DNA sequencing and complementation/deletion analysis of the bchA‐puf operon region of Rhodobacter sphaeroides: in vivo mapping of the oxygen‐regulated puf promoter

Hunter, C. Neil; McGlynn, Peter; Ashby, Mark K.; Burgess, J. Grant; Olsen, John D.

doi:10.1111/j.1365-2958.1991.tb01974.x

Cited by 56 publications

(34 citation statements)

References 46 publications

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“…The pufB, pufA, pufL and pufM genes reside within the same operon and are transcribed in the order BALM [6]. There are two additional genes within thepuf operon: upstream of pufB there is pufQ, which has been implicated in bacteriochlorophyll synthesis [7,8], and downstream of pufM there is pufX [9]. The pufX gene encodes a protein of 82 amino acids which has one possible membranespanning helix [9].…”

Section: Introductionmentioning

confidence: 99%

The Rhodobacter sphaeroides PufX protein is not required for photosynthetic competence in the absence of a light harvesting system

1994

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The effects of deletion of the gene encoding the PufX protein from Rhodobacter sphaeroides have been examined using bacterial strains with simplified photosystems. We find that the PufX protein is required for photosynthetic growth in strains which have the LHI antenna complex, but is not required in a reaction centre-only strain, suggesting that the PufX protein does not directly facilitate cyclic electron transfer between the reaction centre and the cytochrome bc, complex. The influence of PufX and carotenoid type on the size of the reaction center/LHl core complex has also been examined in these strains.

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Section: Introductionmentioning

confidence: 99%

The Rhodobacter sphaeroides PufX protein is not required for photosynthetic competence in the absence of a light harvesting system

1994

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“…The search for elements involved in the regulation of Bchl biosynthesis caused attention to be focused on the pufQ open reading frame located at the extreme 5' end of thepuf operon in both R. capsulatus (1,3,4,17,23) and R. sphaeroides (15,22 ThepufQ gene was then altered by site-directed mutagenesis to produce a sequence, 5' of the coding region, which is a consensus ribosome-binding site (RBS) of genes that are well expressed in E. coli (36). An XbaI restriction site was also introduced at the extreme 5' end of the initiation codon to allow facile subcloning of the pufQ gene in the fusion vectors.…”

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Recombinant expression of the pufQ gene of Rhodobacter capsulatus

et al. 1993

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Genetic studies have shown that the expression of the pufQ gene is required for normal levels of bacteriochlorophyll biosynthesis in Rhodobacter capsulatus. Yet, the exact function of the puJQ gene is unknown, and a puJQ gene product has never been isolated. We describe the recombinant overexpression of pufQ in Escherichia coli, as well as the purification and characterization of its gene product, the 74-amino-acid PufQ protein. Site-directed of R. capsulatus into operons and superoperons has recently been reviewed (37). The relative positions of the puf and puh operons and many of the bch and crt genes in the closely related bacterium Rhodobacter sphaeroides are similar to those found for the equivalent genes of R capsulatus (14). However, the distance between the puf and puh operons is some 10 kb shorter (40), and the photosynthetic gene cluster extends to include genes encoding other photosynthetic components, including cytochrome c2 (cycA) and the a-and 1-subunits of the B800-850 light-harvesting complex II (pucB and pucA, respectively, in the puc operon) (40).The biosynthesis of Bchl in both R. capsulatus and R.

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“…The presence of CrtI in the cytoplasm of E. coli may be due to the fact that there is so much of it in the cell that it cannot all become membrane associated. The puc and puf operons, which encode polypeptides of the photosynthetic apparatus in R. sphaeroides and R. capsulatus, have been shown to be highly regulated by oxygen and light and are repressed under high-oxygen conditions (16,31,35). The alignment of the sequences of the putative promoters for crtI and crtB with the promoter sequences from the R sphaeroidespufoperon (31) andpuc operon (43) shows a significant degree of homology between these sequences (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Sequences upstream of the transcriptional start of crtI and crtB which show significant homology to the sequences of the promoters from the R. sphaeroides puf operon (31) have been located (Fig. 3).…”

Section: Resultsmentioning

confidence: 99%

Early steps in carotenoid biosynthesis: sequences and transcriptional analysis of the crtI and crtB genes of Rhodobacter sphaeroides and overexpression and reactivation of crtI in Escherichia coli and R. sphaeroides

et al. 1994

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In the purple photosynthetic bacterium Rhodobacter sphaeroides, the desaturation of phytoene has already been implicated in the assembly of the light-harvesting 2 complex (H. P. Lang and C. N. Hunter, Biochem. J. 298:197-205, 1994). The phytoene synthase and desaturase enzymes mediate the first steps specific for carotenoid biosynthesis up to and including the synthesis of the colored carotenoid neurosporene. In this report, we present the DNA and deduced amino acid sequences of the genes encoding these proteins, namely, crtB and crtI, from R. sphaeroides and present evidence for the existence of a crtIB operon. Both genes have been shown to possess putative puc and puf operon-like promoter sequences, and oxygen regulation and the point of initiation of the crtI transcript have been demonstrated. The complete crtI gene has been overexpressed in Escherichia coli and R. sphaeroides and shown to catalyze three desaturations of phytoene to give neurosporene. This activity was showti to be ATP dependent, and the cofactor requirement was investigated by using a spectroscopic assay for in vitro carotenogenic activity. Although the crtI and crtB genes have been sequenced from a number of different organisms, the transcriptional organization and regulation of these genes have not been analyzed in detail. In this report, we have located the transcription initiation point and have shown that R. sphaeroides possesses an oxygen-regulated CrtI-type phytoene desaturase gene that forms a transcriptional operon with crtB.Carotenoid pigments are unsaturated hydrocarbons that are produced in the general isoprenoid pathway. The first step that is specific for carotenoid biosynthesis is the condensation of two molecules of geranylgeranyl pyrophosphate to yield phytoene (10). This reaction is mediated by the CrtB, or phytoene synthase, enzyme (12,21,47,55). Phytoene has only a short chromophore of three conjugated double bonds and is therefore colorless and incapable of photoprotection. Its conversion into colored carotenoids requires a series of desaturation reactions to extend the chromophore (10), and these are mediated by the phytoene desaturase (CrtI) enzyme (28).Phytoene synthase and desaturase genes have been isolated and sequenced from a variety of bacteria and fungi (see reference 53 for a review; 46) as well as from cyanobacteria (12, 13) and higher plants (7,8,49 thesis as light-gathering pigments. This role is carried out efficiently as a result of specific attachment to reaction center and light-harvesting pigment proteins. A complete description of the role of carotenoids in the assembly and function of photosynthetic complexes and the coordination of pigment biosynthetic pathways with membrane protein insertion requires a high level of structural and spectroscopic information as well as the availability of molecular genetic techniques. Rhodobacter sphaeroides is an ideal model system for studies of light-harvesting processes since it is genetically well defined and there is a crystallographically determined structure ...

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DNA sequencing and complementation/deletion analysis of the bchA‐puf operon region of Rhodobacter sphaeroides: in vivo mapping of the oxygen‐regulated puf promoter

Cited by 56 publications

References 46 publications

The Rhodobacter sphaeroides PufX protein is not required for photosynthetic competence in the absence of a light harvesting system

The Rhodobacter sphaeroides PufX protein is not required for photosynthetic competence in the absence of a light harvesting system

Recombinant expression of the pufQ gene of Rhodobacter capsulatus

Early steps in carotenoid biosynthesis: sequences and transcriptional analysis of the crtI and crtB genes of Rhodobacter sphaeroides and overexpression and reactivation of crtI in Escherichia coli and R. sphaeroides

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