Rampant horizontal transfer and duplication of rubisco genes in eubacteria and plastids

Previous work has shown that molecular phylogenies of plastids, cyanobacteria, and proteobacteria based on the rubisco (ribulose-1,5- bisphosphate carboxylase/oxygenase) genes rbcL and rbcS are incongruent with molecular phylogenies based on other genes and are also incompatible with structural and biochemical information. Although it has been much speculated that this is the consequence of a single horizontal gene transfer (of a proteobacterial or mitochondrial rubisco operon into plastids of rhodophytic and chromophytic algae), neither this hypothesis nor the alternative hypothesis of ancient gene duplication have been examined in detail. We have conducted phylogenetic analyses of all available bacterial rbcL sequences, and representative plastid sequences, in order to explore these alternative hypothesis and fully examine the complexity of rubisco gene evolution. The rbcL phylogeny reveals a surprising number of gene relationships that are fundamentally incongruent with organismal relationships as inferred from multiple lines of other molecular evidence. On the order of six horizontal gene transfers are implied by the form I (L8S8) rbcL phylogeny, two between cyanobacteria and proteobacteria, one between proteobacteria and plastids, and three within proteobacteria. Alternatively, a single ancient duplication of the form I rubisco operon, followed by repeated and pervasive differential loss of one operon or the other, would account for much of this incongruity. In all probability, the rubisco operon has undergone multiple events of both horizontal gene transfer and gene duplication in different lineages.      

The Monophyletic Origin of the Peridinin‐, and Fucoxanthin‐Containing Dinoflagellate Plastid Through Tertiary Replacement

The dinoflagellates contain diverse plastids of uncertain origin. To determine the origin of the peridinin‐ and fucoxanthin‐containing dinoflagellate plastid, we sequenced the plastid‐encoded psaA, psbA, and rbcL genes from various red and dinoflagellate algae. The psbA gene phylogeny, which was made from a dataset of 15 dinoflagellates, 22 rhodophytes, five cryptophytes, seven haptophytes, seven stramenopiles, two chlorophytes, and a glaucophyte as the outgroup, supports monophyly of the peridinin‐, and fucoxanthin‐containing dinoflagellates, as a sister group to the haptophytes. The monophyletic relationship with the haptophytes is recovered in the psbA + psaA phylogeny, with stronger support. The rubisco tree utilized the ‘Form I’ red algal type of rbcL and included fucoxanthin‐containing dinoflagellates. The dinoflagellate + haptophyte sister relationship is also recovered in this analysis. Peridinium foliaceum is shown to group with the diatoms in all the phylogenies. Based on our analyses of plastid sequences, we postulate that: (1) the plastid of peridinin‐, and fucoxanthin‐containing dinoflagellates originated from a common ancestor; (2) the ancestral dinoflagellate acquired its plastid from a haptophyte though a tertiary plastid replacement; (3) ‘Form II’ rubisco replaced the ancestral rbcL after the divergence of the peridinin‐, and fucoxanthin‐containing dinoflagellates; and (4) we confirm that the plastid of P. foliaceum originated from a Stramenopiles endosymbiont.     

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.     

Photometric method for routine determination of kcat and carbamylation of rubisco

Ribulose bisphosphate carboxylase (rubisco) is the first enzyme in photosynthetic CO₂ assimilation. It is also the single largest sink for nitrogen in plants. Several parameters of rubisco activity are often measured including initial activity upon extraction, degree of carbamylation, catalytic constant of the enzyme (k_cat), and the total amount of enzyme present in a leaf. We report here improvements of the photometric assay of rubisco in which rubisco activity is coupled to NADH oxidation which is continuously monitored in a photometer. The initial lag usually found in this assay was eliminated by assaying rubisco activity at pH 8.0 instead of 8.2, using a large amount of phosphoglycerate kinase, and adding monovalent cations to the assay buffer. We found that when using the photometric assay, the ratio of activity found initially upon extraction divided by the activity after incubating with CO₂ and Mg²⁺ reflects the degree of carbamylation as determined by ¹⁴carboxyarabinitol bisphosphate/¹²carboxyarabinitol bisphosphate competition. We developed methods for measuring the catalytic constant of rubisco as well as the total amount of enzyme present using the photometric assay and carboxyarabinitol 1,5-bisphosphate. We believe that the photometric assay for activity will prove more useful than the ¹⁴CO₂ assay in many studies.Abbreviations CA1P 2-carboxyarabinitol 1-phosphate - GAP glyceraldehyde 3-phosphate - OD optical density - PGA 3-phosphoglycerate - rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose 1,5-bisphosphate     

Small-angle X-ray study on the structure of ribulose-1,5-bisphosphate carboxylase-oxygenase from Rhodospirillum rubrum

The quaternary structure of ribulose-1,5-bisphosphate carboxylase-oxygenase (rubisco) from Rhodospirillum rubrum, an enzyme consisting of two large subunits, L₂, was investigated by small-angle X-ray scattering. In the presence of HCO ₃ ^- and Mg²⁺, rubisco is in the active state and displays a radius of gyration of 2.96 nm, a maximum diameter of 9.5 nm and a volume of 170 nm³. A model is presented where the subunits are arranged back-to-back, rotated relative to each other by 90°, and shifted by 1.3 nm. Upon inactivation by removal of HCO ₃ ^- and Mg²⁺, the model swells slightly without any distinct changes in configuration. This contrasts with our previous observations with rubisco from Alcaligenes eutrophus, an enzyme composed of small (S) and large (L) subunits, L₈S₈, where inactivation gives rise to substantial changes in configuration.Abbreviations RuBP Ribulose-1,5-bisphosphate - 3-PGA 3-phosphoglyceric acid     

Autotrophic,sulfur-oxidizing actinobacteria in acidic environments

Some novel actinobacteria from geothermal environments were shown to grow autotrophically with sulfur as an energy source. These bacteria have not been formally named and are referred to here as “Acidithiomicrobium” species, as the first of the acidophilic actinobacteria observed to grow on sulfur. They are related to Acidimicrobium ferrooxidans with which they share a capacity for ferrous iron oxidation. Ribulose bisphosphate carboxylase/oxygenase (RuBisCO) is active in CO₂ fixation by Acidimicrobium ferrooxidans, which appears to have acquired its RuBisCO-encoding genes from the proteobacterium Acidithiobacillus ferrooxidans or its ancestor. This lateral transfer of RuBisCO genes between a proteobacterium and an actinobacterium would add to those noted previously among proteobacteria, between proteobacteria and cyanobacteria and between proteobacteria and plastids. “Acidithiomicrobium” has RuBisCO-encoding genes which are most closely related to those of Acidimicrobium ferrooxidans and Acidithiobacillus ferrooxidans, and has additional RuBisCO genes of a different lineage. 16S rRNA gene sequences from “Acidithiomicrobium” species dominated clone banks of the genes extracted from mixed cultures of moderate thermophiles growing on copper sulfide and polymetallic sulfide ores in ore leaching columns.     

Phylogeny of Dinoflagellate Plastid Genes Recently Transferred to the Nucleus Supports a Common Ancestry with Red Algal Plastid Genes

It is generally accepted that peridinin-containing dinoflagellate plastids are derived from red alga, but whether they are secondary plastids equivalent to plastids of stramenopiles, haptophytes, or cryptophytes, or are tertiary plastids derived from one of the other secondary plastids, has not yet been completely resolved. As secondary plastids, plastid gene phylogeny should mirror that of nuclear genes, while incongruence in the two phylogenies should be anticipated if their origin was as tertiary plastids. We have analyzed the phylogeny of plastid-encoded genes from Lingulodinium as well as that of nuclear-encoded dinoflagellate homologues of plastid-encoded genes conserved in all other plastid genome sequences. Our analyses place the dinoflagellate, stramenopile, haptophyte, and cryptophyte plastids firmly in the red algal lineage, and in particular, the close relationship between stramenopile plastid genes and their dinoflagellate nuclear-encoded homologues is consistent with the hypothesis that red algal-type plastids have arisen only once in evolution.     

Origins of plastids and glyceraldehyde-3-phosphate dehydrogenase genes in the green-colored dinoflagellate Lepidodinium chlorophorum

The dinoflagellate Lepidodinium chlorophorum possesses "green" plastids containing chlorophylls a and b (Chl a+b), unlike most dinoflagellate plastids with Chl a+c plus a carotenoid peridinin (peridinin-containing plastids). In the present study we determined 8 plastid-encoded genes from Lepidodinium to investigate the origin of the Chl a+b-containing dinoflagellate plastids. The plastid-encoded gene phylogeny clearly showed that Lepidodinium plastids were derived from a member of Chlorophyta, consistent with pigment composition. We also isolated three different glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes from Lepidodinium-one encoding the putative cytosolic "GapC" enzyme and the remaining two showing affinities to the "plastid-targeted GapC" genes. In a GAPDH phylogeny, one of the plastid-targeted GapC-like sequences robustly grouped with those of dinoflagellates bearing peridinin-containing plastids, while the other was nested in a clade of the homologues of haptophytes and dinoflagellate genera Karenia and Karlodinium bearing "haptophyte-derived" plastids. Since neither host nor plastid phylogeny suggested an evolutionary connection between Lepidodinium and Karenia/Karlodinium, a lateral transfer of a plastid-targeted GapC gene most likely took place from a haptophyte or a dinoflagellate with haptophyte-derived plastids to Lepidodinium. The plastid-targeted GapC data can be considered as an evidence for the single origin of plastids in haptophytes, cryptophytes, stramenopiles, and alveolates. However, in the light of Lepidodinium GAPDH data, we need to closely examine whether the monophyly of the plastids in the above lineages inferred from plastid-targeted GapC genes truly reflects that of the host lineages.     

Acclimation of rice to changing atmospheric carbon dioxide concentration

Abstract. The effects were studied of season-long (75 and 88d) exposure of rice (Oryza sativa L. cv. IR-30) to a range of atmospheric CO₂ concentrations in outdoor, computer-controlled, environment chambers under natural solar radiation. The CO₂ concentrations were maintained at 160, 250, 330, 500, 660 and 900μmol mol^-1 air. Photosynthesis increased with increasing growth CO₂ concentrations up to 500u.mol moP1, but levelled off at higher CO₂ values. Specific leaf area also increased significantly with increasing CO₂. Although leaf dry weight and leaf area index increased, the overall response was not statistically significant. Leaf nitrogen content dropped slightly with elevated CO₂, but the response was not statistically significant. The specific activity of ribulose bisphosphate carboxylase/oxygenase (rubisco) declined significantly over the CO₂ concentration range 160 to 900μmol mol^-1. When expressed on a leaf area basis, rubisco activity decreased by 66%. This was accompanied by a 32% decrease in the amount of rubisco protein as a fraction of the total soluble leaf protein, and by 60% on a leaf area basis. For leaves in the dark, the total rubisco activity (CO₂/Mg²⁺-activated) was reduced by more than 60%. This indicates that rice accumulated an inhibitor in the dark, probably 2-car-boxyarabinitol 1-phosphate (CA-1-P). However, the inhibitor did not seem to be involved in the acclimation response. The degree of carbamylation of the rubisco enzyme was unchanged by the CO2 growth regime, except at 900 [μmol mol^-1 where it was reduced by 24%. The acclimation of rice to different atmospheric CO₂ conditions involved the modulation of both the activity and amount of rubisco protein in the leaf.     

Activation of Ribulosebisphosphate Carboxylase/Oxygenase at Physiological CO(2) and Ribulosebisphosphate Concentrations by Rubisco Activase

The enzyme-catalyzed activation of ribulosebisphosphate carboxylase/oxygenase (rubisco) was investigated in an illuminated reconstituted system containing thylakoid membranes, rubisco, ribulosebisphosphate (RuBP), MgCl₂, carbonic anhydrase, catalase, the artificial electron acceptor pyocyanine, and partially purified rubisco activase. Optimal conditions for light-induced rubisco activation were found to include 100 micrograms per milliliter rubisco, 300 micrograms per milliliter rubisco activase, 3 millimolar RuBP, and 6 millimolar free Mg²⁺ at pH 8.2. The half-time for rubisco activation was 2 minutes, and was 4 minutes for rubisco deactivation. The rate of rubisco deactivation was identical in the presence and absence of activase. The K_act(CO₂) of rubisco activation in the reconstituted system was 4 micromolar CO₂, compared to a K_act(CO₂) of 25 to 30 micromolar CO₂ for the previously reported spontaneous CO₂/Mg²⁺ activation mechanism. The activation process characterized here explains the high degree of rubisco activation at the physiological concentrations of 10 micromolar CO₂ and 2 to 4 millimolar RuBP found in intact leaves, conditions which lead to almost complete deactivation of rubisco in vitro.     

Catalysis of Ribulosebisphosphate Carboxylase/Oxygenase Activation by the Product of a Rubisco Activase cDNA Clone Expressed in Escherichia coli

Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) activase activity was obtained from a partially purified extract of Escherichia coli transformed with a 1.6-kilobase spinach (Spinacia oleracea L.) cDNA clone. This activity was ATP-dependent. Catalysis of rubisco activation by spinach and cloned rubisco activase was accompanied by the same extent of carboxyarabinitol bisphosphate-trapped ¹⁴CO₂ as occurred in spontaneous activation, indicating that rubisco carbamylation is one facet of the rubisco activase reaction. The CO₂ concentration required for one-half maximal rubisco activase activity was about 8 micromolar CO₂. These observations are consistent with the postulated role of rubisco activase in regulating rubisco activity in vivo.     

Ribulose-1,5-bisphosphate carboxylase/oxygenase activase protein prevents the in vitro decline in activity of ribulose-1,5-bisphosphate carboxylase/oxygenase

The rate of CO₂ fixation by ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) following addition of ribulose 1,5-bisphosphate (RuBP) to fully activated enzyme, declined with first-order kinetics, resulting in 50% loss of rubisco activity after 10 to 12 minutes. This in vitro decline in rubisco activity, termed fall-over, was prevented if purified rubisco activase protein and ATP were added, allowing linear rates of CO₂ fixation for up to 20 minutes. Rubisco activase could also stimulate rubisco activity if added after fallover had occurred. Gel filtration of the RuBP-rubisco complex to remove unbound RuBP allowed full activation of the enzyme, but the inhibition of activated rubisco during fallover was only partially reversed by gel filtration. Addition of alkaline phosphatase completely restored rubisco activity following fallover. The results suggest that fallover is not caused by binding of RuBP to decarbamylated enzyme, but results from binding of a phosphorylated inhibitor to the active site of rubisco. The inhibitor may be a contaminant in preparations of RuBP or may be formed on the active site but is apparently removed from the enzyme in the presence of the rubisco activase protein.     

Concentrations of dimethylsulphoniopropionate and activities of dimethylsulphide-producing enzymes in batch cultures of nine dinoflagellate species

Dinoflagellates are recognised as one of the major phytoplankton groups that produce dimethylsulphoniopropionate (DMSP), the precursor of the marine trace gas dimethylsulphide (DMS) which has climate-cooling potential. To improve the prospects for including dinoflagellates in global climate models that include DMSP-related processes, we increased the data base for this group by measuring DMSP, DMS-producing enzyme activity (DPEA), carbon, nitrogen and Chl a in nine clonal dinoflagellate cultures (1 heterotrophic and 8 phototrophic strains). Growth rates ranged from 0.11 to 1.92?day^?1 with the highest value being for the heterotroph Crypthecodinium cohnii. Overall, we observed two orders of magnitude variability in DMSP content (11–364?mM) and detected DPEA in five of the nine strains (0.61–59.73?fmol?cell^?1?h^?1). Cell volume varied between 454 and 18,439?μm³ and whilst C and N content were proportional to the cell volume, DMSP content was not. The first DMSP measurements for a dinoflagellate from Antarctic waters and a species with diatom-like plastids are included. Lower DMSP concentrations were found in three small athecate species and a dinoflagellate with haptophyte-like plastids. The highest concentrations and production rates tended to be in globally distributed dinoflagellates and the heterotroph. Photosynthetic species that are distributed in temperate to tropical waters showed low DMSP concentrations and production rates and the polar representative showed moderate concentration and a low production rate. Estuarine species had the lowest concentrations and production rates. These data should help refine the inclusion of dinoflagellates as a functional group in future global climate models.     

Genome fragmentation is not confined to the peridinin plastid in dinoflagellates

When plastids are transferred between eukaryote lineages through series of endosymbiosis, their environment changes dramatically. Comparison of dinoflagellate plastids that originated from different algal groups has revealed convergent evolution, suggesting that the host environment mainly influences the evolution of the newly acquired organelle. Recently the genome from the anomalously pigmented dinoflagellate Karlodinium veneficum plastid was uncovered as a conventional chromosome. To determine if this haptophyte-derived plastid contains additional chromosomal fragments that resemble the mini-circles of the peridin-containing plastids, we have investigated its genome by in-depth sequencing using 454 pyrosequencing technology, PCR and clone library analysis. Sequence analyses show several genes with significantly higher copy numbers than present in the chromosome. These genes are most likely extrachromosomal fragments, and the ones with highest copy numbers include genes encoding the chaperone DnaK(Hsp70), the rubisco large subunit (rbcL), and two tRNAs (trnE and trnM). In addition, some photosystem genes such as psaB, psaA, psbB and psbD are overrepresented. Most of the dnaK and rbcL sequences are found as shortened or fragmented gene sequences, typically missing the 3'-terminal portion. Both dnaK and rbcL are associated with a common sequence element consisting of about 120 bp of highly conserved AT-rich sequence followed by a trnE gene, possibly serving as a control region. Decatenation assays and Southern blot analysis indicate that the extrachromosomal plastid sequences do not have the same organization or lengths as the minicircles of the peridinin dinoflagellates. The fragmentation of the haptophyte-derived plastid genome K. veneficum suggests that it is likely a sign of a host-driven process shaping the plastid genomes of dinoflagellates.     

Fates of Evolutionarily Distinct,Plastid‐type Glyceraldehyde 3‐phosphate Dehydrogenase Genes in Kareniacean Dinoflagellates

The ancestral kareniacean dinoflagellate has undergone tertiary endosymbiosis, in which the original plastid is replaced by a haptophyte endosymbiont. During this plastid replacement, the endosymbiont genes were most likely flowed into the host dinoflagellate genome (endosymbiotic gene transfer or EGT). Such EGT may have generated the redundancy of functionally homologous genes in the host genome—one has resided in the host genome prior to the haptophyte endosymbiosis, while the other transferred from the endosymbiont genome. However, it remains to be well understood how evolutionarily distinct but functionally homologous genes were dealt in the dinoflagellate genomes bearing haptophyte‐derived plastids. To model the gene evolution after EGT in plastid replacement, we here compared the characteristics of the two evolutionally distinct genes encoding plastid‐type glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) in Karenia brevis and K. mikimotoi bearing haptophyte‐derived tertiary plastids: “gapC1h” acquired from the haptophyte endosymbiont and “gapC1p” inherited from the ancestral dinoflagellate. Our experiments consistently and clearly demonstrated that, in the two species examined, the principal plastid‐type GAPDH is encoded by gapC1h rather than gapC1p. We here propose an evolutionary scheme resolving the EGT‐derived redundancy of genes involved in plastid function and maintenance in the nuclear genomes of dinoflagellates that have undergone plastid replacements. Although K. brevis and K. mikimotoi are closely related to each other, the statuses of the two evolutionarily distinct gapC1 genes in the two Karenia species correspond to different steps in the proposed scheme.     

Plastid evolution: gene transfer and the maintenance of 'stolen' organelles

Many heterotrophic organisms sequester plastids from prey algae and temporarily utilize their photosynthetic capacity. A recent article in BMC Genomics reveals that the dinoflagellate Dinophysis acuminata has acquired photosynthesis-related genes by horizontal gene transfer, which might explain its ability to retain 'stolen' plastids for extended periods of time.     

DOES DAPI DETECT CYTOPLASMIC DNA?

Ultrastructural observations have revealed that plastids are present in orchid pollen tubes, but the DNA-binding fluorochrome 4',6-diamidino-2-phenylindole (DAPI) does not localize any DNA in the pollen tube plastids at optimum binding and flourescence conditions. However, the plastids do contain DNA since the gene coding for the large subunit of rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase, rbcL) has been amplified by the polymerase chain reaction from orchid pollen tubes. It is therefore concluded that DAPI is an unreliable fluorochrome for detecting plastid DNA.     

Regulation of Ribulose-1,5-Bisphosphate Carboxylase Activity in Response to Changing Partial Pressure of O(2) and Light in Phaseolus vulgaris

The regulation of ribulose-1,5-bisphosphate (RuBP) carboxylase (rubisco) activity in Phaseolus vulgaris was studied under moderate CO₂ and high light, conditions in which photosynthesis in C₃ plants can be insensitive to changes in O₂ partial pressure. Steady state RuBP concentrations were higher, the calculated rate of RuBP use was lower and the activation state of rubisco was lower in low O₂ relative to values observed in normal O₂. It is suggested that the reduced activity of rubisco observed here is related to feedback effects which occur when the rate of net CO₂ assimilation approaches the maximum capacity for starch and sucrose synthesis (triose phosphate utilization). The activation state of rubisco was independent of O₂ partial pressure when light or CO₂ was limiting for photosynthesis. Reduced activity of rubisco was also observed at limiting light. However, in this species light dependent changes in the concentration of an inhibitor of rubisco controlled the apparent V_max of rubisco in low light while changes in the CO₂-Mg²⁺ dependent activation of rubisco controlled the apparent V_max in high light.