F. Robert Tabita

Synthesis of Catalytically Active Form III Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase in Archaea

Ribulose 1,5 bisphosphate carboxylase/oxygenase (RubisCO) catalyzes the biological reduction and assimilation of carbon dioxide gas to organic carbon; it is the key enzyme responsible for the bulk of organic matter found on earth. Until recently it was believed that there are only two forms of RubisCO, form I and form II. However, the recent completion of several genome-sequencing projects uncovered open reading frames resembling RubisCO in the third domain of life, the archaea. Previous work and homology comparisons suggest that these enzymes represent a third form of RubisCO, form III. While earlier work indicated that two structurally distinct recombinant archaeal RubisCO proteins catalyzed bona fide RubisCO reactions, it was not established that the rbcL genes of anaerobic archaea can be transcribed and translated to an active enzyme in the native organisms. In this report, it is shown not only that Methanococcus jannaschii, Archaeoglobus fulgidus, Methanosarcina acetivorans, and Methanosarcina barkeri possess open reading frames with the residues required for catalysis but also that the RubisCO protein from these archaea accumulates in an active form under normal growth conditions. In addition, the form III RubisCO gene (rbcL) from M. acetivorans was shown to complement RubisCO deletion strains of Rhodobacter capsulatus and Rhodobacter sphaeroides under both photoheterotrophic and photoautotrophic growth conditions. These studies thus indicate for the first time that archaeal form III RubisCO functions in a physiologically significant fashion to fix CO(2). Furthermore, recombinant M. jannaschii, M. acetivorans, and A. fulgidus RubisCO possess unique properties with respect to quaternary structure, temperature optima, and activity in the presence of molecular oxygen compared to the previously described Thermococcus kodakaraensis and halophile proteins.

Modified Pathway To Synthesize Ribulose 1,5-Bisphosphate in Methanogenic Archaea

Several sequencing projects unexpectedly uncovered the presence of genes that encode ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RubisCO) in anaerobic archaea. RubisCO is the key enzyme of the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway, a scheme that does not appear to contribute greatly, if at all, to net CO2 assimilation in these organisms. Recombinant forms of the archaeal enzymes do, however, catalyze a bona fide RuBP-dependent CO2 fixation reaction, and it was recently shown that Methanocaldococcus (Methanococcus) jannaschii and other anaerobic archaea synthesize catalytically active RubisCO in vivo. To complete the CBB pathway, there is a need for an enzyme, i.e., phosphoribulokinase (PRK), to catalyze the formation of RuBP, the substrate for the RubisCO reaction. Homology searches, as well as direct enzymatic assays with M. jannaschii, failed to reveal the presence of PRK. The apparent lack of PRK raised the possibility that either there is an alternative pathway to generate RuBP or RubisCO might use an alternative substrate in vivo. In the present study, direct enzymatic assays performed with alternative substrates and extracts of M. jannsachii provided evidence for a previously uncharacterized pathway for RuBP synthesis from 5-phospho-D-ribose-1-pyrophosphate (PRPP) in M. jannaschii and other methanogenic archaea. Proteins and genes involved in the catalytic conversion of PRPP to RuBP were identified in M. jannaschii (Mj0601) and Methanosarcina acetivorans (Ma2851), and recombinant Ma2851 was active in extracts of Escherichia coli. Thus, in this work we identified a novel means to synthesize the CO2 acceptor and substrate for RubisCO in the absence of a detectable kinase, such as PRK. We suggest that the conversion of PRPP to RuBP might be an evolutional link between purine recycling pathways and the CBB scheme.

Nitrogen and ammonia assimilation in the cyanobacteria: regulation of glutamine synthetase.

Abstract Glutamine synthetase, the first enzyme of the ammonia assimilatory pathway, has been purified from Anabaena sp. CA by use of established procedures and by affinity chromatography as a final step. No adenylylation system controlling glutamine synthetase activity was found. The enzyme shows a marked specificity for Mg 2+ in the biosynthetic assay and Mn 2+ in the transferase assay. Under physiological conditions, Co 2+ produces a large stimulatory effect on the Mg 2+ -dependent biosynthetic activity. The enzyme is inhibited by the feedback modifiers l -alanine, glycine, l -serine, l -aspartate, and 5′-AMP. Inhibition by l -serine and l -aspartate is linear, noncompetitive with respect to l -glutamate with apparent K i values of 3 and 13 m m , respectively. Cumulative inhibition is seen with mixtures of l -serine, l -aspartate, and 5′-AMP. The results indicate that, in vivo , divalent cation availability and the presence of feedback inhibitors may play the dominant role in regulating glutamine synthetase activity and hence ammonia assimilation in nitrogen-fixing cyanobacteria.

Isolation and characterization of a marine Anabaena sp. capable of rapid growth on molecular nitrogen

A marine filamentous cyanobacterium capable of rapid growth under N2-fixing conditions has been isolated from the Texas Gulf Coast. This organism appears to be an Anabaena sp. and has been given the strain designation CA. Cultures grown on mineral salts medium bubbled with 1% CO2-enriched air at 42°C show a growth rate of 5.6±0.1 generations per day with molecular nitrogen as the sole nitrogen source. This growth rate is higher than any other reported in the literature to date for heterocystous cyanobacteria growing on N2. Under similar growth conditions, 7.5 mM NH4Cl yields a growth rate of 6.6±0.1 generations per day while 7.5 mM KNO3 allows for a growth rate of 5.8±0.4 generations-day. Nitrogen-fixation rates, as measured by acetylene reduction, show maximum activity values in the range of 50–100 nmoles ethylene produced/minxmg protein. These values compare favorably with those obtained from heterotrophic bacteria and are much higher than values reported for other cyanobacteria. Growth experiments indicate that the organism requires relatively high levels of sodium and grows maximally at 42°C. Because of its high growth rate on N2, this newly isolated organism appears ideal for studying nitrogen metabolism and heterocyst development among the cyanobacteria.

Inhibition of D-ribulose 1,5-bisphosphate carboxylase by pyridoxal 5′-phosphate☆

Abstract Homogeneous D-ribulose 1,5-bisphosphate carboxylase from Rhodospirillum rubrum , Chlamydomonas reinhardtii , and Hydrogenomonas eutropha are inhibited by low concentrations of pyridoxal 5′-phosphate. In the case of the enzyme from Rhodospirillum rubrum , this inhibition is strongly antagonized by the substrate, D-ribulose 1,5-bisphosphate. These results suggest that pyridoxal 5′-phosphate may act close to or at the ribulose 1,5-bisphosphate binding site of the enzyme from Rhodospirillum rubrum .

Facile assay of enzymes unique to the Calvin cycle in intact cells, with special reference to ribulose 1,5-bisphosphate carboxylase☆

Abstract A procedure for the facile measurement, in intact cells, of two enzymes unique to the Calvin cycle, ribulose 1,5-bisphosphate carboxylase and phosphoribulokinase, is described. The procedure involved a simple toluene treatment to render phototrophic cells permeable to the necessary substrates, effectors, and cofactors. Whole-cell ribulose 1,5-bisphosphate carboxylase activity quantitatively approximates the activity obtained in cell-free extracts. In addition, the activity measured with toluene-treated whole cells results in a stoichiometric carboxylation of ribulose 1,5-bisphosphate to phosphoglyceric acid. The assay procedures described are most convenient for determining enzyme levels as a function of growth. Moreover, such an assay should open the way to further studies on the regulation of CO 2 assimilation by direct measurement of the enzymes concerned within the cell.

Effector-Mediated Interaction of CbbRI and CbbRII Regulators with Target Sequences in Rhodobacter capsulatus

In Rhodobacter capsulatus, genes encoding enzymes of the Calvin-Benson-Bassham reductive pentose phosphate pathway are located in the cbb(I) and cbb(II) operons. Each operon contains a divergently transcribed LysR-type transcriptional activator (CbbR(I) and CbbR(II)) that regulates the expression of its cognate cbb promoter in response to an as yet unidentified effector molecule(s). Both CbbR(I) and CbbR(II) were purified, and the ability of a variety of potential effector molecules to induce changes in their DNA binding properties at their target promoters was assessed. The responses of CbbR(I) and CbbR(II) to potential effectors were not identical. In gel mobility shift assays, the affinity of both CbbR(I) and CbbR(II) for their target promoters was enhanced in the presence of ribulose-1,5-bisphosphate (RuBP), phosphoenolpyruvate, 3-phosphoglycerate, 2-phosphoglycolate. ATP, 2-phosphoglycerate, and KH(2)PO(4) were found to enhance only CbbR(I) binding, while fructose-1,6-bisphosphate enhanced the binding of only CbbR(II). The DNase I footprint of CbbR(I) was reduced in the presence of RuBP, while reductions in the CbbR(II) DNase I footprint were induced by fructose-1,6-bisphosphate, 3-phosphoglycerate, and KH(2)PO(4). The current in vitro results plus recent in vivo studies suggest that CbbR-mediated regulation of cbb transcription is controlled by multiple metabolic signals in R. capsulatus. This control reflects not only intracellular levels of Calvin-Benson-Bassham cycle metabolic intermediates but also the fixed (organic) carbon status and energy charge of the cell.

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

The gene encoding the form II ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPC/O) from Rhodopseudomonas (R.) sphaeroides has been identified on a 3-kb EcoRI fragment and cloned into a broad-host-range, high-copy-number plasmid, using the gene from Rhodospirillum (Rs.) rubrum as a hybridization probe. Subclones of the gene from R. sphaeroides in pBR322 and pUC8 show substantial levels of expression and enzymatic activity in whole cells and crude cell extracts of Escherichia coli. This enzymatic activity has been shown to be similar in many respects to that of the protein purified from R. sphaeroides.

Isolation and characterization of heterocysts from Anabaena sp. strain CA

A comparative study has been made on the pigment composition and nitrogenase activity of whole filaments and isolated beterocysts from a mutant strain of Anabaena CA. The whole cell absorption spectra of intact filaments and isolated heterocysts showed close resemblance especially between 550–700 nm region. On a quantitative basis the chlorophyll a content was found almost equal between the vegetative cell and heterocyst but the c-phycocyanin content in the heterocyst was about 1/2 that of the vegetative cell. The purification of the phycobiliprotein on DEAE-cellulose showed the presence of c-phycocyanin (γmax 615 nm) and allophycocyanin (γmax 645 nm, shoulder 620 nm). Isolated heterocysts under H2 showed acetylene reduction rates of 57 nmol C2H4/mg dry wt·min (342 μmol C2H4/mg chl a·h), whereas intact filaments reduced at the rate of 18 nmol C2H4/mg dry wt·min (108 μmol C2H4/mg chl a·h). This rate accounts for 30% recovery of nitrogenase activity in isolated heterocysts compared to whole filaments. The activity was strictly light dependent and was linear under H2 for more than 3 h. Addition of as little as 5% H2 under argon stimulated the C2H2 reductionseveral fold. The acetylene reduction (nitrogenase activity) also showed tolerance to 5% added O2 either under H2 or argon. The results suggest that the heterocyst of Anabaena CA-V is different in some characteristics (viz., higher endogenous C2H2 reduction rate, prolonged activity and higher levels of phycobiliproteins) than those reported in other Anabaena species.

A novel three-protein two-component system provides a regulatory twist on an established circuit to modulate expression of the cbbI region of Rhodopseudomonas palustris CGA010.

A novel two-component system has been identified in the cbb(I) region of the nonsulfur purple photosynthetic bacterium Rhodopseudomonas palustris. Genes encoding this system, here designated cbbRRS, are juxtaposed between the divergently transcribed transcription activator gene, cbbR, and the form I ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) genes, cbbLS. The three genes of the cbbRRS system represent a variation of the well-known two-component signal transduction systems, as there are a transmembrane hybrid sensor kinase and two response regulators, with no apparent DNA binding domain associated with any of the three proteins encoded by these genes. In this study, we showed that the membrane-bound full-length kinase undergoes autophosphorylation and transfers phosphate to both response regulators. A soluble, truncated version of the kinase was subsequently prepared and found to catalyze phosphorylation of response regulator 1 but not response regulator 2, implying that conformational changes and/or sequence-specific regions of the kinase are important for discriminating between the two response regulators. Analyses indicated that a complex network of control of gene expression must occur, with CbbR required for the expression of the cbbLS genes but dispensable for the synthesis of form II RubisCO (encoded by cbbM). The CbbRRS proteins specifically affected the activity and accumulation of form I RubisCO (CbbLS), as revealed by analyses of nonpolar, unmarked gene deletions. A tentative model of regulation suggested that changes in the phosphotransfer activity of the sensor kinase, possibly in response to a redox metabolic signal, cause modulation of the activity and synthesis of form I RubisCO.