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 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.     

多能硫杆菌RubisCO基因鉴定以及在大肠杆菌中的表达

多能硫杆菌(Thiobacillus versutus)是兼性化能自养细菌,在生理学和分类学上具有重要的地位,也是研究硫杆菌生理、生化、遗传学的理想材料。该菌通过卡尔文循环固定CO_2,其关键酶是1,5-二磷酸核酮糖羧化酶/加氧酶(简称RubisCO)。我们从多能硫杆菌中分离得到的RubisCO基因片段能够在大肠杆菌细胞中表达,说明自养细菌与异养细菌在基因表达方面是相似的。     

Molecular evolution of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO)

Abstract The evolutionary relationship of the RuBisCO large subunit gene(s) ( rbcL ) of several prokaryotes was examined using the technique of heterologous DNA hybridization. Restriction fragments of cloned rbcL from Anacystis nidulans 6301, Chlamydomonas reinhardtii, Rhodospirillum rubrum , and maize were nick-translated and used as probes. The C. reinhardtii and maize probes hybridized with restriction fragment(s) only from cyanobacteria: Agmenellum quadruplicatum, Fremyella diplosiphon , and Mastigocladus laminosus . In addition, the A. nidulans probe hybridized with restriction fragment(s) from Alcaligenes eutrophus, Chromatium vinosum, Nitrobacter hamburgensis, Paracoccus denitrificans, Pseudomonas oxalaticus, Rhodomicrobium vannielii, Rhodopseudomonas capsulata, Rhodopseudomonas palustris, Rhodopseudomonas sphaeroides, Thiobacillus intermedius, Thiobacillus neapolitanus , and Thiothrix nivea . The elucidated fragment of Rhodopseudomonas species is presumably for the Form I RuBisCO LSU of these organisms. The R. rubrum probe hybridized only to a restriction fragment(s) from R. capsulata, R. palustris, R. sphaeroides, T. neapolitanus , and T. nivea . The fragment(s) of Rhodopseudomonas species is the Form II rbcL of these organisms. The restriction fragments of T. neapolitanus and T. nivea were also different from those elucidated by the A. nidulans probe, suggesting the presence of a second (different) rbcL in these organisms. Positive hybridization was not obtained using any of the probes with DNA from Beggiatoa alba, Chlorobium vibrioforme or Chloroflexus aurantiacus . It appears that all rbcL have evolved from a common ancestor. Our data are consistent with and supportive of the evolutionary scheme for RuBisCO proposed by Akazawa, Takabe, and Kobayashi [1].     

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 .     

Construction of a gene bank and identification of the ribulose bisphosphate carboxylase/oxygenase genes from the nutritionally versatile cyanobacterium Chlorogloeopsis fritschii

A gene bank of the nutritionally versatile, nitrogen-fixing cyanobacterium Chlorogloeopsis fritschii was constructed in Charon 4A. 2,800 recombinants containing 10–20 kbp C. fritschii DNA fragments were screened by Southern hybridization using probes containing the genes for the large (LSU) and small (SSU) subunits of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) from Anacystis nidulans. A single recombinant plaque (CDG1) containing a 10.9 kbp EcoR1 fragment from C. fritschii hybridized to both the LSU and SSU probes, indicating a possible linkage of these RuBisCO genes in C. fritschii. RuBisCO activity and protein were detected in CDG1 lysates of Escherichia coli. Hybridization was also obtained between C. fritschii DNA and the LSU probe from Chlamydomonas reinhardtii, although no homology was detected using the LSU probe from maize or the SSU probe from pea.Abbreviations RuBisCO d-ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBP d-ribulose 1,5-bisphosphate - LSU large subunit of RuBisCO - SSU small subunit of RuBisCO - SDS sodium dodecyl sulphate - DOC deoxycholate     

Isolation and partial characterization of Rhodopseudomonas sphaeroides mutants defective in the regulation of ribulose bisphosphate carboxylase/oxygenase.

Several mutants of Rhodopseudomonas sphaeroides defective in the derepression of the enzyme ribulose 1,5-bisphosphate carboxylase have been isolated by using the unstable Tn5 vectors pJB4JI and pRK340. Transpositional insertion mutants obtained with pJB4JI were demonstrated to be incapable of increasing ribulose 1,5-bisphosphate carboxylase/oxygenase levels when grown on butyrate-bicarbonate medium or under conditions of carbon starvation, whereas the wild-type strain increased activity four- to eightfold. When the wild-type strain was starved for carbon in the presence of chloramphenicol, no derepression was observed. Crude extracts from mutant and wild-type strains had distinct and consistent differences in protein content as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Chromatographic evidence indicated that mutants were defective in the regulation of only one of the two forms of ribulose 1,5-bisphosphate carboxylase/oxygenase synthesized by 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.     

Derepression of the synthesis of D-ribulose 1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum.

The synthesis of ribulose 1,5-bisphosphate carboxylase/oxygenase in Rhodospirillum rubrum was greatly influenced by the conditions of culture. When grown photolithotrophically in an atmosphere containing low levels of CO2 (1.5 to 2%), enzyme synthesis was derepressed, with the result that the enzyme comprised up to 50% of the soluble protein of the cells as determined by immunological quantitation. This response was not observed when R. rubrum was grown photolithotrophically in an atmosphere of 5% CO2 in hydrogen. Similarly, the derepression of ribulose 1,5-bisphosphate carboxylase/oxygenase was observed in photoheterotrophically (butyrate)-grown cultures only after the HCO3- supply was nearly exhausted. The increase in enzyme activity observed in derepressed cultures was not paralleled by an increase in the in vivo CO2 fixation rate. Apparently, R. rubrum derepresses the synthesis of ribulose 1,5-bisphosphate carboxylase/oxygenase when exposed to low CO2 concentrations to scavenge the limited CO2 available to such cultures.     

Ribulose bisphosphate carboxylase of the procaryotic symbiont of a hydrothermal vent tube worm: kinetics, activity and gene hybridization

Abstract The giant tube worm, Riftia pachyptila , which is abundant at deep-sea hydrothermal vents, contains an extremely high density of bacterial symbionts in a specialized 'trophosome' tissue. Although the symbiont has not been cultured, enzymatic studies by others indicate that the symbiont is capable of hydrogen-sulfide- or sulfur-based lithoautotrophy and fixes CO₂ via the Calvin-Benson cycle. Here we report additional findings for a specimen from the Guaymas Basin vent site (Gulf of California, 2000 m). Under assay conditions where activity was proportional to cell-free extract concentration, ribulose bisphosphate carboxylase/oxygenase (RuBisCO) activity was 6.3 nmol CO₂/mg protein per min (30°C). This is within the range observed for non-CO₂ limited cultures of sulfur bacteria. The activity vs. temperature profile suggests that the symbiont is a mesophile and not a thermophile. A substrate saturation curve shows an apparent K _m (with respect to ribulose 1,5-bisphosphate) of 65 μM which is considerably lower than the single previous report for a sulfur bacterial symbiont. Strong hybridization was detected between a gene probe derived from the RuBisCO large subunit gene of Anacystis nidulans and Riftia trophosome DNA. A Rhodospirillum rubrum -derived probe also showed hybridization with the same restriction fragments of symbiont DNA.     

Acyl carrier protein-derived sequence encoded by the chloroplast genome in the marine diatom Cylindrotheca sp. strain N1.

The chloroplast genome of chromophytic and rhodophytic algae differs from the plastid genome of plants and green algae in that it encodes the gene for the small subunit (rbcS) of ribulose 1,5-bisphosphate carboxylase/oxygenase. Hybridization studies indicated that there was a second region of chloroplast DNA from the marine diatom Cylindrotheca sp. strain N1 that strongly hybridized to a previously isolated Cylindrotheca fragment that contained the rbcS gene and flanking sequences. Subsequent determination of the oligonucleotide sequence of this second chloroplast DNA fragment, however, indicated that hybridization was due to identical sequences 3' to the previously cloned Cylindrotheca chloroplast rbcL rbcS genes. Sequences derived from the 5' end of the second chloroplast DNA fragment contained a short open reading frame of 80 amino acids which was found to be highly homologous to bacterial acyl carrier protein and nuclear-encoded acyl carrier protein from plants. Amino acid residues in the environment of Ser-36 of the Escherichia coli protein, which is bound to a 4'-phosphopantetheine moiety, are virtually identical in the Cylindrotheca deduced sequence and all other sources of this protein. Unlike plant acyl carrier-deduced amino acid sequences, there was no leader peptide sequence found for the presumptive Cylindrotheca protein, consistent with the location of this DNA fragment on the chloroplast genome of this organism. DNA encoding the putative acyl carrier protein gene and rbcS thus represent two genes that are chloroplast-encoded in the chromophytic marine diatom Cylindrotheca, a significant departure from the organization of such genes in plants.     

Rice chloroplast DNA: a physical map and the location of the genes for the large subunit of ribulose 1,5-bisphosphate carboxylase and the 32 KD photosystem II reaction center protein

Summary By homogenizing rice leaves in liquid nitrogen, it was possible to isolate intact chloroplasts and, subsequently, pure rice chloroplast DNA from the purified chloroplasts. The DNA was digested by several restriction enzymes and fragments were fractionated by agarose gel electrophoresis. The sum of the fragment sizes generated by the restriction enzymes showed that the total length of the DNA is 130 kb. A circular physical map of fragments, generated by digestion with SalI, PstI, and PvuII, has been constructed. The circular DNA contains two inverted repeats of about 20 kb separated by a large, single copy region of about 75 kb and a short, single copy region of about 15 kb. The location of the gene for the large subunit of ribulose 1,5-bisphosphate carboxylase (Fraction I protein) and the 32 KD photosystem II reaction center gene were determined by using as probes tobacco chloroplast DNAs containing these genes. Rice chloroplast DNA differs from chloroplast DNAs of wheat and corn as well as from dicot chloroplast DNAs by having the 32 KD gene located 20 kb removed from the end of an inverted repeat instead of close to the end, as in other plants.     

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.     

An oligonucleotide primer system for amplification of the ribulose-1,5-bisphosphate carboxylase/oxygenase genes of bacteria of various taxonomic groups

Based on the analysis of GenBank nucleotide sequences of the cbbL and cbbM genes, coding for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPC), the key enzyme of the Calvin cycle, a primer system was designed that allows about 800-bp-long fragments of these genes to be PCR-ampliflied in various photo- and chemotrophic bacteria. The efficiency of the designed primer system in detection of RuBPC genes was demonstrated in PCR with DNA of taxonomically diverse bacteria possessing RuBPC genes with a known primary structure. Nucleotide sequences of RuBPC gene fragments of bacteria belonging to the genera Acidithiobacillus. Ectothiorhodospira, Magnetospirillum, Methylocapsa, Thioalkalispira, Rhodobacter, and Rhodospirillum were determined to be deposited with GenBank and to be translated into amino acid sequences and subjected to phylogenetic analysis.     

Structural differences in the catalytic subunits of form I and form II ribulose 1,5-bisphosphate carboxylase/oxygenase from Rhodopseudomonas sphaeroides.

There are significant differences in the large subunits of form I and form II ribulose 1,5-bisphosphate carboxylase/oxygenase isolated from Rhodopseudomonas sphaeroides. Two-dimensional peptide mapping of carboxymethylated large subunits clearly indicates that there are differences in the primary structure of the two proteins. These results are supported by limited proteolysis with three different proteases and by subsequent analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These data, in conjunction with immunological studies and investigations on the regulation of the two enzymes, support the conclusion that the large subunits of form I and form II ribulose 1,5-bisphosphate carboxylase/oxygenase may be different gene products.     

Expression of endogenous and foreign ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) genes in a RubisCO deletion mutant of Rhodobacter sphaeroides.

A Rhodobacter sphaeroides ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) deletion strain was constructed that was complemented by plasmids containing either the form I or form II CO2 fixation gene cluster. This strain was also complemented by genes encoding foreign RubisCO enzymes expressed from a Rhodospirillum rubrum RubisCO promoter. In R. sphaeroides, the R. rubrum promoter was regulated, resulting in variable levels of disparate RubisCO molecules under different growth conditions. Photosynthetic growth of the R. sphaeroides deletion strain complemented with cyanobacterial RubisCO revealed physiological properties reflective of the unique cellular environment of the cyanobacterial enzyme. The R. sphaeroides RubisCO deletion strain and R. rubrum promoter system may be used to assess the properties of mutagenized proteins in vivo, as well as provide a potential means to select for altered RubisCO molecules after random mutagenesis of entire genes or gene regions encoding RubisCO enzymes.     

Immunological determination of phosphoenolpyruvate carboxylase and the large and small subunits of ribulose 1,5-bisphosphate carboxylase in leaves of the c(4) plant pearl millet

The light-dependent development of the photosynthetic apparatus in the first leaf of the C₄ plant pearl millet (Pennisetum americanum) was monitored by immunologically determining the concentration of phospho-enolpyruvate carboxylase and ribulose 1,5-bisphosphate carboxylase. A competitive enzyme-linked immunosorbent assay procedure using antibodies to the monomeric subunit of phosphoenolpyruvate carboxylase and the large and small subunit of ribulose 1,5-bisphosphate carboxylase was used to quantitate the amounts of these polypeptides in the first leaf of etiolated seedlings and etiolated seedlings exposed to light for varying periods of time. Phosphoenolpyruvate carboxylase was present in etiolated tissue; however, light stimulated its synthesis nearly 23-fold. Maximum accumulation of phosphoenolpyruvate carboxylase occurred approximately 4 days after etiolated plants were placed in the light. Both the large subunit and the small subunit of ribulose 1,5-bisphosphate carboxylase were present in leaves of etiolated seedlings. Light also stimulated the synthesis of both of these polypeptides, but at different rates. In etiolated leaves there was approximately a 3-fold molar excess of the small subunit to large subunit. Exposure of the etiolated leaves to light resulted in the molar ratio of the large subunit to the small subunit increasing to approximately 0.72. These data indicate that the net synthesis of these two polypeptides is not coordinately regulated at all times.     

香蕉rbcS基因启动子的克隆及序列分析

以巴西香蕉为材料,根据已经获得的香蕉1,5-二磷酸核酮糖羧化/加氧酶小亚基基因的全长cDNA序列设计1对专一引物,通过PCR扩增得到了香蕉1,5-二磷酸核酮糖羧化/加氧酶小亚基的基因组全长,序列长811 bp,含有2个内含子。根据其基因组序列设计引物,采用SEFA-PCR方法,以总DNA为模板克隆了香蕉1,5-二磷酸核酮糖羧化/加氧酶小亚基基因的启动子序列,长1 681 bp。用PLACE软件分析发现该序列具有启动子的基本元件TATA-box、CAAT-box,包含多个胁迫诱导元件,如光诱导元件、赤霉素、低温诱导元件、昼夜节律调控元件等。该序列的克隆与分析为进一步研究香蕉1,5-二磷酸核酮糖羧化/加氧酶小亚基基因的表达调控奠定了基础。     

Expression and regulation of Bradyrhizobium japonicum and Xanthobacter flavus CO2 fixation genes in a photosynthetic bacterial host.

Calvin cycle carbon dioxide fixation genes encoded on DNA fragments from two nonphotosynthetic, chemolithoautotrophic bacteria, Bradyrhizobium japonicum and Xanthobacter flavus, were found to complement and support photosynthetic growth of a ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) deletion mutant of the purple nonsulfur bacterium Rhodobacter sphaeroides. The regulation of RubisCO expression was analyzed in the complemented R. sphaeroides RubisCO deletion mutant. Distinct differences in the regulation of RubisCO synthesis were revealed when the complemented R. sphaeroides strains were cultured under photolithoautotrophic and photoheterotrophic growth conditions, e.g., a reversal in the normal pattern of RubisCO gene expression. These studies suggest that sequences and molecular signals which regulate the expression of diverse RubisCO genes may be probed by using the R. sphaeroides complementation system.