The CbbQO-type rubisco activases encoded in carboxysome gene clusters can activate carboxysomal form IA rubiscos

The CO₂-fixing enzyme rubisco is responsible for almost all carbon fixation. This process frequently requires rubisco activase (Rca) machinery, which couples ATP hydrolysis to the removal of inhibitory sugar phosphates, including the rubisco substrate ribulose 1,5-bisphosphate (RuBP). Rubisco is sometimes compartmentalized in carboxysomes, bacterial microcompartments that enable a carbon dioxide concentrating mechanism (CCM). Characterized carboxysomal rubiscos, however, are not prone to inhibition, and often no activase machinery is associated with these enzymes. Here, we characterize two carboxysomal rubiscos of the form IA^C clade that are associated with CbbQO-type Rcas. These enzymes release RuBP at a much lower rate than the canonical carboxysomal rubisco from Synechococcus PCC6301. We found that CbbQO-type Rcas encoded in carboxysome gene clusters can remove RuBP and the tight-binding transition state analog carboxy-arabinitol 1,5-bisphosphate from cognate rubiscos. The Acidithiobacillus ferrooxidans genome encodes two form IA rubiscos associated with two sets of cbbQ and cbbO genes. We show that the two CbbQO activase systems display specificity for the rubisco enzyme encoded in the same gene cluster, and this property can be switched by substituting the C-terminal three residues of the large subunit. Our findings indicate that the kinetic and inhibitory properties of proteobacterial form IA rubiscos are diverse and predict that Rcas may be necessary for some α-carboxysomal CCMs. These findings will have implications for efforts aiming to introduce biophysical CCMs into plants and other hosts for improvement of carbon fixation of crops.     

Uptake and utilization of inorganic carbon by cyanobacteria

In the cyanobacteria, mechanisms exist that allow photosynthetic CO₂ reduction to proceed efficiently even at very low levels of inorganic carbon. These inducible, active transport mechanisms enable the cyanobacteria to accumulate large internal concentrations of inorganic carbon that may be up to 1000-fold higher than the external concentration. As a result, the external concentration of inorganic carbon required to saturate cyanobacterial photosynthesis in vivo is orders of magnitude lower than that required to saturate the principal enzyme (ribulose bisphosphate carboxylase) involved in the fixation reactions. Since CO₂ is the substrate for carbon fixation, the cyanobacteria somehow perform the neat trick of concentrating this small, membrane permeable molecule at the site of CO₂ fixation. In this review, we will describe the biochemical and physiological experiments that have outlined the phenomenon of inorganic carbon accumulation, relate more recent genetic and molecular biological observations that attempt to define the constituents involved in this process, and discuss a speculative theory that suggests a unified view of inorganic carbon utilization by the cyanobacteria.Abbreviations C_i Inorganic carbon - H-cells Cells grown under high CO₂ - L-cells Cells grown under low CO₂ - RuBP Ribulose-1,5-bisphosphate - WT Wild type     

Construction of a Synechocystic PCC6803 mutant suitable for the study of variant hexadecameric ribulose bisphosphate carboxylase/oxygenase enzymes

The cyanobacterium Synechocystis PCC6803 was chosen as a target organism for construction of a suitable photosynthetic host to enable selection of variant plant-like ribulose bisphosphate carboxylase/oxygenase (Rubisco) enzymes. The DNA region containing the operon encoding Rubisco (rbc) was cloned, sequenced and used for the construction of a transformation vector bearing flanking sequences to the rbc genes. This vector was utilized for the construction of a cyanobacterial rbc null mutant in which the entire sequence comprising both rbc genes, was replaced by the Rhodospirillum rubrum rbcL gene linked to a chloramphenicol resistance gene. Chloramphenicol-resistant colonies, Syn6803rbc, were detected within 8 days when grown under 5% CO₂ in air. These transformants were unable to grow in air (0.03% CO₂). Analysis of their genome and Rubisco protein confirmed the site of the mutation at the rbc locus, and indicated that the mutation had segregated throughout all of the chromosome copies, consequently producing only the bacterial type of the enzyme. In addition, no carboxysome structures could be detected in the new mutant. Successful restoration of the wild-type rbc locus, using vectors bearing the rbc operon flanked by additional sequences at both termini, could only be achieved upon incubating the transformed cells under 5% CO₂ in air prior to their transferring to air. The yield of restored transformants was proportionally related to the length of those sequences flanking the rbc operon which participate in the homologous recombination. The Syn6803rbc mutant is amenable for the introduction of in vitro mutagenized rbc genes into the rbc locus, aiming at the genetic modification of the hexadecameric type Rubisco.Abbreviations Cm^r chloramphenicol resistance - Km^r kanamycin resistance - HCR high CO₂ requirer - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - SSC sodium chloride and sodium citrate - wt wild-type     

The CO2concentrating mechanism in cyanobactiria and microalgae

Over the past 10 years it has become clear that cyanobacteria and microalgae possess mechanisms for actively acquiring inorganic carbon from the external medium and are able to use this to elevate the CO₂ concentration around the active site of the primary photosynthetic carboxylating enzyme, ribulose bisphosphate carboxylase-oxygenase (Rubisco). This results in a vastly enhanced photosynthetic affinity for inorganic carbon (C_i) and improved photosynthetic efficiency. The CO₂ concentrating mechanism is dependent on the existence of membrane bound C_i transport systems, and a microenvironment within the cell where the accumulated C_i can be used to elevate CO₂ at the site of Rubisco. Evidence presented in this review suggests that in cyanobacteria this is achieved by the packaging of Rubisco and carbonic anhydrase (CA) into discrete structures, which are termed carboxysomes. Analogous structures in microalgae, termed pyrenoids, may perform a similar function. The recovery and analysis of high-CO₂-requiring mutants has greatly advanced our understanding of the mechanisms and genes underlying these systems, especially in cyanobacteria, and this review places particular emphasis on the contribution made by molecular genetic approaches.     

Evidence that changes in the growth conditions affect the relative distribution of diether lipids in haloalkaliphilic archaebacteria

Abstract 90% of the carbonic anhydrase (CA) activity recovered from Chlorogloeopsis fritschii cells, when broken under conditions which favour the isolation of carboxysomes, is particulate. Subsequent sucrose density gradient centrifugation of the carboxysome-containing pellet produced a sharp band of CA, well separated from the carboxysomes and thylakoids. The implications of these findings for the possible functions of carboxysomes and location of CA are discussed     

CARBOXYSOME CONTENT OF SYNECHOCOCCUS LEOPOLIENSIS (CYANOPHYTA) IN RESPONSE TO INORGANIC CARBON1

The carboxysome content of chemostat grown Synechococcus leopoliensis (Racib.) Komarek increases under inorganic carbon limitation. At growth rates of ca. 85%μ_max the carboxysome content (±SE) was 0.57 ± 0.09 carboxysomes·cell section^?1. Under severe carbon limitation (ca. 13%μ_max) this increased to 3.4 · 0.3 carboxysomes·cell section^?1. Corresponding to this change is a three order of magnitude decrease in the half-saturation constant of photosynthesis for dissolved inorganic carbon. Nitrogen and phosphorus limitation had no effect on carboxysome content or the kinetics of photosynthesis with respect to inorganic carbon. These results are discussed in light of the apparent lack of photorespiration in these organisms.     

CYTOCHEMICAL STUDIES ON PROCHLOROPHYTES: LOCALIZATION OF DNA AND RIBULOSE 1,5-BISPHOSPHATE CARBOXYLASE-OXYGENASE1

We studied the distribution of the DNA-containing region and the ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCo) content of polyhedral bodies in three different prochlorophyte cell types in a search for broad evolutionary affinities of these chlorophyll b-containing prokaryotes. DNA was localized by DAPI staining and electron microscopy utilizing monoclonal anti-DNA antibody 2C-10 plus a secondary antibody labeled with colloidal gold. Antibodies against the large RuBisCo subunit from a higher plant raised in rabbits were used to localize RuBisCo in polyhedral bodies. We studied Prochloron Lewin cells from two different didemnid ascidian hosts (Lissoclinum patella and Didemnum molle) collected in Palau, West Caroline Islands, and cells of Prochlorothrix hollandica Burger-Wiersma, Stal, and Mur grown in laboratory culture. Cells of the blue-green alga Anabaena 7120 were studied for comparison. The DNA distribution was markedly different in the two Prochloron cell types. The thylakoids in cells from L. patella were concentrically arranged around a large central vacuole; the DNA-containing stromal areas appeared in thin sections as a concentric arcs between the thylakoid stacks. The central vacuole was lacking in cells from D. molle, and the thylakoid stacks and strands of DNA-containing stroma showed a more haphazard arrangement. In the filamentous Prochlorothrix the DNA-containing stroma was largely limited to a central nucleoid structure running the length of the cell. Although the DNA arrangements in Prochloron might be considered “chloroplast-like” since DNA-containing stroma is distributed, as in chloroplasts, in scattered sites among photosynthetic membranes, this is not so in Prochlorothrix, where there is an axial nucleoid, as in many filamentous cyanobacteria. Our anti-RuBisCo antibodies were selectively bound to the polyhedral bodies of all three cell types, indicating that Prochloron and Prochlorothrix, like many other autotrophic prokaryotes, possess typical carboxysomes.     

聚球藻Synechococcus sp.PCC7942高CO_2需求突变株的筛选及其超微结构观察(英文)

以单细胞蓝藻聚球藻Synechococcussp.PCC7942为材料,利用甲基磺酸乙酯(EMS)进行化学诱变获得了一个高CO2 需求突变株。它能在 4%CO2 下生长而不能在空气中生长。对突变株的初检表明:其回复突变率约为 10 -7。该突变株从高CO2 条件下转到空气中后,细胞在 2～ 3d内逐渐趋于死亡;其光合作用对外源无机碳的依赖性高于野生型细胞,碳酸酐酶活性也低于野生型细胞。在超微结构水平,突变株细胞内出现了不同类型的异常羧体:有的为棒状;有的为不规则状;有的为 空羧体",而且,类囊体周围糖原颗粒增多。进一步说明该突变株在CO2 吸收和利用功能上有缺陷。此外,对低碳条件对羧体的诱导及羧体的生物发生也作了一些探讨     

ISOLATION OF ccmKLMN GENES FROM THE MARINE CYANOBACTERIUM, SYNECHOCOCCUS SP. PCC7002 (CYANOPHYCEAE), AND EVIDENCE THAT CcmM IS ESSENTIAL FOR CARBOXYSOME ASSEMBLY

A high CO₂ requiring mutant of the marine cyanobacterium Synechococcus PCC7002 was generated using a random gene-tagging procedure. This mutant demonstrated a reduced photosynthetic affinity for inorganic carbon (C_i) and accumulated high internal levels of C_i that could not be used for photosynthesis. Analysis of the mutant genomic DNA showed that the mutagenesis had disrupted a cluster of genes involved in the cyanobacterial CO₂ concentrating mechanism (CCM), the so-called ccm genes. These characteristics are consistent with a cyanobacterial mutant with defects in carboxysome assembly and/or functioning. Further genomic analyses indicated that the genes of the Synechococcus PCC7002 operon, ccmKLMN , are structurally similar to those of two closely related cyanobacteria, Synechococcus PCC7942 and Synechocystis PCC6803. The Synechococcus PCC7002 ccmM gene, which encodes a polypeptide with a predicted size of 70 kDa, was the direct target of the mutagenesis event. The CcmM protein has two distinct regions: an N-terminal region that shows similarity to an archaeon gamma carbonic anhydrase and a C-terminal region that contains repeated domains demonstrating sequence similarity to the small subunit of Rubisco. Physiological analysis of a ccmM -defined mutant showed that these cells were essentially identical to the original mutant; they required high CO₂ concentrations for growth, they had a low photosynthetic affinity for C_i, and they internalized C_i to high levels. Moreover, ultrastructural examination showed that both the original and the defined mutants lack carboxysomes. Thus, our results demonstrate that the ccmM gene of Synechococcus PCC7002 encodes a polypeptide that is essential for carboxysome assembly and therefore for proper functioning of the cyanobacterial CCM.