A eukaryotic BLUF domain mediates light-dependent gene expression in the purple bacterium Rhodobacter sphaeroides 2.4.1 Yuchen Han, Stephan Braatsch, Lisa Osterloh*, and Gabriele Klug† Institut fu¨r Mikrobiologie und Molekularbiologie, Universita¨t Giessen, Heinrich-Buff-Ring 26-32, D-35392 Giessen, Germany Edited by Winslow R. Briggs, Carnegie Institution of Washington, Stanford, CA, and approved July 1, 2004 (received for review May 19, 2004)
The flavin-binding BLUF domain functions as a blue-light receptor in eukaryotes and bacteria. In the photoreceptor protein photoactivated 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 PAC␣1-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 ⬇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.
roteobacteria of the genus Rhodobacter 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. Rhodobacter 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 Rhodobacter species, and several redox-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 Rhodobacter cells, which are already pigmented. When grown chemotrophically at an intermediate oxygen concentration (98 ⫾ 25 M dissolved oxygen), blue light was shown 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, little 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 described as part of a major redox signal chain (9) controlling, together with the PrrB兾PrrA two-component system, Fnr and thioredoxin 1, the oxygen-dependent expression of photosynthesis genes (5). The high puf and puc transcript levels of wild-type cells in the dark and their strong decrease after blue-light irradiation at intermediate oxygen tension depend on AppA (7). Thus, the AppA protein not only responds to an oxygen-dependent redox signal but is also a blue-light photoreceptor (7, 8, 10). The AppA primary structure consists of an N-terminal flavinadenine dinucleotide binding domain (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 redox status by
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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 redoxdependent disulfide–dithiol switch (8). Under aerobic conditions, oxidized PpsR binds to the promoter regions 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 domains 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 domain 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 redox signal independently of light, at intermediate oxygen concentrations light determines whether AppA releases the repressing effect of PpsR (7). To date, AppA is the only known protein that transduces and integrates light signals and redox signals. The BLUF domain also occurs in several other bacterial proteins, mainly in cyanobacteria and ␣-proteobacteria (12), but the function of these other bacterial BLUF domain proteins has not been elucidated. Four BLUF domains 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 domains belong to the ␣-subunit of the enzyme PAC␣ and two to the PAC subunit. The BLUF domains of the R. sphaeroides AppA and the E. gracilis PAC proteins share an identity of 28–32%. We fused the PAC␣1-BLUF domain to the C-terminal domain of the AppA protein (Table 1) to test whether the BLUF domain represents a module, which can mediate a light response in different molecular and cellular environments. In addition, we expressed the AppA BLUF domain or the AppA C-terminal domain 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. Methods 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兾Ag electrode (Micro Oxygen Sensor 501, UMS, Meiningen,
This paper was submitted directly (Track II) to the PNAS office. Abbreviations: BChl, bacteriochlorophyll; PAC, photo-activated adenylyl cyclase. *Present address: Institut fu¨r Molekularbiologie und Tumorforschung, Philipps-Universita¨t Marburg, Emil-Mannkopff-Strasse 2, D-35037 Marburg, Germany. †To
whom correspondence should be addressed. E-mail:
[email protected].
© 2004 by The National Academy of Sciences of the USA
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Table 1. Light- and redox-dependent puc expression and BChl contents of APP11-derived strains Regulatory function Strain
AppA domain structure
Light, % inhibition
Redox
n.d.
n.d.
rel. Bchl, M兾OD660 ⱕ0.01
1
APP11
2
APP11(p484-Nco5)
73
⫹
0.64 ⫾ 0.02
3
APP11(pRK4BLUF-E.g.)
85
⫹
0.56 ⫾ 0.02
4
APP11(pBBRAppA170)
n.d.
n.d.
5
APP11(p484-Nco5⌬)
0
⫹
0.60 ⫾ 0.05
6
APP11(pBBRAppA170) (p484-Nco5⌬)
60
⫹
0.61 ⫾ 0.03
ⱕ0.01
The parental strain APP11 is impaired in the production of both photopigments and structural protein components of the photosystem because of an insertional inactivation of the chromosomal appA gene (19). A schematic alignment of AppA proteins expressed from the listed strains is shown. AppA-mediated light- and redox-dependent puc expression levels are summarized, as detected by Northern blot analyses. Relative BChl concentrations shown represent the mean ⫾ SE of three independent measurements of cultures grown in low oxygen (pO2 ⱕ 3 M). puc inhibition under semiaerobic conditions in % ⫽ 100 ⫻ (1 ⫺ signal level in light-irradiated cells兾signal level in dark cells). ⫹, Significant increase in puc signal intensity due to a decrease in oxygen concentration; n.d., no detectable puc signal under all oxygen concentrations tested.
Germany). To analyze AppA-dependent light-signaling characteristics, strains were irradiated with blue light (max 400 nm; fluence rate 20 mol䡠m⫺2䡠s⫺1) in the presence of 104 ⫾ 24 M dissolved oxygen, as described in ref. 7. To analyze the redox-dependent functions, the concentration of dissolved oxygen was decreased from 200 M to ⱕ3 M in dark-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 described in ref. 20. When required, antibiotics were used at the following concentrations: gentamycin, 10 g䡠ml⫺1; kanamycin, 25 g䡠ml⫺1; spectinomycin, 10 g䡠ml⫺1; streptomycin, 100 g䡠ml⫺1 (E. coli) or 25 g䡠ml⫺1 (R. sphaeroides); tetracycline, 20 g䡠ml⫺1 (E. coli) or 2 g䡠ml⫺1 (R. sphaeroides); ampicillin, 200 g䡠ml⫺1 (E. coli); trimethoprim, 50 g䡠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-domain PAC␣1 from E. gracilis was PCR-amplified (primer pair 5⬘CCGCTCGAGAAGGGAGGAGAAACC-3⬘兾5⬘-TGCTCTAGAGTGGGAGTCTTTCATGTG-3⬘) from pGEMPAC␣ (contains the coding sequence of PAC␣1) and cloned into p484Nco50 (contains wild-type appA with its own promoter), replacing appA codons 7–450. Subsequently, a DNA fragment encoding the Cterminal AppA domain 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 pRK4BLUFE.g. DNA sequencing was performed to reveal in-frame fusion. Han et al.
Because of the cloning procedure, additional amino acids serine and arginine were introduced between PAC␣1 and the C-terminal AppA domain at positions 113 and 114 of the hybrid protein. Plasmids pGEMPAC␣ 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 ⍀-resistance cartridge from pHP45⍀ (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 codons 1–168 was constructed by PCR with primers 5⬘CGGCGGAAGCTTAATCCGAGGTC-3⬘ and 5⬘-TGTCCGTCTAGACGGGGGTATC-3⬘. The reverse primer introduced a stop codon 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 described 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, Berthold, Nashua, NH). The mean value of 10 data around the maximum of the peak was used as the luminescence output. All readings were normalized to the optical density of the cultures at 660 nm. Measurements were performed three times on independent cultures. Spectroscopy. Absorbance spectroscopy was performed on a spec-
trophotometer (Lambda 12, PerkinElmer). R. sphaeroides cell PNAS 兩 August 17, 2004 兩 vol. 101 兩 no. 33 兩 12307
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No.
Fig. 1. Absorbance spectra of exponential APP11-derived strains listed in Table 1 grown under low oxygen tension (pO2 ⱕ 3 M). 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.
extracts were obtained by sonication of cells grown to an OD660 of 0.7–0.9 under low oxygen concentration (pO2 ⱕ 3 M) in the dark. Spectral analyses were performed on crude cell-free lysates. All samples contained 600 g of protein per ml as determined by the Bradford method (26). Photopigments were extracted with acetone-methanol (7:2 vol兾vol) from cell pellets, and the bacteriochlorophyll (BChl) concentration was calculated by using an extinction coefficient at 770 nm of 76 mM⫺1䡠cm⫺1 (27). Results and Discussion A Hybrid Protein Consisting of the PAC␣1-BLUF Domain 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 targets. As a consequence of the strong repression of photosynthesis genes by PpsR, the cells are virtually unpigmented, even when grown in the presence of ⱕ3 M 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 redox-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) showed normal light-dependent repression of puc mRNA levels (Fig. 3 C and D). Rhodobacter cells expressing the PAC␣1-AppA hybrid protein [strain 3: APP11(pRK4BLUF-E.g.)] exhibit 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 redox-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 showed 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 M dissolved oxygen, strain APP11(pRK4BLUF-E.g.) showed ⬇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 Rhodobacter and has the V. harveyi luxAB genes transcriptionally fused to the puc promoter region, followed by the pucBA genes. In the presence of the wild-type AppA protein [strain 2: APP11(p484-Nco5)] luciferase activity drops after illumination, approaching a very low level that is also observed in strain APP11 (data not shown) or a strain only expressing the BLUF domain [strain 4: A PP11(pBBRAppA170)] (Fig. 3C). The luciferase assays performed with strains expressing the PAC␣1-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 region present on the reporter plasmid. Average repression rates during blue-light illumination of both semiaerobically grown cultures were 80% (Fig.
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 M to ⱕ3 M. 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. 12308 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0403547101
Han et al.
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Fig. 3. Kinetics of puf and puc expression in R. sphaeroides APP11 strains caused by blue-light irradiation. Cells grown at 104 ⫾ 24 M 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 (䊐) and puf (F) mRNA levels were plotted. Inhibition in % ⫽ 100 ⫻ (1 ⫺ mRNA level in light irradiated cells兾mRNA level in dark cells). (C) Luciferase-activity assays for puc expression in strains APP11(pBBRAppA170) (䊐) and APP11(p484-Nco5) (F). The relative light units (RLU䡠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.) (䊐) and APP11(p484-Nco5) (F). 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.
3D), ref lecting well the 73– 85% inhibition determined in RNA gel blots (Fig. 3 A and B) (7). Our data reveal that the eukaryotic BLUF domain of the PAC␣1-AppA fusion protein can fully replace the AppA BLUF domain from R. sphaeroides in light signaling and does not interfere with the redox 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 domains involved in signaling were identified (e.g., Per-Arnt-Sim domains, His kinase domains, Ser-Thr kinase domains) in both kingdoms. Furthermore, homologous light receptor proteins (microbial rhodopsins) 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 domains or targeting proteins to specific sites in a cell. These hybrid proteins normally consist of domains from proteins of the same or related species; otherwise the protein Han et al.
domains stem from species of the same kingdom of life (31, 32). One exception was the fusion of an archaeal light-signaling domain to a eubacterial chemotaxis protein domain. The archaeal NpSRII domain and part of the NpHtrII domain 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 domain of a eukaryotic organism can fully replace its homologue in a prokaryotic cell. This result is especially remarkable because the output domains of the PAC␣1 and the AppA protein show no homology and are functionally clearly different. The eukaryotic BLUF domain 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 domain creates a light-dependent output signal, which can be recognized and processed by different protein domains fused to BLUF. The amino acids conserved between the Euglena PAC proteins and the R. sphaeroides AppA protein (12, 18) PNAS 兩 August 17, 2004 兩 vol. 101 兩 no. 33 兩 12309
Fig. 4. Kinetics of puf and puc expression in R. sphaeroides APP11(pBBRAppA170)(p484-Nco5⌬) caused by blue-light irradiation. Cells grown at 104 ⫾ 24 M 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 (e) and puf (F) inhibition. The evaluation was performed as described in the Fig. 3B legend.
define the BLUF sequence sufficient to generate this lightdependent signal and to transduce it to the output domains. The C-Terminal Domain of AppA Is Sufficient for Redox Regulation and Is Required Together with the BLUF Domain for Light Signaling. To
better understand the functions of the AppA domains in redox and light signaling, we separately expressed either the BLUF domain or the C-terminal domain in R. sphaeroides (Table 1, strains 4 and 5). The absorption spectrum and the relative BChl concentration of the strain harboring the AppA BLUF domain [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 domain is not stable when expressed separately. However, as we show in the next paragraph, the separated BLUF domain is able to transmit the blue-light signal when expressed together with the C-terminal domain of AppA, which requires expression of a stable protein. Our results show that the BLUF domain alone is not able to release the repressing effect of PpsR. Strain APP11(p484-Nco5⌬) harbors the C-terminal domain of AppA (Table 1, strain 5). Its BChl concentration and absorption spectrum is identical to that of control strain APP11(p484Nco5), 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 redoxdependent increase of the puc mRNA levels (Fig. 2). puc expression was, however, independent of blue light (data not shown). We conclude that the C-terminal domain of AppA is sufficient for redox regulation but not for light regulation. Because the separated BLUF domain is unable to transmit the light signal and to release the PpsR repressing effect, we suggest that the C-terminal AppA domain interacts with the PpsR repressor protein and that the BLUF domain influences this interaction in dependence of blue light.
Signal Transmission by AppA Does Not Require Covalent Linkage of the BLUF Domain and the C-Terminal Domain. The results obtained
with the hybrid AppA protein containing the Euglena BLUF domain suggest that the BLUF domain is able to signal to different output domains. Some bacteria encode proteins only consisting of the BLUF domain (12), but the function of these
12310 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0403547101
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 domain of AppA and the C-terminal AppA domain in R. sphaeroides strain APP11(pBBRAppA170)(p484-Nco5⌬) (Table 1, strain 6). This strain showed 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 domains, we conclude that the BLUF domain functions as a module that can transduce a light-dependent signal to a C-terminally fused output domain or to a separately expressed protein. Our observation would open the possibility that one BLUF domain protein could transfer a signal to different partner proteins. Although this makes the BLUF domain a versatile module to perceive and transmit light-dependent signals, Rhodobacter capsulatus, a close relative of R. sphaeroides, lacks BLUF domain proteins, including AppA (7). Because Rhodopseudomonas palustris, another member of the Rhodospirillaceae, encodes an AppA homologue (12), it is likely that R. capsulatus lost this signaling pathway, possibly concomitantly with the acquirement of additional defense systems against reactive oxygen species. The differences in systems involved in the oxidative stress response in R. sphaeroides and R. capsulatus support this view (34, 35). BLUF domains, with the exception of Euglena, have not been predicted from eukaryotic genomes. The reason that the BLUF domain was successfully adopted as a light-signaling module by the Euglena line but not by the other eukaryotes sequenced remains elusive. Bacterial BLUF domains are often linked to domains involved in c-di-GMP metabolism (12). It is conceivable that BLUF domains were lost in higher eukaryotes concomitantly with domains involved in c-di-GMP metabolism and signaling through c-di-GMP but that other blue-light photoreceptors of prokaryotic origin, such as cryptochromes and phototropins, evolved further. We thank M. Watanabe, M. Iseki (National Institute for Basic Biology and M. Gomelsky for providing strains and plasmids; A. Ja¨ger for technical assistance; and M. Nassal for helpful comments on the manuscript. This work was supported by Deutsche Forschungsgemeinschaft Grant KL 563兾15-1,2. Han et al.
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