Isolation of the Rhodopseudomonas sphaeroides form I ribulose 1,5-bisphosphate carboxylase/oxygenase large and small subunit genes and expression of the active hexadecameric enzyme in Escherichia coli

A library of cloned Rhodopseudomonas sphaeroides DNA was screened by colony hybridization for form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPC/O) sequences using heterologous RuBPC/O probes. A recombinant plasmid was identified that hybridized to both the Anacystis nidulans and the R. sphaeroides form II RuBPC/O genes. Subcloning of a hybridizing 4-kb SmaI fragment allowed expression of active enzyme in Escherichia coli that was identical to form I RuBPC/O based on polyacrylamide gel electrophoresis and Western immunoblot analysis.     

Isolation of plasmid DNA sequences that complement Rhodobacter sphaeroides mutants deficient in the capacity for CO2-dependent growth

Mutants deficient in the proper regulation and derepression of ribulose-1.5-bisphosphate carboxylase oxygenase (RuBPC/O) in Rhodobacter sphaeroides were isolated by ethyl methanesulphonate (EMS) and Tn5 mutagenesis of a recA parental strain. Mutants were identified by their ability to grow under conditions where the organism requires basal levels of RuBPC C/O for growth yet fail to grow under conditions which require derepression of the enzyme (Aut-). The newly isolated Aut- mutants exhibited phenotypes distinguishable from the previously isolated Aut- mutant, strain KW25/11. Rocket immunoelectrophoretic examination of RuBPC/O levels revealed marked variance in the ability of mutants to derepress form I and form II RuBPC/O in the absence of exogenous carbon. Evidence that some of the mutants possessed different mutations was substantiated by complementation of the EMS-generated mutants by entirely different genes isolated from a genomic library of R. sphaeroides constructed in the broad-host-range cosmid vector pVK102. Southern hybridization analysis of the complementing library isolates showed the complementing genes to be normally carried on the endogenous plasmids of R. sphaeroides. The gene complementing mutant strain KW25/11 was mapped by Tn5 insertional inactivation and the complementing region found to reside on a 1.5 kb PstJ. BamHI fragment. Complemented strains were unable to match wild-type levels of RuBPC/O under conditions requiring derepression of the enzyme, except for mutant strain EMS45. The Aut- phenotype, represented by the mutants isolated in this study, stems from a deficiency in some aspect of photoautotrophic growth.     

Oxygen regulation of ribulose 1,5-bisphosphate carboxylase activity in Rhodospirillum rubrum.

The carboxylase activity of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPC/O) decreased when an anaerobic culture of Rhodospirillum rubrum was exposed to atmospheric levels of oxygen. From 70 to 80% of the activity was lost within 12 to 24 h. Inactivation was apparent when the enzyme was assayed in situ (in whole cells) and when activity was measured in dialyzed crude extracts. The quantity of enzyme protein, as estimated from sodium dodecyl sulfate-polyacrylamide gels or as quantified immunologically, did not decrease within 24 h of exposure to air. Following extended exposure to aerobic conditions (48 to 72 h), degradation of enzyme occurred. These results indicate that the inactivation of RuBPC/O in R. rubrum may be due to an alteration or modification of the preformed enzyme, followed by eventual degradation of the inactive enzyme. When shifted back to anaerobic conditions (under an argon atmosphere), the RuBPC/O activity increased rapidly. This increase appeared to be due to de novo synthesis of enzyme. The increase in activity was not observed when the culture was maintained in the dark or in the absence of a suitable carbon source. Thus, the oxygen-mediated inactivation of RuBPC/O appeared to be due to some form of irreversible modification. The cloned R. rubrum RuBPC/O gene, expressed in Escherichia coli, yielded functional enzyme that was not affected by oxygen, indicating that inactivation in R. rubrum is mediated by a gene product(s) not found in E. coli.     

Depression of hydrogenase during limitation of electron donors and derepression of ribulosebisphosphate carboxylase during carbon limitation of Alcaligenes eutrophus

Alcaligenes eutrophus did not form the key enzymes of autotrophic metabolism, the soluble and particulate hydrogenases and ribulosebisphosphate carboxylase (RuBPC), during heterotrophic growth on succinate in batch cultures. During succinate-limited growth in a chemostat, high activities of both hydrogenases were observed. With decreasing dilution rate (D) the steady-state hydrogenase activity (H) followed first-order kinetics, expressed as follows: H = Hmax .e-alpha.D. An identical correlation was observed when autotrophic growth in a chemostat was limited by molecular hydrogen. During autotrophic growth under oxygen or carbon dioxide limitation, the activity if the soluble hydrogenase was low. These data suggested that hydrogenase formation depended on the availability of reducing equivalents to the cells. RuBPC activities were not correlated with the hydrogenase activities. During succinate-limited growth, RuBPC appeared at intermediate activities. During autotrophic growth in a carbon dioxide-limited chemostat, RuBPC was highly derepressed. RuBPC activity was not detected in cells that suffered from energy limitation with a surplus of carbon, as in a heterotrophic oxygen-limited chemostat, nor was it detected in cells limited in carbon and energy, as in the case of complete exhaustion of a heterotrophic substrate. From these data I concluded that RuBPC formation in A. eutrophus depends on two conditions, namely, carbon starvation and an excess of reducing equivalents.     

Organization of phosphoribulokinase and ribulose bisphosphate carboxylase/oxygenase genes in Rhodopseudomonas (Rhodobacter) sphaeroides.

A heterologous phosphoribulokinase (PRK) gene probe was used to analyze two recombinant plasmids isolated from a Rhodopseudomonas (Rhodobacter) sphaeroides gene library. These plasmids were previously shown to carry the genes for form I and form II ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPC/O). Southern blot hybridization analysis indicated that there were two PRK genes linked to the RuBPC/O coding sequences. Restriction mapping showed the arrangement of the duplicate sets of PRK and RuBPC/O to be distinct. Subcloning of the hybridizing PRK sequences downstream of the lac promoter of pUC8 allowed expression of the two PRK enzymes in Escherichia coli. Analysis of the purified proteins by sodium dodecyl sulfate-slab gel electrophoresis revealed polypeptides with molecular weights of 32,000 and 34,000 corresponding to the form I and form II PRKs, respectively. Preliminary experiments on sensitivity to NADH regulation suggested that the two PRK enzymes differ in catalytic properties.     

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.     

Activation of ribulose 1,5-bisphosphate carboxylase from Rhodopseudomonas sphaeroides: probable role of the small subunit.

The activation properties of the form I and form II ribulose 1,5-bisphosphate carboxylases from Rhodopseudomonas sphaeroides were examined. Both enzymes have a requirement of Mg2+ for optimal activity. Mn2+, Ni2+, and Co2+ can also support activity of the form I enzyme, whereas only Mn2+ can substitute for Mg2+ with the form II enzyme. The effect of different preincubations on the carboxylase reaction was also examined. Both enzymes exhibited a lag when preincubated with other than Mg2+ and CO2 before assay, but the lag was much more pronounced and the rate of the reaction was slower with the form I enzyme under these conditions. Activation of the form I carboxylase By Mg2+ and CO2 occurred more rapidly than that of the form II enzyme. The results obtained with the two distinct forms of carboxylase from R. sphaeroides, as well as studies with the spinach and Rhodospirillum rubrum enzymes, thus indicate that the presence of the small subunit affects the rate of activation by Mg2+ and CO2 as well as the rate of reactivation of ribulose bisphosphate-inactivated enzyme.     

Reversible inactivation and characterization of purified inactivated form I ribulose 1,5-bisphosphate carboxylase/oxygenase of Rhodobacter sphaeroides.

Form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) from Rhodobacter sphaeroides is inactivated upon the addition of organic acids to photolithoautotrophically grown cultures. Activity recovers after the dissipation of the organic acid from the culture. The inactivation process depends on both the concentration of the organic compound and the nitrogen status of the cells. The inactivated RubisCO has been purified and was shown to exhibit mobility on both nondenaturing and sodium dodecyl sulfate gels different from that of the active enzyme prepared from cells not treated with organic acids. However, the Michaelis constants for ribulose 1,5-bisphosphate and CO2 or O2 were not dramatically altered. Purified inactivated RubisCO could be activated in vitro by increasing the temperature or the levels of Mg(II), and this activation was accompanied by changes in the electrophoretic mobility of the protein. When foreign bacterial RubisCO genes were expressed in an R. sphaeroides host strain lacking the ability to synthesize endogenous RubisCO, only slight inactivation of RubisCO activity was attained.     

Transposon mutagenesis and physiological analysis of strains containing inactivated form I and form II ribulose bisphosphate carboxylase/oxygenase genes in Rhodobacter sphaeroides.

Strains of Rhodobacter sphaeroides (Rhodopseudomonas sphaeroides) were constructed such that either the gene encoding form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPC-O) or the gene encoding form II RuBPC-O was inactivated. Both strains were capable of photoheterotrophic growth with malate as the electron donor, with only slight differences in growth rate and overall carboxylase specific activity compared with the wild-type strain. Photolithotrophic growth with 1.5% CO2 in hydrogen was also possible for R. sphaeroides strains containing only one of the two RuBPC-O enzyme forms, although the differences in growth rates between wild-type and carboxylase mutant strains were greater under these conditions. These results indicate that the two forms of RuBPC-O are independently regulated. In addition, the regulatory system governing RuBPC-O synthesis may, in some cases, compensate for the lack of the missing enzyme.     

Interaction between ribulose 1,5-bisphosphate carboxylase/oxygenase activity and the ammonia assimilatory system of Rhodobacter sphaeroides.

The levels of form I and form II ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) from Rhodobacter sphaeroides were found to depend on the concentration of ammonia supplied to photolithoautotrophically grown cultures. Under conditions in which the cells rapidly depleted the available ammonia, the level of in situ RubisCO activity decreased to less than 5% maximum activity; even at its maximum level under these conditions, the RubisCO activity was only 5% of the activity obtained from cultures supplied with saturating levels of ammonia. When cells were incubated with somewhat higher but not saturating amounts of ammonia, in situ RubisCO activity decreased immediately after the cells depleted the cultures of ammonia. The decrease in activity was not due to any detectable degradation of RubisCO protein, indicative of some mechanism to regulate the activity of the enzyme in response to the intracellular levels of assimilated ammonia. Furthermore, under conditions optimum for RubisCO inactivation, in situ RubisCO activity in permeabilized whole cells greatly exceeded the levels of enzymatic activity determined in vitro in cell extracts. Blockage of ammonia assimilation by inhibition of glutamine synthetase with methionine sulfoximine prevented the recovery of form I RubisCO from pyruvate-mediated inactivation, suggesting the presence of regulatory mechanisms common to both CO2 fixation and ammonia assimilation.     

Oxygen-dependent inactivation of ribulose 1,5-bisphosphate carboxylase/oxygenase in crude extracts of Rhodospirillum rubrum and establishment of a model inactivation system with purified enzyme.

Ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBPC/O) was inactivated in crude extracts of Rhodospirillum rubrum under atmospheric levels of oxygen; no inactivation occurred under an atmosphere of argon. RuBP carboxylase activity did not decrease in dialyzed extracts, indicating that a dialyzable factor was required for inactivation. The inactivation was inhibited by catalase. Purified RuBPC/O is relatively oxygen stable, as no loss of activity was observed after 4 h under an oxygen atmosphere. The aerobic inactivation catalyzed by endogenous factors in crude extracts was mimicked by using a model system containing purified enzyme, ascorbate, and FeSO4 or FeCl3. Dithiothreitol was found to substitute for ascorbate in the model system. Preincubation of the purified enzyme with RuBP led to enhanced inactivation, whereas Mg2+ and HCO3- significantly protected against inactivation. Unlike the inactivation catalyzed by endogenous factors from extracts of R. rubrum, inactivation in the model system was not inhibited by catalase. It is proposed that ascorbate and iron, in the presence of oxygen, generate a reactive oxygen species which reacts with a residue at the activation site, rendering the enzyme inactive.     

Photosynthetic CO2 fixation and ribulose bisphosphate carboxylase/oxygenase activity of Nostoc sp. strain UCD 7801 in symbiotic association with Anthoceros punctatus.

The cyanobacterium Nostoc sp. strain UCD 7801, immediately after separation from pure cultures of a reconstituted symbiotic association with the bryophyte Anthoceros punctatus, exhibited a rate of light-dependent CO2 fixation that was eightfold lower than that measured in the free-living growth state. Ribulose bisphosphate carboxylase/oxygenase (RuBPC/O) specific activity was also eightfold lower in cell extracts of symbiotic strain 7801 relative to that in free-living cultures. The in vitro activity from symbiotic strain 7801 could not be increased by incubation under the standard RuBPC/O activation conditions. Polyclonal antibodies against the RuBPC/O large subunit were used in an enzyme-linked immunosorbent assay to determine that RuBPC/O accounted for 4.3 and 5.2% of the total protein in cell extracts of strain 7801 grown in symbiotic and free-living states, respectively. The results imply that the regulation of RuBPC/O activity in the symbiotic growth state is by a posttranslational mechanism rather than by an alteration in RuBPC/O protein synthesis. The amount of carboxyarabinitol bisphosphate required to irreversibly inhibit RuBPC/O activity of sybiotic cell extracts was 80% of that required for extracts of free-living cultures. This result indicates that any covalent modification of RuBPC/O in symbiotically associated Nostoc cells did not interfere with the ribulose bisphosphate binding site, since inactive enzyme also bound carboxyarabinitol bisphosphate.     

Independent regulation of synthesis of form I and form II ribulose bisphosphate carboxylase-oxygenase in Rhodopseudomonas sphaeroides.

Ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBPC-O) activity was greatly enhanced when Rhodopseudomonas sphaeroides was grown in a mineral salts medium supplied with 1.5% CO2 in hydrogen. Analysis of cell extracts by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that cells growing on 1.5% CO2 in H2 specifically accumulated RuBPC-O polypeptides. Quantitative immunological determinations revealed that accumulation of form I and form II RuBPC-O closely correlates with the increase of specific activity. However, the two enzymes appeared to be derepressed at different levels. Upon transfer from heterotrophic to autotrophic (1.5% CO2) growth conditions, the intracellular form I RuBPC-O concentration was augmented 17-fold, whereas the form II RuBPC-O content increased only fourfold. As a result, the form I-form II ratio changed from 0.5 to about 2.0. Since this change in the RuBPC-O ratio occurred in the early stage of growth, it suggests that form I RuBPC-O is required for growth under drastic CO2 limitation. The difference in the extent of derepression of form I and form II RuBPC-O also indicates that the synthesis of each enzyme is regulated somewhat independently of the other.     

Nucleotide and deduced amino acid sequence of the Rhodobacter sphaeroides gene encoding form II ribulose-1,5-bisphosphate carboxylase/oxygenase and comparison with other deduced form I and II sequences

The nucleotide sequence for the Rhodobacter sphaeroides form II ribulose 1,5-bisphosphate carboxylase/oxygenase was determined. The deduced product is highly homologous with the form II-like enzyme of Rhodospirillum rubrum , but appears to be more distantly related to the large subunit of the L₈S₈ enzyme found in autotrophic bacteria, cyanobacteria and higher plants. Several regions highly conserved among L₈S₈ and L_X enzymes correspond with regions previously implicated in catalytic activity and subunit interactions. An imperfect palindrome and a stem loop structure were identified in the 5' and 3' flanking sequences, respectively, of R. sphaeroides rbpL .     

Control of autotrophic carbon assimilation in Alcaligenes eutrophus by inactivation and reactivation of phosphoribulokinase

Phosphoribulokinase in Alcaligenes eutrophus was partially inactivated when an autotrophic culture was shifted to heterotrophic growth with pyruvate as the sole source of carbon and energy. A similar response was observed on addition of various organic substrates to autotrophic cultures during the transition to mixotrophic growth. The extent of inactivation depended on the added substrate. Pyruvate or lactate caused the strongest inactivation among the tested substrates. Up to 75% of the phosphoribulokinase activity found in the autotrophic cells was lost within 30 min after supplementation of the cultures with either of these two substrates. This loss of enzyme activity was not the result of degradation of enzyme protein. Inactivation of phosphoribulokinase was accompanied by a decrease in the CO2 fixation rate of the cells. Reactivation of the enzyme occurred after exhaustion of pyruvate from the medium. Neither inactivation nor reactivation required de novo protein synthesis; however, continued energy conversion was necessary for the inactivation to occur. We suggest that the pyruvate metabolism of A. eutrophus is involved in these regulatory processes which act on phosphoribulokinase. They appear to contribute to the control of autotrophic CO2 assimilation in this organism.     

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.     

Characterization of antiserum directed against form II ribulose 1,5-bisphosphate carboxylase from Rhodopseudomonas sphaeroides.

Antiserum directed against form II ribulose 1,5-bisphosphate carboxylase from Rhodopseudomonas sphaeroides showed no cross-reactivity towards the form I enzyme as evidenced by a lack of immunopreciptation. In addition, this antiserum failed to inhibit form I enzymatic activity.     

Rhodobacter sphaeroides uses a reductive route via propionyl coenzyme A to assimilate 3-hydroxypropionate

3-Hydroxypropionate is a product or intermediate of the carbon metabolism of organisms from all three domains of life. However, little is known about how carbon derived from 3-hydroxypropionate is assimilated by organisms that can utilize this C(3) compound as a carbon source. This work uses the model bacterium Rhodobacter sphaeroides to begin to elucidate how 3-hydroxypropionate can be incorporated into cell constituents. To this end, a quantitative assay for 3-hydroxypropionate was developed by using recombinant propionyl coenzyme A (propionyl-CoA) synthase from Chloroflexus aurantiacus. Using this assay, we demonstrate that R. sphaeroides can utilize 3-hydroxypropionate as the sole carbon source and energy source. We establish that acetyl-CoA is not the exclusive entry point for 3-hydroxypropionate into the central carbon metabolism and that the reductive conversion of 3-hydroxypropionate to propionyl-CoA is a necessary route for the assimilation of this molecule by R. sphaeroides. Our conclusion is based on the following findings: (i) crotonyl-CoA carboxylase/reductase, a key enzyme of the ethylmalonyl-CoA pathway for acetyl-CoA assimilation, was not essential for growth with 3-hydroxypropionate, as demonstrated by mutant analyses and enzyme activity measurements; (ii) the reductive conversion of 3-hydroxypropionate or acrylate to propionyl-CoA was detected in cell extracts of R. sphaeroides grown with 3-hydroxypropionate, and both activities were upregulated compared to the activities of succinate-grown cells; and (iii) the inactivation of acuI, encoding a candidate acrylyl-CoA reductase, resulted in a 3-hydroxypropionate-negative growth phenotype.     

Chemoautotrophic growth of hydrogen-uptake-positive strains of Rhizobium japonicum.

Recently reported research from this laboratory has demonstrated the autotrophic growth of certain hydrogen-uptake-positive strains of Rhizobium japonicum and defined minimal conditions for such growth. Ribulose 1,5-bisphosphate carboxylase has been detected in autotrophically growing cells, but at low specific activity. Moreover, growth rates were low, and growth ceased at low cell densities. We report here improved autotrophic growth rates of R. japonicum SR through the use of a modified mineral salts/vitamins medium and a programmed increase in oxygen tension as autotrophic growth proceeds. Under these conditions, ribulose, 1,5-biphosphate carboxylase activity increased greater than 10-fold and crude-extract-uptake-hydrogenase activities were from 20 to 47 times those heretofore reported for free-living R. japonicum. It is likely that previous assays for these enzymes were done on preparations of cells in which their synthesis had been partially repressed. The contribution of CO2 fixation to organic carbon accumulation in autotrophic cells was assessed as sufficient to support observed growth. Enzymological determination of the product of carbon fixation has established a stoichiometric ratio of 1.9 mol of 3-phosphoglycerate per mol of CO2 fixed and unequivocally assigns the role of carbon fixation catalysis to ribulose 1,5-bisphosphate carboxylase. Ammonium served best as a nitrogen source, nitrate was less effective, and gaseous nitrogen would not support autotrophic growth. Ecological, evolutionary, and practical considerations of autotrophy in the rhizobia are briefly discussed in the light of our findings.     

Reductive pentose phosphate-independent CO2 fixation in Rhodobacter sphaeroides and evidence that ribulose bisphosphate carboxylase/oxygenase activity serves to maintain the redox balance of the cell.

Whole-cell CO2 fixation and ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) activity were determined in Rhodobacter sphaeroides wild-type and mutant strains. There is no obvious difference in the levels of whole-cell CO2 fixation for the wild type, a form I RubisCO deletion mutant, and a form II RubisCO deletion mutant. No ribulose 1,5-bisphosphate-dependent CO2 fixation was detected in a form I-form II RubisCO double-deletion mutant (strain 16) or strain 16PHC, a derivative from strain 16 which was selected for the ability to grow photoheterotrophically with CO2 as an electron acceptor. However, significant levels of whole-cell CO2 fixation were detected in both strains 16 and 16PHC. Strain 16PHC exhibited CO2 fixation rates significantly higher than those of strain 16; the rates found for strain 16PHC were 30% of the level found in photoheterotrophically grown wild-type strain HR containing both form I and form II RubisCO and 10% of the level of the wild-type strain grown photolithoautotrophically. Strain 16PHC could not grow photolithoautotrophically in a CO2-H2 atmosphere; however, CO2 fixation catalyzed by photoheterotrophically grown strain 16PHC was repressed by addition of the alternate electron acceptor dimethyl sulfoxide. Dimethyl sulfoxide addition also influenced RubisCO activity under photolithoautotrophic conditions; 40 to 70% of the RubisCO activity was reduced without significantly influencing growth. Strain 16PHC and strain 16 contain nearly equivalent but low levels of pyruvate carboxylase, indicating that CO2 fixation enzymes other than pyruvate carboxylase contribute to the ability of strain 16PHC to grow with CO2 as an electron acceptor.