Filamentous cyanobacteria of the genus Anabaena contain a unique open reading frame, rbcX, which is juxtaposed and cotranscribed with the genes (rbcL and rbcS) encoding form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). Plasmid constructions containing the genes from Anabaena sp. strain CA were prepared, and expression studies in Escherichia coli indicated that the product of the rbcX gene mimicked the ability of chaperonin proteins to facilitate the proper folding of recombinant RubisCO proteins. The purified recombinant Anabaena sp. strain CA RubisCO, much like the RubisCO enzymes from other cyanobacteria, was shown not to undergo inhibition of activity during a time course experiment, and the properties of this chaperoned recombinant protein appear to be consistent with those of the enzyme isolated from the native organism.Ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RubisCO) is an important catalyst which serves as the cornerstone enzyme of the Calvin-Benson-Bassham reductive pentose phosphate pathway of carbon dioxide assimilation. Two major structural forms of this enzyme, form I and form II, have been described, the catalytic (large) subunits of which are only slightly related (25). Unlike the form II enzyme, the form I RubisCO is also comprised of a second polypeptide, the small subunit. In cyanobacteria, the large and small subunits are encoded by the rbcL and rbcS genes, respectively, while in proteobacteria, these proteins are encoded by the cbbL and cbbS genes, which often are found associated with many additional structural and regulatory genes of the Calvin-BensonBassham pathway (8). Recently it was found that the genes in and around the rbcL and rbcS genes of three species of Anabaena are organized differently from the corresponding genes in other cyanobacteria (15,16). Specifically, a gene encoding RubisCO activase (rca) is located downstream from rbcS (16), with another open reading frame, rbcX, juxtaposed and cotranscribed with the rbcL and rbcS genes of Anabaena sp. strain PCC 7120 (12) as well as Anabaena variabilis and Anabaena sp. strain CA (16). Transcription of the rbcLXS operon is differentially regulated relative to rca expression in Anabaena sp. strain CA (16), with rca transcription preceding the appearance of rbc message after a dark-to-light shift. These studies suggest that RubisCO activase might play an important physiological role. Indeed, since various phosphorylated intermediates often accumulate in cyanobacteria in the dark, by analogy to eukaryotic systems (20), the early transcription of rca might signify the need of the organism to use RubisCO activase to catalyze the removal of such metabolic inhibitors (23) from RubisCO. Inasmuch as it is difficult to obtain from recombinant and native sources, large amounts of RubisCO required to examine the role of RubisCO activase in mediating the catalytic properties of Anabaena RubisCO (2, 12, 23), elucidation of the factors which affect the recovery of active recombinant enzyme is crucial. Previous difficul...
There was no discernible effect after incubating recombinant Anabaena Rubisco and carboxyarabinitol 1-phosphate with the product of the Anabaena rca gene. Since the unactivated cyanobacterial Rubisco is not readily inhibited by ribulose 1,5-bisphosphate and fallover is not observed, a genetic basis for the function of the Rubisco activase-like gene (rca) was sought. The monocistronic rca gene was inactivated in vivo and resulting mutant strains of A. variabilis were found to be incapable of synthesizing immunologically detected RCA protein. The requirement for the product of the rca gene in the light was further examined by measuring Rubisco activity in permeabilized whole cells of wild-type and rca mutant strains at different light intensities. In a 1% CO2-air atmosphere, inactivation of rca reduced the ability of A. variabilis to elevate Rubisco activity under high light (73 micromol quanta m(-2) s(-1)), but had little effect under low light (8 micromol m(-2) s(-1)). For air-grown cultures, differences in the rates exhibited by the wild-type and rca mutant to fully activate Rubisco during a whole-cell assay were enhanced by increases in light intensity. The significance of the rca mutation was underlined by effects on growth as, unlike the wild-type, growth rates did not increase after cells transferred from low to high light intensities. Higher exogenous CO2 concentrations (1%) were required to sustain a normal growth rate for the A. variabilis rca mutant. When grown in air levels of CO2, the rca mutant not only needed longer times to double in cell density but also exhibited greatly diminished Rubisco activity compared with the wild-type strain. Despite the unusual properties of cyanobacterial Rubisco, these results suggest a physiological role for the product of the rca gene in maximizing the activity of Rubisco in heterocystous cyanobacteria.
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