Analysis of the genes forming the distal parts of the two cbb CO2 fixation operons from Alcaligenes eutrophus

In the facultative chemoautotroph Alcaligenes eutrophus H16, most of the genes (cbb genes) encoding enzymes of the Calvin carbon reduction cycle are organized within two highly homologous cbb operons, one located on the chromosome and the other on the megaplasmid pHG1. Nucleotide sequencing of the promoter-distal part of the operons revealed three open reading frames, designated cbbG, cbbK, and cbbA. Similarity searches in databases and heterologous expressions of the subcloned genes in Escherichia coli identified them as genes encoding the Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, and a class II fructose-1,6-bisphosphate aldolase, respectively. The aldolase could be grouped together with the enzymes from Rhodobacter sphaeroides and Bacillus subtilis as a new subtype of class II aldolases. A phenotypic complementation analysis with a cbb operon mutant of A. eutrophus showed that the cbbG product is essential for autotrophic growth of the organism, whereas the products of cbbK and cbbA can apparently be substituted by isoenzymes encoded elsewhere on the chromosome. No or only low constitutive promoter activity was associated with cbbK and cbbA, respectively, confirming the two genes as parts of the cbb operon. Downstream of cbbA, the very high overall nucleotide sequence identity (about 94%) prevailing throughout the two cbb operons discontinues, suggesting that cbbA is the most promoter-distal gene of the operon.Abbreviations FBA Fructose 1,6-bisphosphate aldolase - GAPDH Glyceraldehyde-3-phosphate dehydrogenase - PRK Phosphoribulokinase - RubisCO Ribulose-1,5-bisphosphate carboxylase/oxygenase     

Isolation and analysis of the genes for cytochrome c oxidase in Paracoccus denitrificans

Synthetic oligonucleotide probes were used to clone two loci from the chromosomal DNA of Paracoccus denitrificans that contain the genes for cytochrome c oxidase (cytochrome aa₃). One locus seems to contain four or five genes probably forming an operon. Two of these code for the oxidase subunits II and III. Three open reading frames are found between the COII and COIII genes. The other locus codes for the subunit I. A short open reading frame is found upstream of this gene. All three subunits of the Paracoccus enzyme show remarkable homology to the corresponding subunits of the mitochondrial cytochrome oxidase. Possible protein products of the open reading frames have not yet been identified.     

Biochemical and Biophysical Characterization of the Two Isoforms of cbb3-Type Cytochrome c Oxidase from Pseudomonas stutzeri

The cbb₃-type cytochrome c oxidases (cbb₃-CcOs) are members of the heme-copper oxidase superfamily that couple the reduction of oxygen to translocation of protons across the membrane. The cbb₃-CcOs are present only in bacteria and play a primary role in microaerobic respiration, being essential for nitrogen-fixing endosymbionts and for some human pathogens. As frequently observed in Pseudomonads, Pseudomonas stutzeri contains two independent ccoNO(Q)P operons encoding the two cbb₃ isoforms, Cbb₃-1 and Cbb₃-2. While the crystal structure of Cbb₃-1 from P. stutzeri was determined recently and cbb₃-CcOs from other organisms were characterized functionally, less emphasis has been placed on the isoform-specific differences between the cbb₃-CcOs. In this work, both isoforms were homologously expressed in P. stutzeri strains from which the genomic version of the respective operon was deleted. We purified both cbb₃ isoforms separately by affinity chromatography and increased the yield of Cbb₃-2 to a similar level as Cbb₃-1 by replacing its native promoter. Mass spectrometry, UV-visible (UV-Vis) spectroscopy, differential scanning calorimetry, as well as oxygen reductase and catalase activity measurements were employed to characterize both cbb₃ isoforms. Differences were found concerning the thermal stability and the presence of subunit CcoQ. However, no significant differences between the two isoforms were observed otherwise. Interestingly, a surprisingly high turnover of at least 2,000 electrons s⁻¹ and a high Michaelis-Menten constant (K_m ∼ 3.6 mM) using ascorbate–N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride (TMPD) as the electron donor were characteristic for both P. stutzeri cbb₃-CcOs. Our work provides the basis for further mutagenesis studies of each of the two cbb₃ isoforms specifically.     

The Calvin cycle enzyme pentose-5-phosphate 3-epimerase is encoded within the cfx operons of the chemoautotroph Alcaligenes eutrophus.

Several genes (cfx genes) encoding Calvin cycle enzymes in Alcaligenes eutrophus are organized in two highly homologous operons comprising at least 11 kb. One cfx operon is located on the chromosome; the other is located on megaplasmid pHG1 of the organism (B. Bowien, U. Windh?vel, J.-G. Yoo, R. Bednarski, and B. Kusian, FEMS Microbiol. Rev. 87:445-450, 1990). Corresponding regions of about 2.7 kb from within the operons were sequenced. Three open reading frames, designated cfxX (954 bp), cfxY (765 bp), and cfxE (726 bp), were detected at equivalent positions in the two sequences. The nucleotide identity of the sequences amounted to 94%. Heterologous expression of the subcloned pHG1-encoded open reading frames in Escherichia coli suggested that they were functional genes. The observed sizes of the gene products CfxX (35 kDa), CfxY (27 kDa), and CfxE (25.5 kDa) closely corresponded to the values calculated on the basis of the sequence information. E. coli clones harboring the cfxE gene showed up to about 19-fold-higher activities of pentose-5-phosphate 3-epimerase (PPE; EC 5.1.3.1) than did reference clones, suggesting that cfxE encodes PPE, another Calvin cycle enzyme. These data agree with the finding that in A. eutrophus, PPE activity is significantly enhanced under autotrophic growth conditions which lead to a derepression of the cfx operons. No functions could be assigned to CfxX and CfxY.     

Genetic evidence for 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) as a negative effector of cytochrome terminal oxidase cbb 3 production in Rhizobium etli

A Rhizobium etli Tn5mob-induced mutant (CFN035) exhibits an enhanced capacity to oxidize N,N,N′,N′, tetramethyl-p -phenylenediamine (TMPD), a presumptive indicator of elevated cytochrome c terminal oxidase activity. Sequencing of the mutated gene in CFN035 revealed that it codes for the amidophosphoribosyl transferase enzyme (PurF) that catalyzes the first step in the purine biosynthetic pathway. Two c-type cytochromes with molecular weights of 32 and 27?kDa were produced in strain CFN035, which also produced a novel CO-reactive cytochrome (absorbance trough at 553?nm), in contrast to strain CE3 which produced a single 32?kDa c-type protein and did not produce the 553?nm CO-reactive cytochrome. A wild-type R. etli strain that expresses the Bradyrhizobium japonicum fixNOQP genes, which code for the symbiotic cytochrome terminal oxidase cbb ₃, produced similar absorbance spectra (a trough at 553?nm in CO-difference spectra) and two c -type proteins similar in size to those of strain CFN035, suggesting that CFN035 also produces the cbb ₃ terminal oxidase. The expression of a R. etli fixN-lacZ gene fusion was measured in several R. etli mutants affected in different steps of the purine biosynthetic pathway. Our analysis showed that purF, purD, purQ, purL, purY, purK and purE mutants expressed three-fold higher levels of the fixNOQP operon than the wild-type strain. The derepressed expression of fixN was not observed in a purH mutant. The purH gene product catalyzes the conversion of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to 5-formaminoimidazole-4-carboxamide ribonucleotide (FAICAR) and inosine. Supplementation with AICA riboside lowered the levels of fixN expression in the purF mutants. These data are consistent with the possibility that AICAR, or a closely related metabolite, is a negative effector of the production of the symbiotic terminal oxidase cbb ₃ in R. etli.     

Identification of a gene required for the oxygen-regulated formation of the photosynthetic apparatus of Rhodobacter capsulatus

The pigment-binding proteins of Rhodobacter capsulatus are encoded by the polycistronic puf and puc operons. Both operons show higher expression under low oxygen tension than under high oxygen tension in the wild-type strain. The Tn5 mutant strain AH2 shows only low levels of puf and puc mRNA under high and low oxygen tension, indicating that it lacks a gene product required for stimulation of puf and puc gene expression under low oxygen tension. The formation of wild-type levels of photosynthetic complexes and normal oxygen regulation could be restored by the expression in trans of a 1.7 kb fragment of the R. capsulatus wild-type chromosome or by addition of 10μg I^-1 vitamin B₁₂ to the growth medium. An open reading frame of 798 nucleotides containing the Tn5 insertion was identified on the 1.7kb fragment. This open reading frame shows no homology to known genes and has a remarkably high GC content of 76%.     

Oenothera mitochondrial orf454, a gene involved in cytochrome c biogenesis corresponds to orf169 and orf322 of Marchantia

We have characterized a mitochondrial gene in Oenothera, designated orf454, capable of encoding a component of the cytochrome c biogenesis system. This open reading frame is interrupted by an intron of 941 nucleotides showing high similarity to a group II intron residing in the rpl2 gene. RNA editing, which is observed at 18 cytidine positions within the orf454 reading frame, improves the similarity to protein-coding sequences in bacteria and higher plants and removes the last 16 amino acids. orf454 also shows high sequence similarity to two overlapping reading frames (orf169 and orf322) of Marchantia mitochondria. These ORFs belong to an operon-like cluster of genes in the liverwort that is not conserved in Oenothera mitochondria. However, in bacteria these reading frames are organized like the Marchantia gene cluster. It has been shown by genetical analysis in Rhodobacter capsulatus that these genes are essential for cytochrome c biogenesis. Genes of bacterial operons — ccl1 in Rhodobacter and yejR and nrfE in Escherichia coli — show high sequence similarity to the mitochondrial reading frames orf577 and orf454 of Oenothera. orf454, which we describe here, is homologous to the C-terminal region of these bacterial genes, while the previously described orf577 is homologous to the N-terminal region.     

Genetic evidence for 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) as a negative effector of cytochrome terminal oxidase cbb 3 production in Rhizobium etli

A Rhizobium etli Tn5mob-induced mutant (CFN035) exhibits an enhanced capacity to oxidize N,N,N′,N′, tetramethyl-p -phenylenediamine (TMPD), a presumptive indicator of elevated cytochrome c terminal oxidase activity. Sequencing of the mutated gene in CFN035 revealed that it codes for the amidophosphoribosyl transferase enzyme (PurF) that catalyzes the first step in the purine biosynthetic pathway. Two c-type cytochromes with molecular weights of 32 and 27 kDa were produced in strain CFN035, which also produced a novel CO-reactive cytochrome (absorbance trough at 553 nm), in contrast to strain CE3 which produced a single 32 kDa c-type protein and did not produce the 553 nm CO-reactive cytochrome. A wild-type R. etli strain that expresses the Bradyrhizobium japonicum fixNOQP genes, which code for the symbiotic cytochrome terminal oxidase cbb ₃, produced similar absorbance spectra (a trough at 553 nm in CO-difference spectra) and two c -type proteins similar in size to those of strain CFN035, suggesting that CFN035 also produces the cbb ₃ terminal oxidase. The expression of a R. etli fixN-lacZ gene fusion was measured in several R. etli mutants affected in different steps of the purine biosynthetic pathway. Our analysis showed that purF, purD, purQ, purL, purY, purK and purE mutants expressed three-fold higher levels of the fixNOQP operon than the wild-type strain. The derepressed expression of fixN was not observed in a purH mutant. The purH gene product catalyzes the conversion of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to 5-formaminoimidazole-4-carboxamide ribonucleotide (FAICAR) and inosine. Supplementation with AICA riboside lowered the levels of fixN expression in the purF mutants. These data are consistent with the possibility that AICAR, or a closely related metabolite, is a negative effector of the production of the symbiotic terminal oxidase cbb ₃ in R. etli. Received: 21 November 1996 / Accepted: 22 January 1997     

Physiological and Biochemical Characteristics and Capacity for Polyhydroxyalkanoates Synthesis in a Glucose-Utilizing Strain of Hydrogen-Oxidizing Bacteria, Ralstonia eutropha B8562

The physiological, biochemical, genetic, and cultural characteristics of the glucose-utilizing mutant strain Ralstonia eutropha B8562 were investigated in comparison with the parent strain R. eutropha B5786. The morphological, cultural, and biochemical characteristics of strain R. eutropha B8562 were similar to those of strain R. eutropha B5786. Genetic analysis revealed differences between the 16S rRNA gene sequences of these strains. The growth characteristics of the mutant using glucose as the sole carbon and energy source were comparable with those of the parent strain grown on fructose. Strain B8562 was characterized by high yields of polyhydroxyalkanoate (PHA) from different carbon sources (CO₂, fructose, and glucose). In batch culture with glucose under nitrogen limitation, PHA accumulation reached 90% of dry weight. In PHA, β-hydroxybutyrate was predominant (over 99 mol %); β-hydroxyvalerate (0.25–0.72 mol %) and β-hydroxyhexanoate (0.008–1.5 mol %) were present as minor components. The strain has prospects as a PHA producer on glucose-containing media.     

Operation of the cbb3-type terminal oxidase in Azotobacter vinelandii

A part of the gene encoding cbb ₃-type cytochrome oxidase CcoN subunit was cloned from Azotobacter vinelandii and a mutant strain of this bacterium with disrupted ccoN gene was constructed. In contrast to the wild type strain, this one is unable to oxidize cytochromes c ₄ and c ₅. Thus, the A. vinelandii respiratory chain is shown to contain cbb ₃-type cytochrome c oxidase. It is also shown that the activity of this enzyme is not necessary for diazotrophic growth of A. vinelandii at high oxygen concentrations.