Autotrophic CO 2 fixation via the Calvin carbon reduction cycle in Alcaligenes eutrophus H16 is genetically determined by two highly homologous cbb operons, one of which is located on the chromosome and the other on megaplasmid pHG1 of the organism. An activator gene, cbbR, lies in divergent orientation only 167 bp upstream of the chromosomal operon and controls the expression of both cbb operons. The two 5 -terminal genes of the operons, cbbLS, coding for ribulose-1,5-bisphosphate carboxylase/oxygenase, were sequenced. Mapping of the 5 termini of the 2.1-kb cbbLS transcripts by primer extension and by nuclease S1 treatment revealed a single transcriptional start point at the same relative position for the chromosomal and plasmidborne cbb operons. The derived cbb operon promoter showed similarity to 70 -dependent promoters of Escherichia coli. For the 1.4-kb transcripts of cbbR, the transcriptional start points were different in autotrophic and heterotrophic cells. The two corresponding cbbR promoters overlapped the cbb operon promoter and also displayed similarities to 70 -dependent promoters. The deficient cbbR gene located on pHG1 was transcribed as well. A newly constructed double operon fusion vector was used to determine the activities of the cbb promoters. Fusions with fragments carrying the cbb intergenic control regions demonstrated that the cbb operon promoters were strongly regulated in response to autotrophic versus heterotrophic growth conditions. In contrast, the cbbR promoters displayed low constitutive activities. The data suggest that the chromosomal and plasmid-borne cbb promoters of A. eutrophus H16 are functionally equivalent despite minor structural differences.The assimilation of CO 2 in the aerobic, facultatively autotrophic bacterium Alcaligenes eutrophus involves the operation of the Calvin carbon reduction cycle during either lithotrophic growth with hydrogen or organotrophic growth with formate as the sole energy source (11). With the exceptions of the triose-3-phosphate isomerase (EC 5.3.1.1) and pentose-5-phosphate isomerase (EC 5.3.1.6), all enzymes of the cycle, including the CO 2 -fixing ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39; RubisCO), are encoded within a large cbb operon (cbbLSXYEFPTZGKA) encompassing about 12.5 kilobase pairs (kb) (7,41,42). The RubisCO genes cbbLS form the 5Ј-terminal end of the operon. It is of physiological interest that the 2-phosphoglycolate phosphatase (EC 3.1.3.18) gene cbbZ is also part of the operon. The enzyme initiates the conversion of 2-phosphoglycolate, which is produced by the oxygenase activity of RubisCO, to 3-phosphoglycerate via the glycolate pathway (42). The functions of cbbX and cbbY are still unknown. A. eutrophus H16 possesses two copies of the cbb operon, one located on the chromosome and the other on megaplasmid pHG1. Both the chromosomal and the plasmidborne operon were shown to be functional by phenotypic complementation of pHG1-cured mutants deficient in CO 2 assimilation, carrying insertions of transposon Tn5...
Several genes (cfir genes) encoding Calvin cycle enzymes in Akaligenes eutrophus are organized in two highly homologous operons comprising at least 11 kb. One cfxr operon is located on the chromosome; the other is located on megaplasmid pHG1 of the organism (B. Bowien, U. Windhovel, J.-G. Yoo, R. Bednarski, and B. Kusian, FEMS Microbiol. Rev. 87:445-450, 1990 When growing lithoautotrophically with hydrogen or organoautotrophically with formate as an energy source, the facultative chemoautotroph Alcaligenes eutrophus assimilates CO2 via the reactions of the Calvin carbon reduction cycle (7). In strain H16, genes encoding enzymes of this cycle (cfx genes) are organized in two large, highly homologous cfx operons. One copy of the operon is located on the chromosome; the other is located on megaplasmid pHG1 adjacent to the hydrogenase gene cluster (9,12,20). Both operons, which possibly originate from a gene duplication event, are functional and expressed simultaneously. Each of them comprises at least 11 kb (48). The two promoterproximal genes, cfxL and cfxS, encode the L and S subunits, respectively, of the C02-fixing enzyme of the Calvin cycle, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), while the gene coding for glyceraldehyde-3-phosphate dehydrogenase (GAP), cfxG, is the most promoter-distal gene so far identified. Also located within the operons are the genes for fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase (FBP) (cfrF), phosphoribulokinase (PRK) (cfxP), and transketolase (TK) (cfrl) (Fig.
In the facultatively chemoautotrophic bacterium Ralstonia eutropha (formerly Alcaligenes eutrophus), most genes required for CO 2 assimilation via the Calvin cycle are organized within two highly homologous cbb operons located on the chromosome and on megaplasmid pHG1, respectively, of strain H16. These operons are subject to tight control exerted by a promoter upstream of the 5-terminal cbbL gene that is regulated by the activator CbbR. The existence of subpromoters within the operons was now excluded, as determined with lacZ operon fusions to suitable cbb gene fragments in the promoter-probe vector pBK. Nevertheless, marked differential expression of the promoter-proximal ribulose-1,5-bisphosphate carboxylase-oxygenase genes cbbLS and the remaining distal genes occurs within the operons. Computer analysis revealed a potential stem-loop structure immediately downstream of cbbS that was suspected to be involved in the differential gene expression. Nuclease S1 mapping identified a major 3 end and a minor 3 end of the relatively stable cbbLS partial transcript just downstream of this structure. Moreover, operon fusions containing progressively deleted stem-loop structures showed that the structure primarily caused transcriptional termination downstream of cbbS rather than increased the segmental stability of the cbbLS transcript. Premature transcription termination thus represents an important mechanism leading to differential gene expression within the cbb operons.The aerobic, facultatively chemoautotrophic bacterium Ralstonia eutropha (formerly Alcaligenes eutrophus [45]) is able to grow on a wide variety of compounds as sources of carbon and energy. Hydrogen and formate are energy substrates, which support litho-or organoautotrophic growth, respectively, with CO 2 assimilation proceeding via the Calvin carbon reduction cycle (7). In strain H16, two highly homologous cbb operons, one copy located on the chromosome and the other located on megaplasmid pHG1 of the organism, encode most of the Calvin cycle enzymes (5). However, the operons differ in one major structural aspect. Whereas the cbbLS genes coding for the CO 2 -fixing ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) constitute the 5Ј-terminal end, the cbbA gene (fructose-1,6-/sedoheptulose-1,7-bisphosphate aldolase) was believed to form the 3Ј terminus in both operons (34). Only very recently was the chromosomal operon shown to contain an additional distal gene, cbbB c , which encodes a formate dehydrogenase-like protein with no apparent metabolic function (4). The gene of the other key enzyme of the Calvin cycle, cbbP (phosphoribulokinase), is situated in the central part of the large operons encompassing about 12.5 (plasmid) and 15 (chromosome) kbp. Previous findings suggested that the operons are transcribed from a single promoter upstream of cbbL (42). The activity of this promoter is subject to strong regulation by the transcriptional activator protein CbbR, a member of the LysR family of transcriptional regulators. CbbR is the product of t...
The genome of Alcaligenes eutrophus H16 contains two functional, highly homologous clusters of genes (cfx genes) for at least six Calvin cycle enzymes, with one cluster located on the chromosome and the other on megaplasmid pHG1. The genes within each cluster presumably belong to a single large operon of about 11 kilobase pairs (kb) in size. Both operons form a cfx regulon that is controlled by the chromosomally encoded regulatory gene cfxR. The product of cfxR seems to be a transcriptional activator. Occurrence of isoenzymes within the central carbon metabolism of the organism is a consequence of this unusual genetic setup.
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