Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2002 May 14;99(10):7078-83.
doi: 10.1073/pnas.102013099. Epub 2002 Apr 30.

Repression of photosynthesis gene expression by formation of a disulfide bond in CrtJ

Shinji Masuda  1 , Chen Dong, Danielle Swem, Aaron T Setterdahl, David B Knaff, Carl E Bauer
Affiliations

Repression of photosynthesis gene expression by formation of a disulfide bond in CrtJ

Shinji Masuda et al. Proc Natl Acad Sci U S A. .

Abstract

Many species of purple photosynthetic bacteria repress synthesis of their photosystem in the presence of molecular oxygen. The bacterium Rhodobacter capsulatus mediates this process by repressing expression of bacteriochlorophyll, carotenoid, and light-harvesting genes via the aerobic repressor, CrtJ. In this study, we demonstrate that CrtJ forms an intramolecular disulfide bond in vitro and in vivo when exposed to oxygen. Mutational and sulfhydryl-specific chemical modification studies indicate that formation of a disulfide bond is critical for CrtJ binding to its target promoters. Analysis of the redox states of aerobically and anaerobically grown cells indicates that they have similar redox states of approximately -200 mV, thereby demonstrating that a change in midpoint potential is not responsible for disulfide bond formation. In vivo and in vitro analyses indicate that disulfide bond formation in CrtJ is insensitive to the addition of hydrogen peroxide but is sensitive to molecular oxygen. These results suggest that disulfide bond formation in CrtJ may differ from the mechanism of disulfide bond formation used by OxyR.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Examination of disulfide bond formation in CrtJ by AMS modification. (A) In lanes 1 and 3, purified CrtJ was either reduced by exposure to β-mercaptoethanol (β-ME) or oxidized by exposure to oxygen for 20 min before AMS treatment, respectively. Lane 2 is untreated CrtJ. (B) Lanes 1 and 3 are TCA extracts from aerobically and anaerobically grown cells, respectively, that were modified by AMS under anaerobic or aerobic conditions. Lane 2 is untreated extracts from aerobically grown cells. The mobility of CrtJ was assayed by Western blot analysis. Note that the gel system was modified slightly between A and B, which increases separation of unmodified CrtJ.
Figure 2
Figure 2
Redox titration of disulfide bond formation in CrtJ. The presence of reactive thiols was measured by analysis of the fluorescence emission level of CrtJ after incubation with mBBr at various redox values under anaerobic (argon) conditions. The filled circles represent the fluorescence emission at pH 8.0 and the open circles is at pH 7.0. The amplitudes are on a scale where the amplitude measured at the most negative redox value is a value of 1.0. The line represents a fit of the data to a two-electron Nernst curve. Eh (mV) denotes actual potentials.
Figure 3
Figure 3
Effect of oxygen on disulfide bond formation in CrtJ in vivo and in vitro. (A) In vivo analysis of CrtJ disulfide bond formation after exposure to oxygen. Lanes 1 and 6 depict the SDS/PAGE migration of CrtJ from anaerobically grown cells that were treated with AMS. Lanes 2 and 3 are of anaerobically grown cells that were exposed to 1.0 mM H2O2 for 10 and 30 min, respectively, before treatment with AMS. Lanes 4 and 5 are of anaerobically grown cells that were exposed to O2 for 10 or 30 min, respectively, before treatment with AMS. (B) In vitro analysis of CrtJ disulfide bond formation after exposure to oxygen. Lanes 3, 4, and 5 show effect of exposure of CrtJ to oxygen for 1, 5, and 10 min before treatment with AMS, respectively. Lanes 6–8 show the effect of a 5 min treatment of CrtJ to 0.01, 0.1, and 1.0 mM H2O2 before treatment with AMS, respectively. Lane 1 is a control that shows the migration of CrtJ that has not been exposed to AMS, whereas lane 2 is a control that shows the migration of reduced CrtJ that has been exposed to AMS. For optimal separation between oxidized and reduced CrtJ, the isolated protein used for this analysis contained a Cys → Ala mutation at position 22, which is a residue that does not undergo disulfide bond formation.
Figure 4
Figure 4
Inhibition of reversible redox responsive DNA-binding of CrtJ by iodoacetamide and site-directed mutagenesis. (A) 32P-labeled bchC promoter probe was incubated with purified wild-type CrtJ under various reaction conditions and then size fractionated by gel electrophoresis. Lane 1, DNA probe only. Lane 2, oxygen oxidized CrtJ incubated with the DNA probe. Lane 3, CrtJ incubated with 20 mM β-mercaptoethanol (β-ME) for 20 min and then oxidized by bubbling with pure oxygen for 20 min before addition of the DNA probe. Lane 4, CrtJ incubated with β-mercaptoethanol for 20 min before adding the DNA probe. Lane 5, CrtJ sequentially treated for 20 min with 20 mM β-mercaptoethanol, 50 mM iodoacetamide (Iodo), and then pure oxygen. Lane 6, oxygen-treated CrtJ was incubated with 50 mM iodoacetamide for 20 min followed by addition of the DNA probe. Lane 7, CrtJ sequentially incubated for 20 min with β-mercaptoethanol followed by 50 mM iodoacetamide. Lanes 2–7 each contained 9 pmol of CrtJ in the DNA-binding reactions. (B) Loss of in vitro DNA-binding activity of Cys mutants. Lane 1 is wild type CrtJ incubated with the bchC DNA probe, lane 2 is C249A mutant CrtJ, lane 3 is C420A mutant CrtJ, and lane 4 is only the bchC promoter probe. Each of the protein preparations were treated with oxygen for 20 min before addition of the DNA probe.
Figure 5
Figure 5
Measurement of aerobic expression of the pucB, crtI, and bchC promoters in the wild-type strain SB1003, the crtJ deletion strain CD2–4, and in the C22A, C249A, and C420A crtJ mutant strains. Reporter plasmids and β-galactosidase assays were as described in ref. . β-galactosidase activity refers to the amount of O-nitrophenyl-β-d-galactoside hydrolyzed per minute per milligram of protein. β-galactosidase activity is the average of three independent assays.
Figure 6
Figure 6
A comparative depiction of CrtJ and OxyR oxidation and reduction cycles that affect DNA-binding and subsequent repression or activation of target genes. GSH indicates reduced glutathione, which, we have demonstrated, is capable of reducing CrtJ disulfide bonds in vitro.
See this image and copyright information in PMC

References

    1. Cohen-Bazire G, Sistrom W R, Stanier R Y. J Cell Comp Physiol. 1957;49:25–68. - PubMed
    1. Bauer C E, Bird T H. Cell. 1996;85:5–8. - PubMed
    1. Bauer C E. In: Regulation of Photosynthesis. Aero E-M, Andersson B R, editors. Dordrecht, The Netherlands: Kluwer Academic; 2001. pp. 67–83.
    1. Ponnampalam S N, Buggy J J, Bauer C E. J Bacteriol. 1995;177:2990–2997. - PMC - PubMed
    1. Ponnampalam S N, Bauer C E. J Biol Chem. 1998;272:18391–18396. - PubMed

Publication types

MeSH terms

LinkOut - more resources