Identification and Structural Analysis of a Novel Carboxysome Shell Protein with Implications for Metabolite Transport

Bacterial microcompartments (BMCs) are polyhedral bodies, composed entirely of proteins, that function as organelles in bacteria; they promote subcellular processes by encapsulating and co-localizing targeted enzymes with their substrates. The best-characterized BMC is the carboxysome, a central part of the carbon-concentrating mechanism that greatly enhances carbon fixation in cyanobacteria and some chemoautotrophs. Here we report the first structural insights into the carboxysome of Prochlorococcus, the numerically dominant cyanobacterium in the world's oligotrophic oceans. Bioinformatic methods, substantiated by analysis of gene expression data, were used to identify a new carboxysome shell component, CsoS1D, in the genome of Prochlorococcus strain MED4; orthologs were subsequently found in all cyanobacteria. Two independent crystal structures of Prochlorococcus MED4 CsoS1D reveal three features not seen in any BMC-domain protein structure solved to date. First, CsoS1D is composed of a fused pair of BMC domains. Second, this double-domain protein trimerizes to form a novel pseudohexameric building block for incorporation into the carboxysome shell, and the trimers further dimerize, forming a two-tiered shell building block. Third, and most strikingly, the large pore formed at the 3-fold axis of symmetry appears to be gated. Each dimer of trimers contains one trimer with an open pore and one whose pore is obstructed due to side-chain conformations of two residues that are invariant among all CsoS1D orthologs. This is the first evidence of the potential for gated transport across the carboxysome shell and reveals a new type of building block for BMC shells.     

Organization, Structure, and Assembly of α-Carboxysomes Determined by Electron Cryotomography of Intact Cells

Carboxysomes are polyhedral inclusion bodies that play a key role in autotrophic metabolism in many bacteria. Using electron cryotomography, we examined carboxysomes in their native states within intact cells of three chemolithoautotrophic bacteria. We found that carboxysomes generally cluster into distinct groups within the cytoplasm, often in the immediate vicinity of polyphosphate granules, and a regular lattice of density frequently connects granules to nearby carboxysomes. Small granular bodies were also seen within carboxysomes. These observations suggest a functional relationship between carboxysomes and polyphosphate granules. Carboxysomes exhibited greater size, shape, and compositional variability in cells than in purified preparations. Finally, we observed carboxysomes in various stages of assembly, as well as filamentous structures that we attribute to misassembled shell protein. Surprisingly, no more than one partial carboxysome was ever observed per cell. Based on these observations, we propose a model for carboxysome assembly in which the shell and the internal RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) lattice form simultaneously, likely guided by specific interactions between shell proteins and RuBisCOs.     

A Non-radioisotopic Anion-exchange Chromatographic Method to Measure the CO2/O2 Specificity Factor for Ribulose Bisphosphate Carboxylase/Oxygenase

A non-radioisotopic anion-exchange ion chromatographic method for measuring the carboxylation/ oxygenation specificity (τ) of ribulose 1, 5-bisphosphate carboxylase/oxygenase (RubisCO) is presented. The assay measures the amounts of fixation products at varying [CO₂]/[O₂] ratios to measure the relative rates of CO₂ and O₂ fixation reactions. The amount of 3-phosphoglycerate (3-PGA) and phosphoglycolate (PG) in the reaction mixture were measured with a conductivity detector and the specific factor was calculated using the following equations: ν_c = ([3-PGA] – [PG])/2 and ν_o = [PG]. By this method, specificity factors for RubisCOs were measured without using radioactive reagents.     

Cloning and expression of the d-ribulose-1,5-bisphosphate carboxylase/oxygenase form II gene from Thiobacillus intermedius in Escherichia coli

Abstract Both form I and II ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) genes were detected in Thiobacillus intermedius by heterologous hybridization using specific probes from Anacystis nidulans and Rhodobacter sphaeroides , respectively. However, only the previously reported from I enzyme could be demonstrated in cells grown under a number of different conditions. The reason(s) why the form II gene is not expressed in T. intermedius is/are not clear at this time. The form II gene was isolated from a lambda library by screening with the Rb. sphaeroides probe. A Sal I fragment from this clone was ligated into pUC8 and transformed into Escherichia coli DH5α. Subclones pTi20IIA and pTi20IIB representing both orientations relative to the lac promoter were isolated. Low levels of RuBisCO activity were detected in both induced and non-induced pTi20IIA indicating the probable expression from a T. intermedius promoter. Induced pTi20IIB produced much higher levels of enzyme activity. Analysis of cell-free extracts using sucrose density gradients confirmed the expression of a form II RuBisCO similar in size to that found in Rhodobacter capsulatus . Other Calvin cycle genes were not clustered with either the form I or form II genes.     

Variation in photosynthetic electron transport capacity in vivo and its effects on the light modulation of ribulose bisphosphate carboxylase

Photosynthetic electron transport capacity was varied in vivo in sugar beets using iron deficiency, and its effects on the light modulation of ribulose bisphosphate carboxylase (RuBPCase) studied. Three treatment groups corresponding to decreasing amounts of thylakoids per leaf area were examined: iron sufficient (control), moderately iron-stressed, and severely iron-stressed. Reduction in electron transport capacity in vivo was correlated with a substantial decrease in the level of RuBPCase activation, even at saturating irradiances. These results indicate a direct relationship between RuBPCase activation and photosynthetic electron transport. In addition, our data suggest that the activation of RuBPCase could not solely account for the increases in the photosynthetic rate at high irradiances; RuBPCase reached maximal activation at irradiances well below light saturation for net photosynthesis.Abbreviations Chl chlorophyll - FeCN ferricyanide - FBPase fructose 1,6-bisphosphatase - RuBP ribulose 1,5-bisphosphate - RuBPCase ribulose 1,5-bisphosphate carboxylase - SBPase sedoheptulose 1,7-bisphosphatase     

A mechanistic model for photosynthesis based on the multisubstrate ordered reaction of ribulose 1,5 bisphosphate carboxylase

Abstract. A mechanistic model of photosynthesis is developed based on the characteristics of ribulose 1,5-bisphosphate (RuBP) carboxylase and the assimilation of CO₂ as an ordered reaction with RuBP binding before CO₂. An equation is derived which considers the effects of light (for regeneration of RuBP) and CO₂. Taking values for the maximum turnover of RuBP carboxylase at substrate saturation, the maximum carboxylation efficiency (maximum increase in rate per increase in CO₂ concentration) and the minimum quantum requirement for the C₃ pathway, photosynthesis in the absence of photorespiration is simulated. In the model, at varying concentrations of CO₂, the efficiency of light utilization approaches a maximum value as photon flux density decreases. Similarly, with a given maximum carboxyation capacity, at varying photon flux densities the carboxylation efficiency approaches a constant maximum value (equal to V _max/ K _{m CO2}) as CO₂ is decreased. However, a decrease in the state of activation of RuBP carboxylase under low light results in a lower carboxylation efficiency. Limits on the rate of photosynthesis, as the maximum capacity for regeneration of RuBP is reduced relative to carboxylation potential, or as the maximum capacity of the carboxylase varies, are considered.     

HIGH LOCALIZATION OF RIBULOSE-1,5-BISPHOSPHATE CARBOXULASE/OXYGENASE IN THE PYRENOIDS OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA), AS REVEALED BY CRYOFIXATION AND IMMUNOGOLD ELECTRON MICROSCOPY

The distribution of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the chloroplasts of the unicellular green alga Chlamydomonas reinhardtii Dangeard was examined using cryotechnique and conventional fixation for immunogold electron microscopy. Both methods provided essentially identical results, although somewhat higher densities of gold particles indicating Rubisco molecules were recognized in the pyrenoids of cryofixed cells. The gold particles were highly concentrated in the pyrenoid matrix within the chloroplasts. Even when considering the vast difference in volume between the pyrenoid and the rest of the Chloroplast, more than 99% of the total Rubisco labeling in the chloroplast was calculated to be present in the pyrenoid matrix. High localization of Rubisco in the pyrenoid matrix was also recognized regardless of cell age, based on immunofluorescence microscopy of the same en bloc samples. These results are inconsistent with a recent immunocytochemical study employing cryotechnique in which more than 90% of the total Rubisco was recognized in the thylakoid region (thylakoid membranes and stroma) of C. reinhardtii cells. Rubisco highly localized in the pyrenoid matrix may take part in active photosynthetic CO₂ fixation and/or the CO₂ concentrating mechanism .     

Phylogeny of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Genes in Haloalkaliphilic Obligately Autotrophic Sulfur-Oxidizing Bacteria of the Genus <Emphasis Type="Italic">Thioalkalivibrio</Emphasis>

Fragments of genes of the “green-like” form I ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) of eight species of haloalkaliphilic obligately autotrophic sulfur-oxidizing bacteria of the genus Thioalkalivibrio have been revealed and sequenced using previously developed oligonucleotide primers. The data obtained are used for the construction of phylogenetic trees on the basis of nucleotide sequences of RuBisCO genes and their conceptual translations into amino acid sequences. Comparative analysis of the 16S rRNA and RuBisCO gene trees reveals discrepancies between their topologies. According to a RuBisCO gene analysis, the genus Thioalkalivibrio is not monophyletic, and its inner divergence conforms to the significant morphological differences observed between the species. Presumably, horizontal (interspecies) gene transfer was involved in the evolution of the genus Thioalkalivibrio.__________Translated from Mikrobiologiya, Vol. 74, No. 3, 2005, pp. 378–386.Original Russian Text Copyright © 2005 by Tourova, Spiridonova, Berg, Kuznetsov, Sorokin.     

外源海藻糖对PEG渗透胁迫下小麦Rubisco及其活化酶的影响

以抗旱品种‘晋麦47’和干旱敏感品种‘郑引1号’为材料,通过室内水培试验研究了外源海藻糖对PEG渗透胁迫下小麦叶片净光合速率、1,5-二磷酸核酮糖羧化酶/加氧酶(Rubisco)和1,5-二磷酸核酮糖羧化酶/加氧酶活化酶(RCA)含量和相关基因表达特性的影响。结果表明:(1)外源海藻糖和渗透胁迫均能显著增加2个小麦品种叶片海藻糖含量。(2)渗透胁迫显著降低了2个品种小麦叶片的净光合速率,而外源海藻糖能显著缓解受胁迫小麦叶片净光合速率的降低幅度。(3)渗透胁迫仅使‘郑引1号’Rubisco大亚基基因(rbcL)相对表达量及相应蛋白含量显著降低;渗透胁迫显著降低了小麦RCAα和β亚基基因相对表达量,并显著降低RCA蛋白含量,而外源海藻糖不能缓解RCA蛋白含量的降低;渗透胁迫显著降低了Rubisco总活性、初始活性、活化状态及RCA活性,而外源海藻糖则能显著缓解上述酶活性的下降。(4)小麦叶片净光合速率与其rbcL、RCAα和β亚基基因相对表达量及Rubisco总活性、初始活性、活化状态及RCA活性均呈极显著正相关关系。研究发现,在渗透胁迫条件下,外源海藻糖主要从翻译后层面对小麦叶片Rubisco和RCA的活性发挥显著保护作用,从而缓解了小麦净光合速率的降低。     

RuBPCO kinetics and the mechanism of CO2 entry in C3 plants

Abstract. The CO₂ partial pressure in the chloroplasts of intact photosynthetic C₃ leaves is thought to be less than the intercellular CO₂ partial pressure. The intercellular CO₂ partial pressure can be calculated from CO₂ and H₂O gas exchange measurements, whereas the CO₂ partial pressure in the chloroplasts is unknown. The conductance of CO₂ from the intercellular space to the chloroplast stroma and the CO₂ partial pressure in the chloroplast stroma can be calculated if the properties of photosynthetic gas exchange are compared with the kinetics of the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPCO). A discrepancy between gas exchange and RuBPCO kinetics can be attributed to a deviation of CO₂ partial pressure in the chloroplast stroma from that calculated in the intercellular space. This paper is concerned with the following: estimation of the kinetic constants of RuBPCO and their comparison with the CO₂ compensation concentration; their comparison with differential uptake of ¹⁴CO₂ and ¹²CO₂; and their comparison with O₂ dependence of net CO₂ uptake of photosynthetic leaves. Discrepancy between RuBPCO kinetics and gas exchange was found at a temperature of 12.5 °C, a photosynthetic photon flux density (PPFD) of 550 μmol quanta m^?2 s^?1, and an ambient CO₂ partial pressure of 40 Pa. Consistency between RuBPCO kinetics and gas exchange was found if CO₂ partial pressure was decreased, temperature incresed and PPFD decreased. The results suggest that a discrepancy between RuBPCO kinetics and gas exchange is due to a diffusion resistance for CO₂ across the chloroplast envelope which decreases with increasing temperature. At low CO₂ partial pressure, the diffusion resistance appears to be counterbalanced by active CO₂ (or HCO₃) transport with high affinity and low maximum velocity. At low PPFD, CO₂ partial pressure in the chloroplast stroma appears to be in equilibrium with that in the intercellular space due to low CO₂ flux.     

Absence of multiple forms of ribulose 15-bisphosphate carboxylase/oxygenase in mung bean

Ribulose 1,5-bisphosphate carboxylase/oxygenase has been reported to occur in multiple forms in mung bean (Phaseolus aureus) using Sephadex G-200 chromatography. We have isolated this enzyme by identical methodology. The profile from Sephadex G-200 chromatography shows only one peak in contrast to the previous report and we find no evidence to corroborate the conclusions. Where V_c, V_o and K_c, K_o represent V_max and Michaelis constants, respectively, the constant V_cK_o/V_oK_c for the single form is 70 at 40 μM CO₂ and 1200 μM O₂.     

Growth of Collagen Fibril Seeds from Embryonic Tendon: Fractured Fibril Ends Nucleate New Tip Growth

Collagen fibrils are the principal tensile element of vertebrate tissues where they occur in the extracellular matrix as spatially organised arrays. A major challenge is to understand how the mechanisms of nucleation, growth and remodelling yield fibrils of tissue-specific diameter and length. Here we have developed a seeding system whereby collagen fibrils were isolated from avian embryonic tendon and added to purified collagen solution, in order to characterise fibril surface nucleation and growth mechanisms. Fragmentation of tendon in liquid nitrogen followed by Dounce homogenisation generated fibril length fragments. Most (> 94%) of the fractured ends of fibrils, which show an abrupt square profile, were found to act as nucleation sites for further growth by molecular accretion. The mechanism of this nucleation and growth process was investigated by transmission electron microscopy, atomic force microscopy and scanning transmission electron microscopy mass mapping. Typically, a single growth spur occurred on the N-terminal end of seed fibrils whilst twin spurs frequently formed on the C-terminal end before merging into a single tip projection. The surface nucleation and growth process generated a smoothly tapered tip that achieved maximum diameter when the axial extension reached ∼ 13 μm. Lateral growth also occurred along the entire length of all seed fibrils that contained tip projections. The data support a model of collagen fibril growth in which the broken ends of fibrils are nucleation sites for propagation in opposite axial directions. The observed fibril growth behaviour has direct relevance to tendon matrix remodelling and repair processes that might involve rupture of collagen fibrils.     

Responses of Penultimate and Flag Leaves of Wheat to Different Nitrogen Supply

Uniculm wheat (Triticum aestivum L.) was grown to maturity at four concentrations of nitrogen corresponding to 3 (N1), 6 (N2), 9 (N3) and 12 (N4) g m^–2. Penultimate and flag leaves were examined throughout the ontogeny. Sub-optimal concentrations of N resulted in sharp decline in both area and dry mass of the leaves. Decline in leaf area was due to fewer mesophyll cells. Net photosynthetic rate (P_N) increased up to full expansion, remained constant for about a week and then declined. P_N, nitrogen, ribulose-1,5-bis-phosphate carboxylase/oxygenase (RuBPCO) amount and activity, chlorophyll and soluble protein contents were similar at all the N concentrations. Both amount and activity of RuBPCO in the flag leaf were about two fold higher as compared to penultimate leaf, but P_N was similar. This indicates the presence of an excess amount of RuBPCO in the flag leaf.