Isolation and characterization of Rhodobacter capsulatus mutants defective in oxygen regulation of the puf operon

Narro, Martha L.; Adams, Camellia; Cohen, Stanley N.

doi:10.1128/jb.172.8.4549-4554.1990

Cited by 37 publications

(19 citation statements)

References 40 publications

(31 reference statements)

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“…Mutational analysis of the puf promoter region has indicated that this promoter may be under control of a repressor, as evidenced by increased aerobic expression of some promoter point mutations (18). There has also been a report of an uncharacterized protein that binds to the puf operon promoter in an oxygen-dependent manner (13,14,15).…”

Section: Discussionmentioning

confidence: 98%

AerR, a Second Aerobic Repressor of Photosynthesis Gene Expression in Rhodobacter capsulatus

et al. 2002

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Open reading frame orf192, which is located immediately upstream of the aerobic repressor gene crtJ, was genetically and biochemically demonstrated to code for a second aerobic repressor (AerR) of photosynthesis gene expression in Rhodobacter capsulatus. Promoter-mapping studies indicate that crtJ has its own promoter but that a significant proportion of crtJ expression is promoted by read-through transcription of orf192 (aerR) transcripts through crtJ. Disruption of aerR resulted in increased photopigment biosynthesis during aerobic growth to a level similar to that of disruption of crtJ. Like that reported for CrtJ, ␤-galactosidase assays of reporter gene expression indicated that disruption of aerR resulted in a two-to threefold increase in aerobic expression of the crtI and pucB operons. However, unlike CrtJ, AerR aerobically represses puf operon expression and does not aerobically repress bchC expression. Gel mobility shift analysis with purified AerR indicates that AerR does not bind to a bchC promoter probe but does bind to the crtI, puc, and puf promoter probes. These results indicate that AerR is a DNA-binding protein that targets genes partially overlapping a subset of genes that are also controlled by CrtJ. We also provide evidence for cooperative binding of AerR and CrtJ to the puc promoter region.Oxygen tension is an important factor regulating synthesis and assembly of the photosynthetic apparatus in photosynthetic bacteria (7). Oxygen suppression of photosystem synthesis is mainly attributed to the activation or inactivation of many transcription factors that regulate photosynthesis gene expression. One well-characterized, oxygen-regulated transcription factor is the aerobic repressor CrtJ, which in the presence of oxygen represses bacteriochlorophyll (bch), carotenoid (crt), and respiratory (29) and light harvesting-II (puc) (22) gene expression. It has recently been demonstrated that exposure of CrtJ to oxygen results in the formation of an intramolecular disulfide bond and that formation of this bond is needed for binding of CrtJ to its target promoters (S. Masuda, C. Dong, D. Swem, and C. E. Bauer, unpublished data). Another wellcharacterized redox regulator is the sensor kinase RegB, which, together with its cognate response regulator RegA, is responsible for the global control of many aerobically and anaerobically regulated cellular processes (3). This includes photosynthesis (17, 26), nitrogen fixation (8), hydrogen utilization (8), carbon fixation (32), respiration (29, 30), and cytochrome biosynthesis (29,30).In this study, we have identified a new transcription factor coded by an open reading frame (orf192) located just upstream of crtJ in the previously sequenced Rhodobacter capsulatus photosynthesis gene cluster (1). Disruption of orf192 indicates that it codes for an aerobic repressor (AerR) of carotenoids and bacteriochlorophyll and for the reaction center and light-harvesting apoproteins. The results of gel mobility shift assays indicate that AerR binds to the pufQ, crtA-crtI, and p...

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Section: Discussionmentioning

confidence: 98%

AerR, a Second Aerobic Repressor of Photosynthesis Gene Expression in Rhodobacter capsulatus

et al. 2002

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“…Vitreoscilla was routinely grown in PYA medium (1% peptone, 1% yeast extract, and 0.02% sodium acetate, pH 7.8) at 25°C. E. coli strains were grown in Luria Broth (1% tryptone, 0.5% yeast extract, and 1% NaCl, pH 7.5) at 37°C, and the oxygen level was adjusted according to the method adopted by Narro et al (28) and as described previously (29). The plasmid, Bluescript KS(ϩ), was obtained from Stratagene and the expression-secretion vector, pTMNO (30), was used for the generation of the ompA-vgb gene fusion.…”

Section: Methodsmentioning

confidence: 99%

Vitreoscilla Hemoglobin

Ramandeep

Hwang

Raje

et al. 2001

Journal of Biological Chemistry

136

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The obligate aerobic bacterium, Vitreoscilla, synthesizes elevated quantities of a homodimeric hemoglobin (VHb) under hypoxic growth conditions. Expression of VHb in heterologous hosts often enhances growth and product formation. A role in facilitating oxygen transfer to the respiratory membranes is one explanation of its cellular function. Immunogold labeling of VHb in both Vitreoscilla and recombinant Escherichia coli bearing the VHb gene clearly indicated that VHb has a cytoplasmic (not periplasmic) localization and is concentrated near the periphery of the cytosolic face of the cell membrane. OmpA signal-peptide VHb fusions were transported into the periplasm in E. coli, but this did not confer any additional growth advantage. The interaction of VHb with respiratory membranes was also studied. The K d values for the binding of VHb to Vitreoscilla and E. coli cell membranes were ϳ5-6 M, a 4 -8-fold higher affinity than those of horse myoglobin and hemoglobin for these same membranes. VHb stimulated the ubiquinol-1 oxidase activity of inverted Vitreoscilla membranes by 68%. The inclusion of Vitreoscilla cytochrome bo in proteoliposomes led to 2.4-and 6-fold increases in VHb binding affinity and binding site number, respectively, relative to control liposomes, suggesting a direct interaction between VHb and cytochrome bo.

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“…1). The pMLN plasmids carry the RK2 replicon, and their construction has been described previously (16). The base pair exchange in the puf upstream region present in plasmid pMLN44 is indicated in Fig.…”

Section: Methodsmentioning

confidence: 99%

“…The arrow above the puf sequence points to the C nucleotide that is changed to a T in pMLN44. The arrows below the puf DNA sequence indicate a dyad symmetry involved in the oxygen regulation of puftranscription (16). The numbering of the puf sequence is with respect to the transcriptional start, which is 0.…”

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confidence: 99%

A base pair transition in a DNA sequence with dyad symmetry upstream of the puf promoter affects transcription of the puc operon in Rhodobacter capsulatus

Klug¹,

Jock²

1991

J Bacteriol

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A DNA sequence with dyad symmetry upstream of the transcriptional start of the Rhodobacter capsulatus puf operon, which encodes pigment-binding proteins of the light-harvesting I complex and of the reaction center, has previously been shown to be a protein-binding site (G. Klug, Mol. Gen. Genet. 226:167-176, 1991). When a low-copy-number plasmid with a base pair transition at position -43 within this dyad symmetry in front of the puf structural genes was transferred into a Rhodobacter strain with the puf operon deleted, different phenotypes occurred during cultivation of the transconjugants and the kinetics of the loss of the wild-type phenotype was dependent on the oxygen tension in the culture. After growth for 150 generations, the different phenotypes were stably inherited. The strains having the wild-type phenotype carried the wild-type puf DNA sequence. The original mutation was still present in the strains that showed lighter color. These strains had less light-harvesting H complex in the membrane and showed lower rates of transcription of the puc operon, which encodes the proteins of this complex. This deregulation of puc expression was due to one or more chromosomally located, secondary mutations, not directly to the mutation present on the plasmid. Thus, a single-base-pair transition in the pufupstream region can result in a deregulation ofpuc expression, suggesting a direct or indirect transcriptional coregulation of both these operons by a common factor.In Rhodobacter capsulatus, two light-harvesting complexes (LHI and LHII) transfer energy to the reaction center (RC), where the photosynthetic energy conversion takes place. The formation of the photosynthetic apparatus is strongly induced when the oxygen tension drops below a certain threshold value (7). A sequential increase of LHI/RC and LHII complexes that is correlated with a sequential increase of LHI/RC-and LHII-specific mRNA, respectively, was reported (12), suggesting a coregulation of the formation of the three types of pigment-protein complexes.The six pigment-binding proteins involved in the formation of these photosynthetic complexes are encoded by two different operons. The genes for the LHII-specific pigmentbinding proteins (genes pucB and pucA) are part of the polycistronic puc operon (19,22) (Fig. 1). The polycistronic pufoperon encodes the pigment-binding proteins of the LHI antenna complex (genes puJR and pufA) and of the reaction center (genes puff and pu.fM) and includes two open reading frames, pufQ and pufX (2,3,21) (Fig. 1).Recently, the promoters of the puf and puc operons were identified (2, 24), and it became possible to study the effect of oxygen on the transcription of LHI/RC and LHII specific genes. An oxygen-regulated promoter was identified about 200 bp upstream of the first open reading frame, pufQ, and it was shown by lacZ fusions that the rate of transcription starting at this promoter was higher when cells were grown under a low oxygen concentration than when they were grown under a high oxygen concentration (1, 2). ...

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Isolation and characterization of Rhodobacter capsulatus mutants defective in oxygen regulation of the puf operon

Cited by 37 publications

References 40 publications

AerR, a Second Aerobic Repressor of Photosynthesis Gene Expression in Rhodobacter capsulatus

AerR, a Second Aerobic Repressor of Photosynthesis Gene Expression in Rhodobacter capsulatus

Vitreoscilla Hemoglobin

A base pair transition in a DNA sequence with dyad symmetry upstream of the puf promoter affects transcription of the puc operon in Rhodobacter capsulatus

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