Identification and organization of carbon dioxide fixation genes in Xanthobacter flavus H4-14

The presence of two sets (rbcL1-rbcS1 and rbcL2-rbcS2) of rbc operons has been demonstrated in Thiobacillus ferrooxidans Fe1 (T. Kusano, T. Takeshima, C. Inoue, and K. Sugawara, J. Bacteriol. 173:7313-7323, 1991). A possible regulatory gene, rbcR, 930 bp long and possibly translated into a 309-amino-acid protein, was found upstream from the rbcL1 gene as a single copy. The gene is located divergently to rbcL1 with a 144-bp intergenic sequence. As in the cases of the Chromatium vinosum RbcR and Alcaligenes eutrophus CfxR, T. ferrooxidans RbcR is thought to be a new member of the LysR family, and these proteins share 46.5 and 42.8% identity, respectively. Gel mobility shift assays showed that T. ferrooxidans RbcR, produced in Escherichia coli, binds specifically to the intergenic sequence between rbcL1 and rbcR. Footprinting and site-directed mutagenesis experiments further demonstrated that RbcR binds to overlapping promoter elements of the rbcR and rbcL1 genes. The above data strongly support the participation of RbcR in regulation of the rbcL1-rbcS1 operon and the rbcR gene in T. ferrooxidans.

Section: Resultssupporting

confidence: 78%

Specific binding of Thiobacillus ferrooxidans RbcR to the intergenic sequence between the rbc operon and the rbcR gene

Kusano

Sugawara

1993

“…strain 6301) was found to be expressed at high levels in this system, as previously described (7); however, only 19% inhibition of RubisCO activity was obtained after the addition of 20 mM pyruvate to the culture. X. flavus (27) and A. eutrophus (1) RubisCO genes were expressed in R sphaeroides 16 by using Xanthomonas and Alcaligenes promoters, respectively (8). After the addition of pyruvate to the cultures, 27% inhibition was obtained for the Xanthomonas enzyme; no inhibition of the Alcaligenes enzyme was found.…”

Section: Resultsmentioning

confidence: 99%

“…R. sphaeroides 16 is a RubisCO-negative strain which lacks the ability to synthesize either form I or form II RubisCO (7). This strain may be complemented to photolithoautotrophic growth by genes encoding foreign RubisCO enzymes expressed from a recently described promoter system (7) or by gene clusters containing foreign RubisCO genes and their original promoters (8 (27). Plasmid pAE312, containing the chromosomally encoded Alcaligenes eutrophus rbcL and rbcS genes and an upstream promoter, was provided by Kjell Andersen (1).…”

Section: Methodsmentioning

confidence: 99%

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

Wang

Tabita

1992

(31,40) and, in addition, reversible binding of the intracellular inhibitor 2-carboxy-arabinitol-1-phosphate to carbamylated RubisCO (3,15). In bacteria, there have been periodic indications that RubisCO might be subject to some form of modification or alteration of its activity in vivo (17,21,34), and recent work with the photosynthetic bacterium Rhodomicrobium vannielii suggests a covalent phosphorylation (26); in this latter study, however, no information relative to how phosphorylation affected RubisCO activity was presented. In other photosynthetic bacteria, particularly Rhodobacter sphaeroides and Rhodobacter capsulatus, two different forms of RubisCO are synthesized (12,13,33). The form I enzyme resembles the enzyme that is widely distributed in plants, algae, and most bacteria and contains both large (catalytic) and small subunits (L8S8); the form II RubisCO is composed only of large subunits which show little homology to form I large subunits (11,14,37). At the molecular level, the form I RubisCO genes of R. sphaeroi-* Corresponding author.des, rbcL and rbcS, and the form II RubisCO gene of R. sphaeroides, rbpL, are found in distinct chromosomal operons (10, 11), and there is evidence to support both independent and interdependent regulation of these genes (6,11,16,19). As for the regulation of RubisCO activity in this organism, it was recently found that the form I enzyme is specifically inactivated when metabolizable organic acids are added to cells growing with CO2 as the sole source of carbon (20).The results of the present investigation further characterize the inactivation of the form I RubisCO of R. sphaeroides both in vivo and in vitro and give further credence to the possibility that this enzyme is subject to a reversible modification of its activity in the cell.

“…One of the mutant strains was subject to an enhanced repression of the cbb operon promoter by the gluconeogenic substrate succinate and in addition failed to grow autotrophically. (9,32,33). A second fructosebisphosphatase gene required for heterotrophic growth is constitutively expressed (31,33).…”

mentioning

confidence: 99%

The Calvin cycle enzyme phosphoglycerate kinase of Xanthobacter flavus required for autotrophic CO2 fixation is not encoded by the cbb operon

Meijer

1994

During autotrophic growth of Xanthobacterflavus, energy derived from the oxidation of hydrogen methanol or formate is used to drive the assimilation of CO2 via the Calvin cycle. The genes encoding the Calvin cycle enzymes are organized in the cbb operon, which is expressed only during autotrophic growth. Although it has been established that the transcriptional activator CbbR is required for the expression of the cbb operon, it is unclear whether CbbR is the only factor contributing to the regulation of the cbb operon. This paper describes the isolation of X. flavus mutants which were affected in the regulation of the cbb operon. One of the mutant strains was subject to an enhanced repression of the cbb operon promoter by the gluconeogenic substrate succinate and in addition failed to grow autotrophically. (9,32,33). A second fructosebisphosphatase gene required for heterotrophic growth is constitutively expressed (31,33).Transcription of the cbb operon is dependent on a LysRtype activator encoded by cbbR which binds to a low-and high-affinity site in the cbb operon promoter (45). Physiological studies have shown that high-level expression of the cbb operon is dependent on the absence of multicarbon substrates and the presence of methanol, formate, or hydrogen (9, 33). Although it has been established that CbbR is important in the transduction of these signals to the transcription apparatus (45), it is unclear how this is accomplished and whether CbbR is the only factor involved. To gain further insight into the molecular mechanism for the regulation of the cbb operon, a method that allowed the isolation of X flavus mutants with altered regulation of the cbb operon promoter was developed.The cbb operon promoter is not active during heterotrophic * Phone: (31)50-632150. Electronic mail address: meyerwg@biol.rug.nl. growth of X. flavus on succinate; however, when X flavus harbors multiple copies of the cbb operon promoter a low level of cbb operon promoter activity is observed during growth on succinate (26,30,32). This property was exploited to isolate X flavus mutants that lacked cbb operon promoter activity under these conditions and in addition failed to grow autotrophically. This paper describes the characterization of one of these mutants, X flavus G7, which was shown to be defective in phosphoglycerate kinase. This enzyme, which catalyzes the phosphorylation of 3-phosphoglycerate to 1,3-diphosphoglycerate, is required both for glycolysis and gluconeogenesis and for CO2 fixation via the Calvin cycle. In this paper, evidence that in contrast with the fructosebisphosphatase isoenzymes, X flavus employs only a single phosphoglycerate kinase that is responsible for both these functions is presented. MATERIALS AND METHODSBacterial strains and plasmids. The bacterial strains and plasmids used in this study are listed in Table 1.Media and growth conditions. Escherichia coli strains were grown on Luria-Bertani medium at 37°C (41). X flavus strains were grown on yeast extract (0.8% [wt/vol]) or in minimal media (27)...