terminal, middle (M)-, and C-terminal region. Repetitive motifs can be identified in the N- and M-regions. Multiple lines of evidence suggest CsoS2 is highly flexible, possibly an intrinsically disordered protein. Based on our results from bioinformatic, biophysical, genetic and biochemical approaches, including peptide array scanning for protein-protein interactions, we propose a model for CsoS2 function and its spatial location in the α-carboxysome. Analogies between the pathway for β-carboxysome biogenesis and our model for α-carboxysome assembly are discussed.

No MeSH data available.