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
. 2004 Aug 17;101(33):12306-11.
doi: 10.1073/pnas.0403547101. Epub 2004 Aug 3.

A eukaryotic BLUF domain mediates light-dependent gene expression in the purple bacterium Rhodobacter sphaeroides 2.4.1

Yuchen Han  1 , Stephan Braatsch, Lisa Osterloh, Gabriele Klug
Affiliations

A eukaryotic BLUF domain mediates light-dependent gene expression in the purple bacterium Rhodobacter sphaeroides 2.4.1

Yuchen Han et al. Proc Natl Acad Sci U S A. .

Abstract

The flavin-binding BLUF domain functions as a blue-light receptor in eukaryotes and bacteria. In the photoreceptor protein photo-activated adenylyl cyclase (PAC) from the flagellate Euglena gracilis, the BLUF domain is linked to an adenylyl cyclase domain. The PAC protein mediates a photophobic response. In the AppA protein of Rhodobacter sphaeroides, the BLUF domain is linked to a downstream domain without similarity to known proteins. AppA functions as a transcriptional antirepressor, controlling photosynthesis gene expression in the purple bacterium R. sphaeroides in response to light and oxygen. We fused the PACalpha1-BLUF domain from Euglena to the C terminus of AppA. Our results show that the hybrid protein is fully functional in light-dependent gene repression in R. sphaeroides, despite only approximately 30% identity between the eukaryotic and the bacterial BLUF domains. Furthermore, the bacterial BLUF domain and the C terminus of AppA can transmit the light signal even when expressed as separated domains. This finding implies that the BLUF domain is fully modular and can relay signals to completely different output domains.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.
Absorbance spectra of exponential APP11-derived strains listed in Table 1 grown under low oxygen tension (pO2 ≤ 3 μM). The absorbance maximum of BChl associated with the light-harvesting complex I is 875 nm, and those of BChl associated with the light-harvesting complex II are 800 and 850 nm. Colored carotenoids absorb in the range of 450–550 nm. Numbers refer to the strain constructs shown in Table 1.
Fig. 2.
Fig. 2.
Redox-dependent function of strain APP11 complemented with plasmid constructs listed in Table 1. During the time course of the experiments, the concentration of dissolved oxygen in the media was decreased from 200 μMto ≤3 μM. Total RNA was isolated at indicated time points, and puc transcript levels were monitored by RNA gel-blot analyses. A 14S rRNA-specific probe (14S rRNA is a product of 23S rRNA in vivo processing) (28) was used to show relative RNA loadings. Numbers refer to the strain constructs shown in Table 1.
Fig. 3.
Fig. 3.
Kinetics of puf and puc expression in R. sphaeroides APP11 strains caused by blue-light irradiation. Cells grown at 104 ± 24 μM dissolved oxygen were shifted from the dark into blue light or kept in the dark. (A) puc and puf expression changes in strain APP11(pRK4BLUF-E.g.), as determined by RNA gel-blot analyses. A 14S rRNA-specific probe was used to show relative RNA loadings. (B) The intensities of APP11(pRK4BLUF-E.g.) mRNA signals were quantified and normalized to the intensities of the rRNA signals. Percent of inhibition of normalized puc (□) and puf (•) mRNA levels were plotted. Inhibition in % = 100 × (1 – mRNA level in light irradiated cells/mRNA level in dark cells). (C) Luciferase-activity assays for puc expression in strains APP11(pBBRAppA170) (□) and APP11(p484-Nco5) (•). The relative light units (RLU·s–1) of light-irradiated cells were plotted after normalization to the optical density of the cultures at 660 nm. Each point and bar shows the mean and the SD, respectively, of three independent experiments. (D) Luciferase-activity assays for puc expression in strains APP11(pRK4BLUF-E.g.) (□) and APP11(p484-Nco5) (•). The relative light units of light-irradiated and dark cells were normalized to the optical density of the cultures at 660 nm and plotted as the percentage of inhibition. Each point and bar shows the mean and the SD, respectively, of three independent experiments.
Fig. 4.
Fig. 4.
Kinetics of puf and puc expression in R. sphaeroides APP11(pBBRAppA170)(p484-Nco5Δ) caused by blue-light irradiation. Cells grown at 104 ± 24 μM dissolved oxygen were shifted from the dark in to blue light or kept in the dark. (A) puc and puf expression changes as determined by RNA gel-blot analyses. A 14S rRNA-specific probe was used to show relative RNA loadings. (B) Quantification of puc (□) and puf (•) inhibition. The evaluation was performed as described in the Fig. 3B legend.
See this image and copyright information in PMC

References

    1. Bauer, C. E., Elsen, S. & Bird, T. H. (1999) Annu. Rev. Microbiol. 53, 495–523. - PubMed
    1. Gregor, J. & Klug, G. (1999) FEMS Microbiol. Lett. 179, 1–9. - PubMed
    1. Gregor, J. & Klug, G. (2002) J. Mol. Microbiol. Biotechnol. 4, 249–253. - PubMed
    1. Oh, J. I. & Kaplan, S. (2000) EMBO J. 19, 4237–4247. - PMC - PubMed
    1. Zeilstra-Ryalls, J. H. & Kaplan, S. (2004) Cell. Mol. Life Sci. 61, 417–436. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources