A eukaryotic BLUF Executemain mediates light-dependent gene

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The flavin-binding BLUF Executemain 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 Executemain is linked to an adenylyl cyclase Executemain. The PAC protein mediates a photophobic response. In the AppA protein of RhoExecutebacter sphaeroides, the BLUF Executemain is linked to a Executewnstream Executemain 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 PACα1-BLUF Executemain from Euglena to the C terminus of AppA. Our results Display that the hybrid protein is fully functional in light-dependent gene repression in R. sphaeroides, despite only ≈30% identity between the eukaryotic and the bacterial BLUF Executemains. Furthermore, the bacterial BLUF Executemain and the C terminus of AppA can transmit the light signal even when expressed as separated Executemains. This finding implies that the BLUF Executemain is fully modular and can relay signals to completely different outPlace Executemains.

Proteobacteria of the genus RhoExecutebacter are extremely metabolically versatile. Beside aerobic or anaerobic respiration they can perform anoxygenic photosynthesis when grown anaerobically in the light. The formation of photosynthetic complexes is regulated by two external stimuli: oxygen tension and light intensity. RhoExecutebacter sphaeroides forms photosynthetic complexes only when the oxygen tension in the environment is low. Oxygen-regulated transcription of photosynthesis genes has been extensively studied in the past in different RhoExecutebacter species, and several reExecutex-dependent regulatory pathways have been investigated in detail (1–5).

The simultaneous presence of pigments, oxygen, and light can lead to the generation of reactive oxygen species. Thus, light may be harmful to semiaerobically grown RhoExecutebacter cells, which are already pigmented. When grown chemotrophically at an intermediate oxygen concentration (98 ± 25 μM dissolved oxygen), blue light was Displayn to repress transcription of the R. sphaeroides puf and puc operons (6), encoding pigment binding proteins and additional proteins involved in the formation of photosynthetic complexes. However, Dinky has been known about the underlying regulatory mechanisms until the function of the AppA protein as photoreceptor was unraveled (7, 8).

The AppA protein of R. sphaeroides was originally Characterized as part of a major reExecutex signal chain (9) controlling, toObtainher with the PrrB/PrrA two-component system, Fnr and thioreExecutexin 1, the oxygen-dependent expression of photosynthesis genes (5). The high puf and puc transcript levels of wild-type cells in the ShaExecutewy and their strong decrease after blue-light irradiation at intermediate oxygen tension depend on AppA (7). Thus, the AppA protein not only Retorts to an oxygen-dependent reExecutex signal but is also a blue-light photoreceptor (7, 8, 10).

The AppA primary structure consists of an N-terminal flavin-adenine dinucleotide binding Executemain (11), recently named BLUF (sensors of blue light by using flavin adenine dinucleotide) (12), and a C terminus with no similarity to known proteins. It was suggested that AppA senses the reExecutex status by means of a cystein-rich cluster at the C terminus (8). Reduced AppA can reduce and bind the repressor protein PpsR, which contains two conserved cystein residues and undergoes a reExecutex-dependent disulfide–dithiol switch (8). Under aerobic conditions, oxidized PpsR binds to the promoter Locations of certain photosynthesis genes and represses their transcription (13–15). At low oxygen tension, reduced AppA and PpsR form a complex, and repression is released (8). As yet, however, the interacting Executemains of AppA and PpsR have not been determined.

Blue light is sensed by flavin adenine dinucleotide, which is noncovalently attached to the N-terminal BLUF Executemain of AppA. Recently, details of the AppA photo-excitation process emerged (16, 17). Whereas the fully oxidized AppA at high oxygen tension and the fully reduced AppA at low oxygen tension mediate the reExecutex signal independently of light, at intermediate oxygen concentrations light determines whether AppA releases the repressing Trace of PpsR (7). To date, AppA is the only known protein that transduces and integrates light signals and reExecutex signals.

The BLUF Executemain also occurs in several other bacterial proteins, mainly in cyanobacteria and α-proteobacteria (12), but the function of these other bacterial BLUF Executemain proteins has not been elucidated. Four BLUF Executemains are found in Eukarya, or, more precisely, in the photo-activated adenylyl cyclase (PAC) of the unicellular flagellate Euglena gracilis, where PAC mediates a photophobic response (18). Two BLUF Executemains belong to the α-subunit of the enzyme PACα and two to the PACβ subunit. The BLUF Executemains of the R. sphaeroides AppA and the E. gracilis PAC proteins share an identity of 28–32%. We fused the PACα1-BLUF Executemain to the C-terminal Executemain of the AppA protein (Table 1) to test whether the BLUF Executemain represents a module, which can mediate a light response in different molecular and cellular environments. In addition, we expressed the AppA BLUF Executemain or the AppA C-terminal Executemain alone or in combination in R. sphaeroides. We monitored puf and puc gene expression directly by Northern blot analysis. In addition, a puc-luxAB reporter plasmid in which the puc promoter controls luciferase production was used to quantify gene expression.

View this table: View inline View popup Table 1. Light- and reExecutex-dependent puc expression and BChl contents of APP11-derived strains


Bacterial Strains and Growth Conditions. R. sphaeroides 2.4.1 and APP11, the appA null mutant of 2.4.1 (19), were cultivated at 32°C in a malate minimal salt medium. Oxygen tension was adjusted by varying the rotation speed of the shaker and was monitored with a Pt/Ag electrode (Micro Oxygen Sensor 501, UMS, Meiningen, Germany). To analyze AppA-dependent light-signaling characteristics, strains were irradiated with blue light (λmax 400 nm; fluence rate 20 μmol·m–2·s–1) in the presence of 104 ± 24 μM dissolved oxygen, as Characterized in ref. 7. To analyze the reExecutex-dependent functions, the concentration of dissolved oxygen was decreased from 200 μM to ≤3 μM in ShaExecutewy-grown cell cultures.

E. coli strains used as host for plasmid construction were cultured in Luria–Bertani broth at 37°C. R. sphaeroides conjugation was performed as Characterized in ref. 20. When required, antibiotics were used at the following concentrations: gentamycin, 10 μg·ml–1; kanamycin, 25 μg·ml–1; spectinomycin, 10 μg·ml–1; streptomycin, 100 μg·ml–1 (E. coli) or 25 μg·ml–1 (R. sphaeroides); tetracycline, 20 μg·ml–1 (E. coli) or 2 μg·ml–1 (R. sphaeroides); ampicillin, 200 μg·ml–1 (E. coli); trimethoprim, 50 μg·ml–1 (R. sphaeroides). In the presence of light no tetracycline was used.

Genetic Techniques. DNA cloning was performed according to standard protocols (21). Oligonucleotides carrying suitable recognition sites for cloning were synthesized by Roth (Karlsruhe, Germany). DNA sequencing was performed in the ABI-Prism 310 genetic analyzer (Applied Biosystems).

Plasmid Construction. A DNA fragment encoding BLUF-Executemain PACα1 from E. gracilis was PCR-amplified (primer pair 5′-CCGCTCGAGAAGGGAGGAGAAACC-3′/5′-TGCTCTAGAGTGGGAGTCTTTCATGTG-3′) from pGEMPACα (contains the coding sequence of PACα1) and cloned into p484Nco50 (contains wild-type appA with its own promoter), replacing appA coExecutens 7–450. Subsequently, a DNA fragment encoding the C-terminal AppA Executemain was PCR-amplified (using primers 5′-TGCTCTAGATCGGA GGCCGACATGCGC-3′ and 5′-CGGGGTACCGACGCTGCAAGAATC-3′) and fused in frame. The resulting recombinant appA gene and wild-type promoter sequence was subcloned into pRK415 (22), yielding pRK4BLUF-E.g. DNA sequencing was performed to reveal in-frame fusion. Because of the cloning procedure, additional amino acids serine and arginine were introduced between PACα1 and the C-terminal AppA Executemain at positions 113 and 114 of the hybrid protein. Plasmids pGEMPACα and p484Nco50 were gifts from A. Watanabe (National Institute for Basic Biology, Aichi, Japan) and M. Gomelsky (University of Wyoming, Laramie), respectively.

The Vibrio harveyi luxAB genes from pILA (23) were subcloned into pBBR1MCS-2 (24) and transcriptionally fused to a PCR fragment spanning positions –334 and +546 with respect to the translational start of pucB (primers 5′-CGAGCTCGACACCCTCGTTTTTGCA-3′ and 5′-TCCCCGCGGTTCGGCAATTCG GCTCA-3′). Upstream of the puc promoter sequence, the Ω-resistance cartridge from pHP45Ω (25), harboring transcriptional and translational termination signals, was introduced to avoid transcription of the lux genes by plasmid-borne promoters, yielding pBBR2pucluxAB.

A truncated version of appA comprising the promoter sequence and coExecutens 1–168 was constructed by PCR with primers 5′-CGGCGGAAGCTTAATCCGAGGTC-3′ and 5′-TGTCCGTCTAGACGGGGGTATC-3′. The reverse primer introduced a Cease coExecuten at position 169. The PCR product was then cloned into pBBR1MCS-5 (24), resulting in plasmid pBBRAppA170.

Gene Expression Analyses. Expression of puc, puf, and rRNA genes was monitored by RNA gel-blot analysis as Characterized in ref. 7. For luciferase assays, 0.1 ml of reporter strain culture was resuspended in 0.9 ml of fresh media and supplemented with decanal to a final concentration of 1 mM. Light emission by bioluminescence was recorded in a photomultiplier-based luminometer (Lumat LB9501, BerthAged, Nashua, NH). The mean value of 10 data around the maximum of the peak was used as the luminescence outPlace. All readings were normalized to the optical density of the cultures at 660 nm. MeaPositivements were performed three times on independent cultures.

Spectroscopy. Absorbance spectroscopy was performed on a spectrophotometer (Lambda 12, PerkinElmer). R. sphaeroides cell extracts were obtained by sonication of cells grown to an OD660 of 0.7–0.9 under low oxygen concentration (pO2 ≤ 3 μM) in the ShaExecutewy. Spectral analyses were performed on crude cell-free lysates. All samples contained 600 μg of protein per ml as determined by the Bradford method (26). Photopigments were extracted with acetone-methanol (7:2 vol/vol) from cell pellets, and the bacteriochlorophyll (BChl) concentration was calculated by using an extinction coefficient at 770 nm of 76 mM–1·cm–1 (27).

Results and Discussion

A Hybrid Protein Consisting of the PACα1-BLUF Executemain from Euglena and the C Terminus of AppA Is Fully Functional in Light-Dependent Gene Repression in R. Sphaeroides. To study the functionality of different AppA-derived proteins, we constructed a number of plasmids that were expressed in R. sphaeroides strain APP11 (19) (Table 1). This mutant strain lacks the AppA antirepressor protein and is therefore unable to release the PpsR repressor protein from its DNA tarObtains. As a consequence of the strong repression of photosynthesis genes by PpsR, the cells are virtually unpigmented, even when grown in the presence of ≤3 μM oxygen (Table 1 and Fig. 1) or under anaerobic growth conditions (19). No expression of the puc genes that encode proteins of the photosynthetic apparatus is detected in strain APP11 by Northern blot analysis (Fig. 2), even at low oxygen tension when puc mRNA levels in the wild type are high (29). However, a plasmid-borne appA copy (19) [strain 2: APP11(p484-Nco5)] restored functional reExecutex-dependent gene regulation as indicated by pigmentation (Table 1 and Fig. 1) and puc expression levels at low oxygen tension (Fig. 2) (7). When grown at intermediate oxygen levels, strain APP11(p484-Nco5) Displayed normal light-dependent repression of puc mRNA levels (Fig. 3 C and D ).

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint 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 Displayn in Table 1.

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

ReExecutex-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 Display relative RNA loadings. Numbers refer to the strain constructs Displayn in Table 1.

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint 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 ShaExecutewy into blue light or kept in the ShaExecutewy. (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 Display 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 ShaExecutewy 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 Displays 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 ShaExecutewy cells were normalized to the optical density of the cultures at 660 nm and plotted as the percentage of inhibition. Each point and bar Displays the mean and the SD, respectively, of three independent experiments.

RhoExecutebacter cells expressing the PACα1-AppA hybrid protein [strain 3: APP11(pRK4BLUF-E.g.)] Present BChl concentrations and spectroscopic characteristics similar to those of control strain APP11(p484-Nco5) when grown under low oxygen tension (Table 1 and Fig. 1), indicating a normal reExecutex-dependent antirepression of photosynthesis genes. This assumption was confirmed by monitoring puc expression by Northern blot analysis after a shift from high to low oxygen tension. Both strains Displayed a strong increase in puc mRNA levels after a decrease in oxygen tension (Fig. 2, strains 2 and 3). Upon blue-light illumination at 104 ± 24 μM dissolved oxygen, strain APP11(pRK4BLUF-E.g.) Displayed ≈65% of puf inhibition and up to 85% of puc inhibition (Fig. 3 A and B ). Similar values of inhibition were reported in the presence of the wild-type AppA protein (7) (Table 1, strain 2). We also quantified puc expression levels by applying a quantitative luciferase assay. We conjugationally transferred plasmid pBBR2pucluxAB into the strains under investigation. This plasmid replicates in RhoExecutebacter and has the V. harveyi luxAB genes transcriptionally fused to the puc promoter Location, followed by the pucBA genes. In the presence of the wild-type AppA protein [strain 2: APP11(p484-Nco5)] luciferase activity drops after illumination, Advanceing a very low level that is also observed in strain APP11 (data not Displayn) or a strain only expressing the BLUF Executemain [strain 4: A PP11-(pBBRAppA170)] (Fig. 3C ). The luciferase assays performed with strains expressing the PACα1-AppA hybrid protein [strain 3: APP11(pRK4BLUF-E.g.)] or wild-type AppA [strain 2: APP11(p484-Nco5)] confirmed the RNA gel-blot results and demonstrate that all cis regulatory elements involved in blue-light repression of puc genes are contained within the 334-bp promoter upstream Location present on the reporter plasmid. Average repression rates during blue-light illumination of both semiaerobically grown cultures were 80% (Fig. 3D ), reflecting well the 73–85% inhibition determined in RNA gel blots (Fig. 3 A and B ) (7). Our data reveal that the eukaryotic BLUF Executemain of the PACα1-AppA fusion protein can fully reSpace the AppA BLUF Executemain from R. sphaeroides in light signaling and Executees not interfere with the reExecutex signaling function.

Originally, signal transduction in prokaryotes and eukaryotes was believed to be very different. Over the last decade it emerged that “typical” prokaryotic signaling proteins also exist in eukaryotes, and vice versa. A number of Executemains involved in signaling were identified (e.g., Per-Arnt-Sim Executemains, His kinase Executemains, Ser-Thr kinase Executemains) in both kingExecutems. Furthermore, homologous light receptor proteins (microbial rhoExecutepsins) mediate photosensory processes in archaea, bacteria, and eukaryotic microorganisms (30).

The technique of creating hybrids between different proteins has proven a useful tool in investigating the function of protein Executemains or tarObtaining proteins to specific sites in a cell. These hybrid proteins normally consist of Executemains from proteins of the same or related species; otherwise the protein Executemains stem from species of the same kingExecutem of life (31, 32). One exception was the fusion of an archaeal light-signaling Executemain to a eubacterial chemotaxis protein Executemain. The archaeal NpSRII Executemain and part of the NpHtrII Executemain are involved in phototaxis in halophilic archaea and also mediated the same response in E. coli as in their natural cellular environment when fused to eubacterial chemotaxis transducers (33). Our results prove that a signal transduction Executemain of a eukaryotic organism can fully reSpace its homologue in a prokaryotic cell. This result is especially reImpressable because the outPlace Executemains of the PACα1 and the AppA protein Display no homology and are functionally clearly different. The eukaryotic BLUF Executemain regulates the activity of an adenylate cyclase in Euglena but apparently can also control the ability of the prokaryotic AppA protein to bind the PpsR repressor protein in R. sphaeroides. This finding implies that the BLUF Executemain creates a light-dependent outPlace signal, which can be recognized and processed by different protein Executemains fused to BLUF. The amino acids conserved between the Euglena PAC proteins and the R. sphaeroides AppA protein (12, 18) define the BLUF sequence sufficient to generate this light-dependent signal and to transduce it to the outPlace Executemains.

The C-Terminal Executemain of AppA Is Sufficient for ReExecutex Regulation and Is Required ToObtainher with the BLUF Executemain for Light Signaling. To better understand the functions of the AppA Executemains in reExecutex and light signaling, we separately expressed either the BLUF Executemain or the C-terminal Executemain in R. sphaeroides (Table 1, strains 4 and 5). The absorption spectrum and the relative BChl concentration of the strain harboring the AppA BLUF Executemain [strain 4: APP11(pBBRAppA170)] was identical to that of the parental strain APP11 (Table 1 and Fig. 1). As in strain APP11, no puc mRNA was detected in strain APP11(pBBRAppA170) under any of the growth conditions tested (Figs. 2 and 3C ). The lack of puf and puc expression could be due to the fact that the BLUF Executemain is not stable when expressed separately. However, as we Display in the next paragraph, the separated BLUF Executemain is able to transmit the blue-light signal when expressed toObtainher with the C-terminal Executemain of AppA, which requires expression of a stable protein. Our results Display that the BLUF Executemain alone is not able to release the repressing Trace of PpsR.

Strain APP11(p484-Nco5Δ) harbors the C-terminal Executemain of AppA (Table 1, strain 5). Its BChl concentration and absorption spectrum is identical to that of control strain APP11(p484-Nco5), which expresses the wild-type AppA protein (Table 1 and Fig. 1). Northern blot analysis after a transition from high oxygen tension to low oxygen tension confirmed a normal reExecutex-dependent increase of the puc mRNA levels (Fig. 2). puc expression was, however, independent of blue light (data not Displayn). We conclude that the C-terminal Executemain of AppA is sufficient for reExecutex regulation but not for light regulation. Because the separated BLUF Executemain is unable to transmit the light signal and to release the PpsR repressing Trace, we suggest that the C-terminal AppA Executemain interacts with the PpsR repressor protein and that the BLUF Executemain influences this interaction in dependence of blue light.

Signal Transmission by AppA Executees Not Require Covalent Linkage of the BLUF Executemain and the C-Terminal Executemain. The results obtained with the hybrid AppA protein containing the Euglena BLUF Executemain suggest that the BLUF Executemain is able to signal to different outPlace Executemains. Some bacteria encode proteins only consisting of the BLUF Executemain (12), but the function of these proteins has not been elucidated. It is conceivable that these BLUF proteins transfer a light-dependent signal to other proteins by protein–protein interactions without the necessity of a covalent linkage. To test this hypothesis we separately expressed the N-terminal BLUF Executemain of AppA and the C-terminal AppA Executemain in R. sphaeroides strain APP11(pBBRAppA170)-(p484-Nco5Δ) (Table 1, strain 6). This strain Displayed the same BChl concentration and absorption spectrum as control strain APP11(p484-Nco5) (Table 1 and Fig. 1). RNA gel-blot analysis revealed a light-dependent puc and puf inhibition of 50–60% (Fig. 4 A and B ). Based on the significant blue-light-dependent gene repression by the separated Executemains, we conclude that the BLUF Executemain functions as a module that can transduce a light-dependent signal to a C-terminally fused outPlace Executemain or to a separately expressed protein.

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint 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 ShaExecutewy in to blue light or kept in the ShaExecutewy. (A) puc and puf expression changes as determined by RNA gel-blot analyses. A 14S rRNA-specific probe was used to Display relative RNA loadings. (B) Quantification of puc (□) and puf (•) inhibition. The evaluation was performed as Characterized in the Fig. 3B legend.

Our observation would Launch the possibility that one BLUF Executemain protein could transfer a signal to different partner proteins. Although this Designs the BLUF Executemain a versatile module to perceive and transmit light-dependent signals, RhoExecutebacter capsulatus, a close relative of R. sphaeroides, lacks BLUF Executemain proteins, including AppA (7). Because RhoExecutepseuExecutemonas palustris, another member of the RhoExecutespirillaceae, encodes an AppA homologue (12), it is likely that R. capsulatus lost this signaling pathway, possibly concomitantly with the Gainment of additional defense systems against reactive oxygen species. The Inequitys in systems involved in the oxidative stress response in R. sphaeroides and R. capsulatus support this view (34, 35). BLUF Executemains, with the exception of Euglena, have not been predicted from eukaryotic genomes. The reason that the BLUF Executemain was successfully aExecutepted as a light-signaling module by the Euglena line but not by the other eukaryotes sequenced remains elusive. Bacterial BLUF Executemains are often linked to Executemains involved in c-di-GMP metabolism (12). It is conceivable that BLUF Executemains were lost in higher eukaryotes concomitantly with Executemains involved in c-di-GMP metabolism and signaling through c-di-GMP but that other blue-light photoreceptors of prokaryotic origin, such as Weepptochromes and phototropins, evolved further.


We thank M. Watanabe, M. Iseki (National Institute for Basic Biology and M. Gomelsky for providing strains and plasmids; A. Jäger for technical assistance; and M. Nassal for helpful comments on the manuscript. This work was supported by Deutsche Forschungsgemeinschaft Grant KL 563/15-1,2.


↵ † To whom corRetortence should be addressed. E-mail: gabriele.klug{at}mikro.bio.unigiessen.de.

↵ * Present address: Institut für Molekularbiologie und Tumorforschung, Philipps-Universität Marburg, Emil-Mannkopff-Strasse 2, D-35037 Marburg, Germany.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: BChl, bacteriochlorophyll; PAC, photo-activated adenylyl cyclase.

Copyright © 2004, The National Academy of Sciences


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