Abstract
The carboxylase activities of crude carboxysome preparations obtained from the wild-type Synechococcus elongatus strain PCC 7942 strain and the mutant defective in the carboxysomal carbonic anhydrase (CA) were compared. The carboxylation reaction required high concentrations of bicarbonate and was not even saturated at 50 mM bicarbonate. With the initial concentrations of 50 mM and 25 mM for bicarbonate and ribulose-1,5-bisphosphate (RuBP), respectively, the initial rate of RuBP carboxylation by the mutant carboxysome (0.22 μmol mg−1 protein min−1) was only 30 % of that observed for the wild-type carboxysomes (0.71 μmol mg−1 protein min−1), indicating the importance of the presence of CA in efficient catalysis by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). While the mutant defective in the ccmLMNO genes, which lacks the carboxysome structure, could grow under aeration with 2 % (v/v) CO2 in air, the mutant defective in ccaA as well as ccmLMNO required 5 % (v/v) CO2 for growth, indicating that the cytoplasmically localized CcaA helped utilization of CO2 by the cytoplasmically localized Rubisco by counteracting the action of the CO2 hydration mechanism. The results predict that overexpression of Rubisco would hardly enhance CO2 fixation by the cyanobacterium at CO2 levels lower than 5 %, unless Rubisco is properly organized into carboxysomes.
Similar content being viewed by others
Abbreviations
- CA:
-
Carbonic anhydrase
- 3-PGA:
-
3-Phosphoglycerate
- RuBP:
-
Ribulose-1,5-bisphosphate
- Rubisco:
-
Ribulose-1,5-bisphosphate carboxylase/oxygenase
- WT:
-
Wild-type
References
Badger MR, Hanson D, Price GD (2002) Evolution and diversity of CO2 concentrating mechanisms in cyanobacteria. Funct Plant Biol 29(2–3):161–173
Cameron JC, Wilson SC, Bernstein SL, Kerfield CA (2013) Biogenesis of a bacterial organelle: the carboxysome assembly pathway. Cell 155(5):1131–1140
Cannon GC, Heinhorst S, Kerfeld CA (2010) Carboxysomal carbonic anhydrases: structure and role in microbial CO2 fixation. Biochim Biophys Acta 1804(2):382–392
Chakrabarti S, Bhattacharya S, Bhattacharya SK (2002) A nonradioactive assay method for determination of enzymatic activity of d-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). J Biochem Biophys Methods 52(3):179–187
Cot SSW, So AKC, Espie GS (2008) A multiprotein bicarbonate dehydration complex essential to carboxysome function in cyanobacteria. J Bacteriol 190(3):936–945
Delwiche CF (1999) Tracing the thread of plastid diversity through the tapestry of life. Am Nat 154:S164–S177
Dou ZC, Heinhorst S, Williams EB, Murin CD, Shively JM, Cannon GC (2008) CO2 fixation kinetics of Halothiobacillus neapolitanus mutant carboxysomes lacking carbonic anhydrase suggest the shell acts as a diffusional barrier for CO2. J Biol Chem 283(16):10377–10384
Elhai J, Wolk CP (1988) A versatile class of positive-selection vectors based on the nonviability of palindrome-containing plasmids that allows cloning into long polylinkers. Gene 68(1):119–138
Fukuzawa H, Suzuki E, Komukai Y, Miyachi S (1992) A gene homologous to chloroplast carbonic-anhydrase (icfA) is essential to photosynthetic carbon-dioxide fixation by Synechococcus PCC7942. Proc Natl Acad Sci USA 89(10):4437–4441
Hanes CS, Isherwood FA (1949) Separation of the phosphoric esters on the filter paper chromatogram. Nature 164(4183):1107–1112
Kuhlemeier CJ, Thomas AAM, van der Ende A, van Leen RW, Borrias WE, van den Hondel CAMJJ, van Arkel GA (1983) A host-vector system for gene cloning in the cyanobacterium Anacystis nidulans R2. Plasmid 10(2):156–163
Laemmli UK (1970) Cleavage of structural proteins during assembly of head of bacteriophage-T4. Nature 227(5259):680–685
Long BM, Badger MR, Whitney SM, Price GD (2007) Analysis of carboxysomes from Synechococcus PCC7942 reveals multiple Rubisco complexes with carboxysomal proteins CcmM and CcaA. J Biol Chem 282(40):29323–29335
Peña KL, Castel SE, de Araujo C, Espie GS, Kimber MS (2010) Structural basis of the oxidative activation of the carboxysomal γ-carbonic anhydrase, CcmM. Proc Natl Acad Sci USA 107(6):2455–2460
Price GD, Badger MR (1989) Isolation and characterization of high CO2-requiring-mutants of the cyanobacterium Synechococcus PCC7942: two phenotypes that accumulate inorganic carbon but are apparently unable to generate CO2 within the carboxysome. Plant Physiol 91(2):514–525
Price GD, Badger MR (1991) Evidence for the role of carboxysomes in the cyanobacterial CO2-concentrating mechanism. Can J Bot 69(5):963–973
Price GD, Coleman JR, Badger MR (1992) Association of carbonic-anhydrase activity with carboxysomes isolated from the cyanobacterium Synechococcus PCC7942. Plant Physiol 100(2):784–793
Price GD, Howitt SM, Harrison K, Badger MR (1993) Analysis of a genomic DNA region from the cyanobacterium Synechococcus sp. strain PCC7942 involved in carboxysome assembly and function. J Bacteriol 175(10):2871–2879
Price GD, Badger MR, Woodger FJ, Long BM (2008) Advances in understanding the cyanobacterial CO2-concentrating-mechanism (CCM): functional components, Ci transporters, diversity, genetic regulation and prospects for engineering into plants. J Exp Bot 59(7):1441–1461
Rae BD, Long BM, Badger MR, Price GD (2013) Functions, compositions, and evolution of the two types of carboxysomes: polyhedral microcompartments that facilitate CO2 fixation in cyanobacteria and some proteobacteria. Microbiol Mol Biol Rev 77(3):357–379
Reinhold L, Zviman M, Kaplan A (1989) A quantitative model for inorganic carbon fluxes and photosynthesis in cyanobacteria. Plant Physiol Biochem 27(6):945–954
Sawaya MR, Cannon GC, Heinhorst S, Tanaka S, Williams EB, Yeates TO, Kerfield CA (2006) The structure of β-carbonic anhydrase from the carboxysomal shell reveals a distinct subclass with one active site for the price of two. J Biol Chem 281(11):7546–7555
So AKC, Espie GS (1998) Cloning, characterization and expression of carbonic anhydrase from the cyanobacterium Synechocystis PCC6803. Plant Mol Biol 37(2):205–215
So AKC, John-McKay M, Espie GS (2002) Characterization of a mutant lacking carboxysomal carbonic anhydrase from the cyanobacterium Synechocystis PCC6803. Planta 214(3):456–467
So AKC, Espie GS, Williams EB, Shively JM, Heinhorst S, Cannon GC (2004) A novel evolutionary lineage of carbonic anhydrase (ε class) is a component of the carboxysome shell. J Bacteriol 186:623–630
Stanier RY, Kunisawa R, Mandel M, Cohen-Bazier G (1971) Purification and properties of unicellular blue-green algae (order Chroococcales). Bacteriol Rev 35(2):171–205
Suzuki E, Fukuzawa H, Miyachi S (1991) Identification of a genomic region that complements a temperature-sensitive, high CO2-requiring mutant of the cyanobacterium, Synechococcus sp. PCC7942. Mol Gen Genet 226(3):401–408
Tabita FR (1999) Microbial ribulose 1,5-bisphosphate carboxylase/oxygenase: a different perspective. Photosynth Res 60(1):1–28
Tyszkiewicz E (1962) An improved solvent system for paper chromatography of phosphate esters. Anal Biochem 3(2):164–166
Williams JGK, Szalay AA (1983) Stable integration of foreign DNA into the chromosome of the cyanobacterium Synechococcus-R2. Gene 24(1):37–51
Yu JW, Price GD, Song L, Badger MR (1992) Isolation of a putative carboxysomal carbonic anhydrase gene from the cyanobacterium Synechococcus PCC7942. Plant Physiol 100(2):794–800
Acknowledgments
This work was supported in part by a Grant-in-Aid for Scientific Research in Innovative Areas (No. 21114003) from the Ministry of Education, Culture, Sports, Science and Technology, Japan; and the CREST program from Japan Science and Technology Agency.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Nishimura, T., Yamaguchi, O., Takatani, N. et al. In vitro and in vivo analyses of the role of the carboxysomal β-type carbonic anhydrase of the cyanobacterium Synechococcus elongatus in carboxylation of ribulose-1,5-bisphosphate. Photosynth Res 121, 151–157 (2014). https://doi.org/10.1007/s11120-014-9986-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11120-014-9986-7