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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. From photochemistry to signaling
  5. LOV and BLUF-mediated physiological responses in prokaryots
  6. New blue light trends
  7. Author Biographies
  8. References
  9. Supporting Information

The knowledge on the mechanisms by which blue light (BL) is sensed by diverse and numerous organisms, and of the physiological responses elicited by the BL photoreceptors, has grown remarkably during the last two decades. The basis for this “blue revival” was set by the identification and molecular characterization of long sought plant BL sensors, employing flavins as chromophores, chiefly cryptochromes and phototropins. The latter photosensors are the foundation members of the so-called light, oxygen, voltage (LOV)-protein family, largely spread among archaea, bacteria, fungi and plants. The accumulation of sequenced microbial genomes during the last years has added the BLUF (Blue Light sensing Using FAD) family to the BL photoreceptors and yielded the opportunity for intense “genome mining,” which has presented to us the intriguing wealth of BL sensing in prokaryotes. In this contribution we provide an update of flavin-based BL sensors of the LOV and BLUF type, from prokaryotic microorganisms, with special emphasis to their light-activation pathways and molecular signal-transduction mechanisms. Rather than being a fully comprehensive review, this research collects the most recent discoveries and aims to unveil and compare signaling pathways and mechanisms of BL sensors.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. From photochemistry to signaling
  5. LOV and BLUF-mediated physiological responses in prokaryots
  6. New blue light trends
  7. Author Biographies
  8. References
  9. Supporting Information

The directional growth of plants toward a blue light (BL) source had been described more than one hundred years ago (1). However, only in recent years the molecular structure of those “phototropin” (phot) photoreceptors that regulate phototropism has been disclosed (2). Many more physiological processes can now be ascribed to BL photoreceptors in plants, including stomata opening, chloroplast movements, leaf expansion, solar tracking and circadian rhythms regulation (1,3,4). The BL photoreceptors in plants that sense a ubiquitous and dominating (vide infra) environmental factor for the living world, presently comprise cryptochromes (Cry) (5,6), phot, ZTL (Zeitlupe), LKP2 (LOV Kelch Protein 2) and FKF1 (Flavin-binding Kelch F-box1) proteins (4). These photoreceptors host riboflavin (RB, vitamin B2) derivatives as chromophores, bound within defined protein domains or subdomains: a photolyase-like domain in Cry binds flavin adenine dinucleotide (FAD) into an all-helix subdomain; the LOV (light, oxygen, voltage) domain of phot and ZTL/LKP2/FKF1, forming a conserved α/β fold of the PAS (PerArntSim) superfamily (7,8), binds flavin mononucleotide (FMN) (2). The BLUF domain (Blue Light sensing Using FAD), the third type of flavin-based photosensing units, is solely found in Euglenoids and Bacteria, and binds FAD. Cry and the phot-related, so-called LOV proteins appear to be ubiquitous, being wide spread in prokaryotes, fungi and lower plants (9,10). Cry are also present in animals (6,11,12), whereas LOV proteins are the sole flavin-based BL sensors occurring also in Archaea (10,13,14).

It is apparent that not only plants, but all organisms have to detect and to respond to environmental stimuli in order to adjust their metabolic activities. Light is a major factor for the adaptation process, be it to escape harmful and deleterious irradiation, in relation to photosynthetic activity or for the entrainment of the circadian clock. The BL spectral region deserves particular attention, since light from this wavelength range not only penetrates deepest into a water column (15), but it also excites with high efficiency ubiquitous photosensitizers, e.g. endogenous metal-free porphyrins and flavins (16). From their excited state, these molecules readily form triplets that, decaying to the singlet ground state, generate singlet oxygen (1OΔ) and other reactive oxygen species with high quantum yield (16). The same RB derivatives found in BL sensors, are potentially very powerful photosensitizers for 1OΔ (ca 50% efficiency in aqueous solution for RB and FMN) (17,18). On the other hand, flavins are extremely versatile, i.e. they undergo one- or two-electron transfer reactions, function as electrophiles and nucleophiles and are frequently involved in enzyme catalysis with the formation of covalent intermediates of flavin and substrates (19,20). Light excitation by a flavin derivative obviously results in charge redistribution and a changed redox potential, giving rise to a variety of possible photochemical reactions, but may also generate potential danger: the high redox potential of the flavin triplet (ca 1.7 V) accounts for the oxidation of several electron rich biological substrates, such as aromatic amino acids and DNA bases, and is responsible for a great part of food degradation under illumination (21). Flavin photoreceptors must therefore be able to minimize the yield of potentially dangerous reaction intermediates, and to maximize the requirements for signaling and for “beneficial” reactions.

After the analysis of the basic requirements for BL sensing in flavin-based receptors, we will follow the molecular journey of signal transduction from the chromophore binding cavity down to interacting partners, based on structural, functional, molecular biology and cellular data. Prominent examples of biological responses driven by BL sensors will be discussed in greater detail. Finally, we will illustrate some novel trends in this research area, notably the applicability of BL sensors in cellular studies and the uncovering of novel proteins by screening environmental samples. We will restrict our survey to LOV and BLUF proteins.

Sensing light: I. The chromophores, RB, FMN and FAD

The principal requirement of a photosensory protein is the presence of a light absorbing chromophore that undergoes physico-chemical and structural changes upon light absorption. The protein moiety hosting this light-activated molecule modulates these photochemical aspects and at the same time constitutes the microenvironment that immediately responds to the photoinduced changes of the chromophore. By this multiple function it obeys an important duty to carry this information to partner domains and/or proteins: namely to carry out the first and essential step in the signal-transduction process.

Riboflavin, i.e. 7,8-dimethyl-10-(1-deoxy-d-ribitol-1-yl)isoalloxazine, is biosynthesized by plants, fungi, bacteria and archaea, whereas it is essential for mammalian cells (vitamin B2) to which it must be provided with the diet (22–24). RB kinase and FAD diphosphorylase convert RB to FMN (riboflavin 5′-phosphate) and FAD, respectively, the two RB derivatives predominantly present in cells. FMN and FAD are cofactors of enzymes, mostly involved in biological redox-systems. Mammalian cells contain as many as approximately 50 different flavoproteins (25). LOV and BLUF domains bind preferentially (but not exclusively) FMN and FAD, respectively. The spectrum shows basically three major π–π* electronic transitions, encompassing the UVB, UVA and the BL region and minor n–π* transitions (26–28) (Fig. 1).

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Figure 1.  (a) The three flavin chromophores of blue light sensors. Riboflavin (RB) consists of a 7,8-dimethyl-isoalloxazine ring linked to d-ribitol. FMN and FAD are the phosphorylated and adenosylated forms of RB, respectively; (b) Absorption spectrum of FMN in water, scaled to show the absorption coefficients at the different wavelengths. This spectrum exemplifies a fully oxidized form of the chromophore and is basically the same for the three derivatives. FMN, flavin mononucleotide; FAD, flavin adenine dinucleotide.

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In the BL region, the maximum absorption coefficient of a fully oxidized flavin ring is ca 12 500 m−1 cm−1 at ca 450 nm (29,30). In this form, RB and FMN are brightly fluorescent with a quantum yield of ca 0.25–0.3 (29,31), and efficiently form triplets with a yield of ca 0.6 (32–34). A recent ab initio theoretical study on the flavin isoalloxazine ring suggests that, in solution, this high yield of intersystem crossing is related to spin-orbit vibronic coupling between the S11(π–π*) state (BL transition) and the triplet state T23(π–π*), and by the intersection of their potential energy hypersurfaces (27). FAD in solution is a special case, such that the singlet excited state of the isoalloxazine ring is quenched by stacking interactions with the adenosine moiety (35), consequently rendering a much lower fluorescence, triplet and singlet oxygen yield (<0.1 [18]).

One electron reduction or triplet formation cause a shift to longer wavelengths for transition I, e.g. around 600 nm for the FMN neutral radical as determined for flavodoxin (36), and 660 nm for the FMN triplet in LOV domains (37,38). Depending on the surrounding microenvironment, the semiquinone species can be stable on a time scale of several milliseconds or even longer (39), thus providing the possibility for a red light-driven or secondary photochemistry. The fully reduced (two electron) form, with loss of a double bond and conjugation, is nonfluorescent and absorbs poorly in the visible region. One or two electron reduction can occur from the ground state or from the excited states (triplets or singlets), obviously with different redox potentials. The presence of dissociable protons on the ring system (at N(1) and N(5)) of fully reduced flavins accounts for the occurrence of anionic and neutral one electron (flavin semiquinones) and two electron (flavin hydroquinones) species, depending of the pH (see ref. (36) and references therein).

Sensing light: II. The protein moieties, LOV and BLUF domains

LOV and BLUF domains are small photosensing modules of ca 100 amino acid building up quite compact α/β structures. Their topology is depicted in Fig. 2. According to a recently proposed nomenclature, we assign the following secondary structure elements for the LOV core (from the N-terminal part): AβBβCαDαEαFαGβHβIβ, and for the BLUF core: β1α1β2β3α2β3 (40–47). In Fig. 3 the absorption spectra of a LOV and a BLUF protein are shown, together with a simplified reaction scheme.

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Figure 2.  Top: topology of (a) LOV and (b) BLUF domains. Secondary structure elements have been named after (117) and (46). Arrows: strands, cylinders: helices. The caps at the N- or C-termini of the domain core are given without color. The caps are variable and may adopt different conformations/geometries in different proteins or/and in a different state (dark or lit). Relative size of loops, strands and helices are not respected. In (a) the N-cap has been labeled α0. Bottom: structure of (c) a LOV (PDB 1V1A [111]) and of (d) a BLUF (PDB 3GFX [45]) domain, highlighting residues involved in signal propagation, in different LOV and BLUF proteins. Numbering is from Asphot1-LOV2 to KpBlrP1, respectively (As = Avena sativa, Kp = Klebsiella pneumoniae); see Tables 2 and 3 for details. We also show residues that participate in the modification of the HB network around the flavin after photoexcitation (N482, N492 and R451 in [c]; K30 and N31 in [d]). Some secondary structure elements, or part of them, have been eliminated in order to give insight into the chromophore cavity.

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image

Figure 3.  Steady-state absorption spectra and proposed simplified reaction scheme for: (a, c) the LOV-protein-YtvA from Bacillus subtilis- and (b, d) the BLUF protein-YcgF from Escherichia coli. For clarity, the numbering of involved amino acids is the same as in Fig. 2 (Asphot1-LOV2 and KpBlrP1, respectively).

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The photochemistry of LOV domains was initially elucidated for phot that bear two such domains in tandem (LOV1 and LOV2) (2,48), and afterward for a variety of bacterial and fungal proteins (10,14,49–55). The photoprocess involves the formation of the so-called FMN-Cys photoadduct, significantly blueshifted with respect to the dark state and nonfluorescent (referred to as LOV390), which is generated via the short μs decay of the FMN triplet state (reviewed in ref. [56]). In LOV390 a covalent bond is formed between the carbon atom at position 4a and the thiol group of a conserved cysteine localized in the Dα-Eα loop. Concomitantly, N(5) becomes protonated in LOV390 (37,57,58). LOV390 reverts in the dark to the unphotolyzed state (LOV447). The timescale for this thermally driven process varies for the LOV domains investigated so far from a few seconds to many hours at room temperature (56,59–62). The quantum yield of LOV390 formation ranges from 0.3 to 0.6 after BL excitation, but drops dramatically for UVA excitation, for still poorly understood reasons (63,64). Despite this well established photocycle, the details both for LOV390 formation from the triplet state and for its reversion are still a matter of debate, as well as the molecular basis of the dramatically different photocycle kinetics in different proteins.

Formation of the adduct involves the breaking of the cysteine SH bond, the establishment of new C(4a)-S and N-H (5)bonds and triplet-to-singlet spin inversion. Basically, two reaction mechanisms have been suggested: (1) an ionic model (65); and (2) a radical-pair mechanism (66,67). FTIR studies (68,69) as well as quantum mechanical considerations (70) disfavor an ionic mechanism but cannot rule it out completely.

Interestingly, in a Cys–Gly mutant of Crphot-LOV1, per se photochemically unable to form the adduct, methylmercaptan is able to penetrate the cavity and to form an adduct with similar spectral features as LOV390 (71). Besides, methylmercaptan and bulkier mercaptans (i.e. unable to penetrate the cavity) are able to reduce the bound FMN, forming a stable FMNH˙ radical absorbing in the red. On the basis of their results, the authors of that study suggest a reaction chain from FMN triplet state, with a flavin-cysteine radical pair as intermediate, for which it is not clear at which step spin inversion occurs (71):

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As for the dark recovery reaction, i.e. breaking of the covalent bond plus deprotonation of N(5) and reprotonation of the reactive cysteine, it is known that its lifetime (τR) is accelerated by imidazole suggesting a base-catalyzed reaction (49,58,62,72,73). Furthermore, pH (38) and deuterium effects (65) indicate that a proton transfer reaction from N(5) is the rate-limiting step for the dark recovery reaction (58). The strain imposed on the adduct by the protein microenvironment provides the driving energy for the C(4a)-S bond splitting (56,63,74). Accordingly, a series of mutations in the vicinity of the chromophore are able to affect the photocycle kinetics by altering steric restrictions or the hydrogen bonds (HB) network that stabilizes the flavin moiety (Fig. 4) (58,63,75–79,236). In Table 1 we give a list of selected mutations affecting strongly τR.

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Figure 4.  (a) Alignment of selected LOV domains (core region), with secondary structure elements derived from crystal structures of the dark-adapted state. The PDB databank accession codes are as follows: 1G28, Acphy3-LOV2 (7); 1N9L, Crphot-LOV1 (110); 2PR5, BsYtvA (81); 2PD7, NcVVD (112); 2VOU, Asphot1-LOV2 (111). The reactive cysteine is in blue; residues in close vicinity of the chromophore are marked with full arrows. Open arrows indicate the E-K couple forming a conserved salt-bridge at the surface of the LOV core (108). Residues showing a strong effect on τR upon mutation (Table 1) are highlighted in green. The lower part of the figure shows a close up of the chromophore binding pocket with the residues mutated as in Table 1 for: (b) Acphy3-LOV2, (c) Asphot1-LOV2, (d) BsYtvA, (e) NcVVD. Figures drawn with DeepView (228).

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Table 1.   Mutations affecting τR (lifetime for the recovery dark reaction) in LOV proteins.
ProteinMutations altering τRLocationτR,WT/τR,mut
  1. Ac = Adiantum capillus veneris; Nc = Neurospora crassa; Bs = Bacillus subtilis; Cr = Chlamydomonas reinhardtii; Pp = Pseudomonas putida.

Acphy3-LOV2F1010 (229)0.1
Asphot1-LOV2V416L; V416L/L496I (58)Aβ; Aβ/Hβ0.1; 0.08
I427V (75)10
N425C (78)Aβ-Bβ loop6.4
Q513N, Q513L (76)1.8; 0.06
NcVVDI74V; I85V; I74V/I85V (58)Aβ; Bβ; Aβ/Bβ25; 23; 640
M135I/M165IFα/Hβ0.1
BsYtvAV28L; I39V (58)Aβ; Bβ0.2; 5.4
I39V/F46H (79)Bβ/Cα75
N94A; N94S (236)45; 21
N104A; N104S (236)2.8; 5.6
Q123N (236)85
R63K (80)10
Crphot-LOV1R58K (63)2.8
PpSB1-LOVR66I (77)100
R61H (77)Eα-Fα loop3
PpSB2-LOVI66R (77)0.1
H61R(77)Eα-Fα loop0.25

The most extensive work on this subject has been performed on the FAD binding, LOV-protein VVD from Neurospora crassa (Nc) by Zoltowski and collaborators (58). Deuterium and pH effects, and a series of mutations have led to the conclusion that main factors determining the recovery kinetics are: (1) acceleration of N(5) deprotonation and/or reduced stability of the flavin N-H bond (5); (2) steric destabilization of the flavin-Cys adduct or steric/electronic factors that favor the flavin-oxidized state over the two electron reduced LOV390. Specific amino acids that control solvent/base accessibility along the flavin side-chain (I74 and C76 on strand Aβ, T83 and I85 on strand Bβ; VVD numbering), and steric/electronic effects at the reface of the isoalloxazine ring (opposite of the reactive cysteine, e.g. F1010 on Hβ in Adiantum capillus veneris (Ac) phy3-LOV2 and M135I/M165I on Fα/Hβ in VVD, seem to be the major determinants for LOV390 lifetime and stabilization (Table 1). These considerations also apply to other LOV proteins (58). In particular two amino acids, I74 and I85 (VVD numbering) have a large effect on solvent accessibility to N(5): I74, being in steric contact with the reactive C108 and the “flipping” glutamine (Q182, vide infra), and I85, positioned between the flavin ring and the solvent channel. Changes at these two positions render the kinetics of VVD sensitive to pH and base catalysis. The corresponding residues in the Bacillus subtilis (Bs) LOV-protein YtvA (34) are V28 and I39 (Table 1) (58).

An interesting approach has been carried out by Jentzsch et al. on SB1-LOV and SB2-LOV, two “LOV-only” proteins in Pseudomonas putida (Pp) (77): the former (PpSB1-LOV) has a very slow photocycle with τRca 2470 min at 20°C, whereas τR for PpSB2-LOV is 137 s. Site-directed mutagenesis combined with microtiter plate-prescreening allowed the identification of residues influencing the dark recovery. The amino acid in position 66 (PpSB1-LOV numbering) strongly influences the dark recovery in both proteins in a correlated manner (Table 1). A homology model of PpSB1-LOV indicates that a rotamer of R66 can come in HB distance to the FMN phosphate, although this is not the case in other known structures of LOV domains (77).

Other residues crucial for the kinetics of the photocycle are those forming a HB network around the chromophore, the mutation of which may have profound effects on the recovery kinetics. Examples are R58 in Chlamydomonas reinhardtii (Cr) phot-LOV1 and R63 in BsYtvA that are H-bonded to the FMN terminal phosphate (Table 1) (80), and substitutions at the conserved N94, N104 and Q123 in BsYtvA (236). These three residues form an extended HB network with N(5), C(4)=O, NH(3) and C(2)=O on the flavin ring (81). In LOV domains, the HBs at these positions undergo changes after blue light absorption, recently studied in detail by means of advanced infrared spectroscopy techniques (68,82,83). In particular HBs at C(2)=O and C(4)=O are weakened upon formation of the adduct, to a somehow different extent in different LOV-systems (73,83–85).

An alternative approach has been recently reported by Mansurova et al., who exchanged the native chromophore against sterically modified flavins via a chromophore exchange protocol (86). In particular 8-isopropyl FMN that introduces a steric constraint within the cavity, accelerates almost three-fold the photocycle of YtvA (86).

Finally, the variable protein regions flanking the LOV core seem to play a role in determining τR, as shown recently for the PpSB-LOV proteins and a LOV kinase from Caulobacter crescentus, most probably by affecting solvent accessibility to the adduct (73,77). These studies are of great importance not only for understanding the reaction mechanism as tuned by the protein cavity, but also for the optimal design of LOV-based photofunctional proteins for biotechnology applications (vide infra).

BLUF domains respond to light excitation with a reversible redshift of the absorption spectrum (87,88), dictated by the rearrangement of the HB-network involving N(5) and O(4), and by the flipping of a glutamine residue in the chromophore vicinity (Q49 in Fig. 2b, protein KpBlrP1, i.e. Klebsiella pneumoniae blue–light-regulated phosphodiesterase [45], for a comparative discussion see also ref. [56]). This peculiar photocycle was first described for AppA, a blue light receptor from Rhodobacter sphaeroides (87), and for a photoactivated adenylate cyclase (PAC) from Euglena gracilis (89) and, afterward, for several other bacterial proteins (40,90–92). All the BLUF proteins up to now investigated show an AppA-like photocycle, with some differences in the dark-state absorption maxima, in the magnitude of the redshift interval and in the recovery lifetime, ranging from a few seconds to several minutes (for a comprehensive presentation of these parameters see [93]). Values between 0.24 and 0.9 have been reported for the photocycle quantum yield of BLUF proteins (reviewed in ref. [56]). Different to LOV domains, triplet formation is just a side reaction for the BLUF photocycle (94), but its yield can be increased by point mutagenesis: in the Thermosynechococcus elongatus TePixD protein, the change Q50N (Q50 corresponds to Q49 of KpBlrP1) results in a well detectable formation of a triplet state (95) and the I66C substitution, bringing a LOV-like cysteine in the vicinity of C(4a), and permits the formation of a LOV390-like photoadduct (96). There is evidence that photoinduced electron transfer reactions occur also in BLUF domains prior to the formation of the photoactivated state, possibly forming short-living radical intermediates (67,97). The signaling state formation seems to proceed via light-driven electron and proton transfer from the conserved tyrosine (Y7 in Fig. 2) to FAD, followed by a hydrogen-bond rearrangement and radical-pair recombination (67). An alternative mechanism was postulated by Domratcheva et al., according to whom Gln tautomerization may occur during the lifetime of the biradical state of the system, then followed by biradical recombination leading to the tautomeric form of Q51 (AppA numering) (98). The possibility of tautomerization was proposed earlier in the FTIR study of Stelling et al. (99). Recent theoretical calculations support the view of tautomerization (100,101).

From photochemistry to signaling

  1. Top of page
  2. Abstract
  3. Introduction
  4. From photochemistry to signaling
  5. LOV and BLUF-mediated physiological responses in prokaryots
  6. New blue light trends
  7. Author Biographies
  8. References
  9. Supporting Information

The photosensing unit continues its work by passing the signal to partner domains (intraprotein pathways) or/and partner proteins (interprotein interaction). These steps are often the most difficult to elucidate, given the general paucity of structural data. Furthermore, the soluble domains of BL sensors have the tendency to forms dimers or oligomers, whose function and regulation by light is only partially understood. Light-regulated phosphorylation is also emerging as an important aspect during signal transduction. Finally, partner proteins, often shared with other signaling pathways, have the duty to integrate the BL inputs within the cellular signal-transduction networks.

Intraprotein pathways of signal propagation

LOV proteins.  In phot, the activity of the effector S/T-kinase domain is enhanced after LOV photoactivation with blue light, resulting in phot autophosphorylation (102–105), a key event in signaling (106). The same effect is observed for bacterial LOV-HisKinases (50,51,107). In other LOV proteins the effector module can be represented by diverse functional motifs, e.g. gene-expression regulators, phosphodiesterases, phosphatases (10,108,109). This poses the question whether LOV domains communicate with effector partners through the same or partially overlapping surfaces, such that the structural basis for light-to-signal transduction is basically the same in the different LOV proteins, with only few residues acting as specific signal transmitters within the different systems. There is increasing experimental evidence that this surface largely involves the β-scaffold of the LOV core (Fig. 2) which on one side hosts residues directly interacting with the isoalloxazine ring of FMN (7,110) and on the other side communicates with helical regions flanking the LOV core (69,111–113). In phot, a conserved glutamine on strand Iβ (Gln513 in Avena sativa phot1-LOV2) participates in light-driven conformational changes of LOV2 (69,76,82,83,114), and in the light activation of phot self-phosphorylation (115), and, in the fungal LOV protein VVD, in promoting light-induced dimerization (112). In B. subtilis YtvA, two acidic aa localized on strand Hβ (E105 and D109) are essential for conveying intraprotein light-triggered conformational changes to the STAS (Sulfate Transporter/Anti-Sigma factor antagonist) domain, considered a secondary switch or/and the effector module of the protein (80). The same two residues are also essential for the in vivo light-regulation of YtvA activity (116) (see Table 2 for a list of residues involved in signal transmission in several LOV proteins).

Table 2.   Residues affecting signal propagation in LOV domains (core region).
 Light-driven changes/effects of mutationsLocation
Acphy3-LOV2F1010, conformational changes (230) F1010L impairs conformational changes (229)
Q1029 (flipping) (230); Q1029L impairs conformational changes (82)
BsYtvAW103, conformational changes (231)
E105L, suppresses intraprotein signal transmission (113); functionally locked in a pseudolit state (116)
L106F, impairs intraprotein signal transmission (80)
D109L suppresses intraprotein signal transmission (80); functionally locked in a pseudodark state (116)
Q123 (flipping) (81)
Asphot1-LOV2W491, conformational changes (111,232)
Removal of interactions for Leu493 with J-helix (I532E), constitutively activates the kinase (118)
Q513 (flipping) (111); Q513L, impairs light-driven conformational changes, pseudodark state (76) Q513N, impairs light-driven conformational changes, pseudolit state (76)
Atphot1-LOV2F556L, diminishes light-driven conformational changes (233)
Q575 (flipping) (111); Q575L: diminishes light-driven conformational changes (233) and attenuates light-induced self-phosphorylation (115)
Atphot2-LOV2Q489 (flipping) (69)
ViviDQ182, (flipping) (112); Q182L impairs light-driven dimerization (112)

Autophosphorylation of phot is enhanced by the light-induced undocking of the so-called Jα-linker helix that connects LOV2 to the kinase domain. In the dark, the Jα-linker is folded as an α-helix and docked to the β-scaffold of LOV2 (117–119). Phot2 also shows light-triggered conformational changes of the Jα-linker, although not as dramatic as in phot1 (114). Nevertheless, we are now facing the intriguing concept, experimentally supported, that light activation barely shifts the β-scaffold docked-undocked equilibrium of the Jα-linker from “mostly docked” (inactive) to “mostly undocked” (active) (120). This means that a fraction of the photoreceptor is functionally active also in the dark state. This aspect is extremely important in the design of optimized photofunctional proteins to be used in biotechnology applications (vide infra and Fig. 5a).

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Figure 5.  Dark-state conformational equilibria (black filling) of protein constructs bearing BLUF and LOV domains and effects of photoactivation (gray filling). (a) Photoexcitation of Asphot1-LOV2 is accompanied by displacement of the Jα-helix from the β-scaffold. New data indicate that actually photoactivation shifts the equilibrium from a “mostly docked” to a “mostly undocked” Jα-helix and that this equilibrium is affected by the presence of a linked effector domain (modified from [120]); (b) Asphot1-LOV2 shows a concentration-dependent monomer–dimer equilibrium in the dark; photoexcitation of the monomer is proposed to induce a transient dimerization, while light activation of the dimer induces a transient dissociation (modified from [144]); (c) the protein VIVID shows in the dark a compact (top) or extended (bottom) conformation. The compact form is probably a partial oxidative modification/degradation. Each of these forms is converted into an extended monomeric lit state that tends to dimerize (modified from [145]) via the N-terminal region, partially helical. The lit state is actually a fast equilibrium between a monomeric and a dimeric form (147); (d) in Escherichia coli YcgF, the monomer–dimer equilibrium of the dark state is temperature-dependent. According to this model, dimerization is promoted by a conformational change of the EAL domain, as a consequence of conformational changes in the linker (modified from ref. [159]).

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An involvement of the Jα-linker in interdomain communication has been demonstrated also in the bacterial protein YtvA (80), however, this function is not connected to light-driven unfolding (113,121). As a whole, the scenario for the early signal propagation steps in LOV-proteins points to a general involvement of the β-sheet/helical cap mechanism, as recently proposed also for BLUF proteins (46), with the involvement of specific residues depending on the different proteins. Also, it is now clear that a conserved “flipping” glutamine, located on strand Iβ and directly interacting with the chromophore (Q123 in YtvA, Q182 in VVD, Q513 in Asphot1-LOV2, Q575 in Atphot1-LOV2), is important both in signal propagation and in the photochemical reactions (e.g. duration of the photocycle, see Tables 1 and 2 and Fig. 2) (69,76,111,112,115,236).

Still we had, until recently, only few hints about the significance of two tandem LOV domains in phot, given that only LOV2 photoexcitation plays a major role in regulating phot1 kinase activation by BL (48,105,122,123). This issue was elegantly addressed by the Christie group by means of a domain-swapping strategy that highlighted the constitutive (not light-regulated) activity of a phot1-LOV1 + LOV1 protein and the need of having a LOV2 adjacent to the Jα-linker in order to inhibit the kinase (124). Thus, although it is possible to exchange LOV domains and to retain the photobiological function, even among different organisms (125,126), LOV1 is not able to replace LOV2 as inhibitor of the kinase, most probably due to a weaker binding of the β-sheet to the Jα-linker (104).

BL-triggered enhancement of autophosphorylation activity in LOV kinases is obviously an important process (50,51,107), but still needs to be linked to the photobiological activity mediated by the receptor. In a bacterial LOV kinase from P. syringae the phospho-acceptor is a fused response regulator, denoting a typical bacterial two-component system (107). For phot, a substrate other than phot itself is to date not known, but several phospho-acceptor sites have been identified (103,105). Atphot1 is phosphorylated in vivo on multiple serine residues, localized in the N-terminus (S58, S185) and in the linker region between LOV1 and LOV2 (S350,S410), with phosphorylation at S185, S350 and S410 being proven to be BL induced (105). Phosphorylation at these sites is not determinant for phototropism and leaf expansion, but may be necessary for maximizing stomatal performance, light-induced relocalization of phot1 and interactions with protein partners (105,124,127).

BLUF proteins.  A recent work, dealing with the BLUF domain of KpBlrP1 (see Figure 2) has summarized our current understanding about signal propagation within BLUF proteins (46). Earlier studies with the Escherichia coli YcgF protein, built of a BLUF domain and a C-terminal EAL domain (a putative phosphodiesterase for cyclic diguanylate, c-di-GMP [128]) and with SyPixD (a short-BLUF protein) suggested that the helical region comprising α3 and α4 (Fig. 2), C-terminal to the BLUF core, is important in signal propagation (129–131). Although a phosphodiesterase activity of YcgF-EAL has been ruled out (132), the importance of the α34 region for protein stability and conformational changes has been confirmed (133). Similarly, the C-terminal helical cap is necessary for the stability of KpBlrP1-BLUF, and the α3α4 loop undergoes significant light-induced conformational changes, as detected by NMR experiments (46). Other regions involved in signal propagation comprise the β4β5 loop and strand β5 (see also Table 3). These elements host T90 and M92 (in KpBlrP1-BLUF, see Fig. 2), residues which are the subject of intense debate regarding their roles in signal activation process for BLUF proteins.

Table 3.   Residues affecting signal propagation in BLUF domains.
 Light-driven changes/effects of mutationsLocation
RsAppaQ63L suppresses photocycle; light insensitivity in vivo (135)β3
W104A biphasic and fast photocycle (137) (AppA1−126); light insensitivity in vivo (135)β4β5 loop
W104F fast photocycle; alteration of in vivo activity (136)β4β5 loop
W104M/M106W much faster photocycle (134,137)β4β5 loop
KpBlrP1L41V interrupts signal propagation (46)β3
Q49, essential for flavin binding and photocycling, several mutations investigated (46)β3
Mutations (e.g. T90W) alter conformational changes (46)β4β5 loop
M92, essential for flavin binding and photocycling, several mutations investigated (46)β4β5 loop
F112L, F112Y, alter light-induced conformational changes (46)α3
V117F, V117L, alter conformational changes (46)α3α4 loop
SySlr1694/SyPixDW91A, retains photobiological activity (158)β4β5 loop
M93A, suppresses signal transmission; functionally locked in a lit state (158)β4β5 loop
TeTll0078/TePixDQ50N, alters photocycle, formation of flavin triplet (95)β3
I66C, forms a LOV-like adduct (96)α2β4 loop
EgPACY60F (F1), loss of photostimulated cyclase activity; no effects for Y472F (F2) (234)β1
W556L (F2), faster photocycle and increased yield (235)β4β5 loop

In AppA-BLUF, the W104A mutation (W104 corresponds to T90 in KpBlrP1) impairs light-induced structural changes in the β-sheet, without appreciable effects on the yield of the photocycle (134). In functional assays with full-length AppA, the W104A mutation locks the protein in a functionally lit-like state (135). The W104F substitution, instead, accelerates the photocycle about 10-fold, altering the in vivo activity of AppA as an antirepressor (136). Recently, spectroscopic studies led to the suggestion that light absorption by the flavin causes partial movement/uncovering of W104, but does not cause dramatic conformational changes that can support a model of W104 movement as a key output signal (137), the so-called “Trp-in” to “Trp-out” previously proposed on the basis of alternative dark and lit-state structures (43,44,98,134,138). Studies with ultraviolet resonance Raman spectroscopy favor a “Trp-in” conformation in the dark state and structural alterations in the light-activated state, but not as extensive as to obtain a “Trp-out” conformation (139). Nevertheless, the role of this residue is subtle: in AppA mutation of Y21 (directly involved in the photocycle) into phenylalanine or cysteine causes a complete loss of AppA antirepressor activity, yet, the W104F/Y21F double mutant shows a wildtype-like behavior (136).

Two other residues, T90 and M92, appear essential for WT-like conformational changes, FAD binding and photocycling in KpBlrP1-BLUF (46). As a whole, the extensive work on KpBlrP1-BLUF (46) raises the possibility that, after BL illumination, the C-terminal helical cap undergoes a reorientation process that might be associated with the conformational changes of β4β5 and α3α4 loops and strand β5 (46).

The oligomeric state of LOV and BLUF proteins and the link to physiology

LOV proteins.  As other PAS domains, LOV domains show, to a different degree, a tendency to dimerize, with or without the assistance or competition of the helical flanking regions (60,81,112,140–143). The role of LOV dimerization in the context of the full-length proteins is largely unexplored, with few exceptions, as well as the solution dynamics of dimers as affected by light activation and their in vivo relevance. Recently, thermal grating experiments have demonstrated that phot1-LOV2 domains can form light-induced dimers (144), but, in a somehow puzzling way, these experiments also yielded evidence that a dimeric state of LOV2 can monomerize in the light (Fig. 5b).

It is clear that, at least in some cases, besides conveying signal propagation, the β-scaffold is also involved in LOV–LOV dimerization (81,113,142,145), whereas in other instances dimerization occurs via the N-terminal cap, as in the crystal structure of light-activated VVD (112) and of the YtvA-LOV domain from Bacillus amyloliquefaciens (146). A further complication arises from the observation that crystallized dimers might have an interface different from solution dimers, as recently demonstrated for VVD by using small-angle X-ray scattering (145); also the helical regions flanking the LOV core may solely assist dimerization, chiefly relying on the β-scaffold surfaces (79,81,145). For VVD, built solely of a LOV core with a helical N-cap, it was demonstrated that in solution two different monomeric dark-state conformations exists, named compact and extended, but both tend to dimerize to the same structure after light activation (Fig. 5c) (145). A combined approach employing size-exclusion chromatography (SEC), equilibrium ultracentrifugation and static and dynamic light scattering showed actually that the lit dimeric structure is in rapid equilibrium with the monomer (Fig. 5c), with residues 39–42 in the N-cap being essential for dimerization (147). It thus appears that in solution dimerization of LOV proteins may be a highly dynamic phenomenon.

For BsYtvA, contrasting data have been obtained: the LOV core, with or without the flanking regions, is certainly constitutively dimeric, engaging the β-scaffold in dimer formation (113,148). Circular dichroism (CD) analysis also indicates that the two β-sheets of the facing monomers become twisted due to the tight interactions, and that the same surface undergoes the most relevant light-driven conformational changes (113). As for the full-length protein, SEC analysis indicates a major peak with ca 1.5–1.6 × MW (113,121), assigned to an elongated monomer (113) or to a homodimer (121,149). Analytical ultracentrifugation studies and small-angle X-ray scattering experiment point to a dimeric structure, not changed by light activation (121). Nevertheless, the MW obtained by all experimental methods employed is always less, in the case of SEC considerably less, than the expected MW for a dimer (121), as in the case of VVD (147), suggesting that at least a fraction of the protein probably is monomeric, and potentially in equilibrium with the dimeric form. We also note that CD experiments indicate that in full-length YtvA the antiparallel β-sheets are not twisted as in the LOV-core dimers, but remain extended and regular. Nevertheless, CD studies have been perfomed at nanomolar concentrations, a condition that could prevent dimerization (113). The study of cross-linked YtvA dimers by SEC (147) should help clarify this point. It is of course crucial to understand, if and how dimerization is relevant for the in vivo activity.

Bimolecular fluorescence complementation analysis has shown that Atphot1 undergoes light-dependent dimerization in vivo, coinciding with a mechanism of light-driven autophosphorylation in trans (124). LOV1 is not necessary for this dimerization, as it occurs also in a phot1 carrying two LOV2 domains. The site(s) of receptor dimerization still need to be defined, and the relevance of the trans phosphorylation for phot signaling remains to be assessed (124).

Finally, we note that again the fungal LOV-photoreceptor system provides us with valuable information on the in vivo relevance of LOV dimerization. It has long been known that white collar 1 (WC-1; see Fig. 4), a Zn-finger LOV protein, is active as a dimer (150). The WC-1 dimer is part of the white-collar complex, WCC that also comprises the nonphotosensing WC-2 and acts together with VVD during photoresponses. VVD is expressed under control of light-activated WCC (151). BL triggers WCC dimerization via the LOV domain of WC-1 (152). Activated WCC induces expression of VVD that inactivates WCC by forming a competing VVD-WCC heterodimer, leading to photoadaptation and acting together with VVD as a negative regulator (152).

BLUF proteins.  AppA-BLUF has been suggested to form a dimer in the dark state (153), in good agreement with SEC analysis (154). Light excitation induces the transient formation of a tetrameric state (153), driven by hydrophobic interactions (155). SEC experiments showed an increase in the apparent MW to 37 kDa in the light-activated state of AppA-BLUF, also indicative of a sharp conformational change, but the data are not compatible with the formation of a tetramer (154). From the dimers within the crystal units, we have a good hint about the dimer interface of AppA-BLUF in solution, conceivably built by the hydrophobic β-scaffold surface (42,156), as recently confirmed by spectroscopic studies on W104 located on strand β5 (137,156). The full protein instead appears to be monomeric in solution, as it is also evident from the higher degree of exposure of W104 (T90 in Figure 2) with respect to AppA-BLUF alone indicating that the same surface is involved in dimerization and intraprotein interactions (137,156). The BLRB protein, a short-BLUF protein, is dimeric in crystals, adopting a similar conformation as AppA-BLUF dimers (40), referred to as AB dimers. The two cyanobacterial short-BLUF proteins TePixD and SyPixD form a 10-subunit complex comprised of two stacked pentameric rings (41,43). These structures show another way how BLUF domains can dimerize in oligomeric complexes: they form β-sheet-interfaced dimers between subunits from separate rings, while within the same ring they form dimers, stabilized via the α34 helices (41,43). From a recent analysis of the interactions of SyPixD with the cognate response regulator PixE, it was demonstrated that the latter drives aggregation of SyPixD dimers, the stable form in solution, into a SyPixD10–PixE5 complex under dark conditions (157). Photoactivation destabilizes the complex into monomers of PixE and dimers of SyPixD (157). The light-driven destabilization is most probably triggered by the conformational changes occurring at strand β5 and at the β45 loop, a region comprising the conserved W91 and M93 residues (157). It is likely that light-induced disassembly of the PixD–PixE complex constitutes the “output signal” that regulates a signal-transduction pathway controlling motility of Synechocystis. Indeed, M93, but not W91, is essential for the biological response, i.e. positive phototaxis, as well as for the light-driven destabilization of the SyPixD10–PixE5 complex (158).

Information is also available for the KpBlrP1-BLUF domain in solution, which appears to be a stable monomer (46), while full-length KpBlrP1 is dimeric (45). A crystal structure is now available for KpBlrP1, and biochemical investigation reveals that light absorption by the BLUF domain of one subunit of the antiparallel homodimer activates in trans the phosphodiesterase activity of the EAL domain of the second subunit via allosteric communication, transmitted through conserved domain–domain interfaces (45). Interestingly, in the full-length protein, dimerization does not occur via BLUF–BLUF interactions, but rather via BLUF–EAL interactions in trans and mostly mediated by α4 which is in turn clamped on the BLUF-domain β-sheet (45).

Finally, a monomer–dimer equilibrium perturbed by light activation was observed for YcgF. By combining SEC and transient-thermal grating, it was shown that YcgF exists in fast and temperature-dependent monomer–dimer equilibrium. Light excitation results in transient dimerization of the monomeric species, with a 20 ms rate constant, as revealed by the sharp decrease of the diffusion coefficient (159) (Figure 5d).

Summarizing the data discussed in these last two sections, we can state for LOV and BLUF domains: (1) the β-sheet undergoes light-driven conformational changes, directly conveyed by interactions with the flavin chromophores; (2) the β-sheet can mediate dimerization, but is also able to interact, more or less tightly, with helical regions flanking the photosensing LOV or BLUF core or, alternatively, can directly interact with an effector domain; (3) the sequences flanking the photosensing LOV or BLUF core can in turn mediate an alternative dimerization and interactions with effector domains, thus mediating light-driven activation of the latter. Unfolding of the Jα-linker in phot might represent just one variation of a general β-sheet/helical cap mechanism (46). We have to admit that there is not a canonical reaction mechanism for either the LOV or for the BLUF domains irrespective of the highly conserved three dimensional structures found for these protein domains.

Molecular partners of BL receptors during signal transduction

LOV proteins.  Up to now much effort has been focussed on elucidating the primary mechanisms underlying LOV-proteins activation by light, but the downstream signaling processes remain largely elusive. Yet several targets have been identified, especially in eukaryoptic organisms. Phot-interacting proteins include: (1) 14-3-3, a family of conserved regulatory molecules expressed in all eukaryotic cells (106,127,160,161); (2) NPH3 (NONPHOTOTROPIC HYPOCOTYL-3), essential for phototropism (162); (3) RPT2 (ROOT PHOTOTROPISM-2) that is closely related to NPH3 and mediates both phototropism and stomatal opening (163) and (4) PKS1 (phytochrome kinase substrate 1) that influences phototropic curvature (164) (reviewed in ref. [4]). Growth responses such as phototropism require auxin and brassinosteroids, but these two factors have been shown to be not involved in the control of chloroplast movements. Also, gene-expression changes are essential for phototropism, and are most probably induced following the asymmetric distribution of auxin that is required for directional growth (165).

Besides the indirect control of gene expression carried out via complex pathways by phot (4), some LOV proteins are equipped with effector modules predicted to directly interact with target genes (10,166), and at least for one class of them, the AUREOCHROMES (AUREO) from photosynthetic stramenopiles algae, such interaction has been demonstrated (167,168).

In the ZTL/LKP2/FKF1 family, the LOV domain is linked to an F-box and six Kelch (beta propeller structures) repeats (169), suggesting that they may act in BL-regulated protein degradation, but interactions with components of ubiquitin-mediated protein turnover is not dependent on the LOV domain (4,170–172). The main partner of ZTL and LKP2 is GIGANTEA (GI) that interacts directly with the LOV domain in a light-dependent way, ultimately leading to regulation of CONSTANS (CO) gene expression, which is necessary for day-length discrimination and photoperiodic flowering (173,174).

For bacterial LOV proteins we only have limited information. Yet the best characterized protein function was found for B. subtilis YtvA (34). The fused STAS domain shows light-modulated, low-affinity GTP- and ATP-binding in vitro (80,175). During the environmental stress pathway, YtvA up-regulates the alternative transcription factor σB through a cascade of Rsb proteins, organized in stressosomes, in a BL-regulated way (149,176,177). This capability strictly depends on signal propagation from the LOV domain to the GTP binding cavity on the STAS domain (80,116), demonstrating that nucleotide binding is physiologically relevant.

BLUF proteins.  The E. coli YcgF protein carries an EAL domain, until recently being postulated to catalyze c-di-GMP hydrolysis in a light-regulated way (90,131,133). Actually it could be shown that YcgF does not hydrolyze or bind c-di-GMP, but rather acts by protein–protein interaction (132). YcgF directly binds to the repressor YcgE, releasing it from its operator DNA upon blue light irradiation. As a consequence, a distinct regulon of eight small proteins involved in biofilm formation is induced. Furthermore, BL and other stress signals are integrated at the level of the YcgF-YcgE system that is strongly induced at low temperature and starvation conditions, and may thus modulate biofilm formation when E. coli has to survive in an extrahost aquatic environment (132). This work not only elucidated the molecular pathway leading to a BL response in E. coli via a BLUF protein, but also showed that in a protein possessing a degenerate EAL domain (128), BL can activate a function other than an enzymatic one. One should not forget that in the protein KpBlrP1 from Klebsiella pneumonia (ortholog to E. coli YcgF, i.e. identically built with a BLUF+EAL architecture), the EAL domain shows a light-activated phosphodiesterase activity (45).

The way how YcgF works is reminiscent of AppA, one of the other few complex bacterial BLUF proteins (the majority of which [ca 87%] are “short-BLUF” without a fused effector domain) (10,88). Present in the facultative photosynthetic bacterium R. sphaeroides, AppA binds, constitutively at low oxygen tension, the repressor protein PpsR, whereas under fully aerobic conditions, PpsR is released from AppA and binds to the promoter of certain photosynthesis genes, repressing their transcription. These responses are light independent, but at intermediate oxygen concentration (∼50% oxygen saturation), light determines whether AppA releases the repressor PpsR (178,179). Thus, AppA integrates and transmits both redox and light signals, the former thanks to the heme-binding SCHICH (Sensor Containing Heme Instead of Cobalamin) domain (180) and probably to a C-terminal cysteine-rich sequence. The function of the AppA/PpsR system is fully coordinated by the PrrB/PrrA system (based on bacteriochlorophyll absorbers) that, at low oxygen concentration, activates photosynthesis gene expression (179,181). The work of these systems in R. sphaeroides is obviously aimed to the maximization of photosynthesis efficiency in conditions when oxidative heterotrophy is impaired, and to minimize waste of energy for the building of photosynthesis complexes and also to reduce photooxidative damages.

LOV and BLUF-mediated physiological responses in prokaryots

  1. Top of page
  2. Abstract
  3. Introduction
  4. From photochemistry to signaling
  5. LOV and BLUF-mediated physiological responses in prokaryots
  6. New blue light trends
  7. Author Biographies
  8. References
  9. Supporting Information

Phototropism, photomorphogenesis and circadian rhythm entrainment in plants have important counterparts in BL-regulated growth patterns and BL/redox regulation of photosynthesis or for infectivity of bacteria, and even for BL killing of pathogens.

LOV proteins: from stress responses to regulation of infection

In eukaryots, the major LOV protein groups are formed by phot and the ZTL/FKF/LKP2 family in plants (3,4), by white collar 1 (WC-1) (182–184), VIVID (185,186) and ENVOY (187) in fungi, and by the recently discovered AUREO in stramenopiles (167,168). Phot act as BL-driven regulators for processes that aim at the optimization of photosynthesis, growing of plants under dim light conditions and minimization of photodamage (2,4). In Arabidopsis thaliana two phot (phot1 and phot2) act redundantly during many responses (phototropism, stomatal opening, leaf expansion, chloroplast accumulation at low light intensity), but are specifically active for others, e.g. phot1 inhibits hypocothyl growth during photomorphogenesis while phot2 is responsible for chloroplast avoidance movement at high light intensities (see refs. [2,4,188] and references therein).

In the green alga C. reinhardtii, however, only one phot is present. This BL photoreceptor regulates the algal sexual lifecycle, namely, it is essential for the light-dependent step in gamete differentiation (189). Furthermore, it modulates the expression of several photosynthesis genes (190). Broadly speaking the two-LOV-domain phot proteins regulate movements, growth, development and reproduction patterns. Proteins belonging to the ZTL/FKF/LKP2 influence instead flowering time by affecting circadian rhythm-controlled gene expression, i.e. they are BL sensors that posttranslationally regulate the circadian clock and photoperiodic flowering (172). The precise role of LKP2 is still to be clarified, but it is certainly involved in the circadian regulation of the central clock (4,171,191).

The LOV-bearing AUREO from the stramenopile Vaucheria frigida not only binds to target DNA as predicted from its domain organization but also regulates gene expression in a BL-dependent way, in other words it is a (directly) BL-regulated transcription factor. The physiological response regulated by AUREO1 is a photomorphogenetic growth pattern, namely BL-induced branching, elegantly demonstrated by means of interference RNA experiments (166,167).

Despite the wide distribution of LOV proteins in the prokaryotic world, including Archaea (13,14,108,109,166), only for few of these proteins a biological role has been established. YtvA from B. subtilis binds GTP and ATP with low affinity within its C-terminal STAS domain (175). This NTP-binding capability is of special interest, as it has been shown that ATP and GTP are important signaling molecules during the sporulation process and nutritional stress responses of B. subtilis (192–194). It has also been demonstrated that YtvA acts as a positive regulator for the general stress transcription factor σB, specifically within the environmental branch, apparently in a BL-regulated way (116,149,176,177,195). BL up-regulation of the stress pathway is suppressed by mutations that affect NTP binding or light-induced signal propagation within YtvA, thus establishing a link between in vitro and in vivo data (80,116).

Recently a link has been established between a LOV kinase and the photoregulation of bacterial pathogenesis of the gram-negative pathogen Brucella abortus: the LOV kinase is required for BL-dependent cellular proliferation in macrophages (51). A similar LOV kinase modulates the capacity for cell adhesion in a BL-dependent way in C. crescentus (50). As the ability of bacterial pathogens to adhere to host cells is often a critical determinant of virulence, this observation might represent an important feature during infection. Bioinformatic analysis of over 600 bacterial genomes reveals that at least one LOV domain protein is present in 13% of all species, including a number of pathogens (Data S1). However, the relation between BL receptors and pathogenicity cannot yet be considered as a general one (10,196).

BLUF proteins: from phototaxis to biofilms

There are still few examples where physiological functions could be unambiguously ascribed to BLUF proteins. Important information has been collected for the PAC proteins of E. gracilis. PAC-α and PAC-β are large proteins, comprising each ca 1000 aa. Each protein (α and β) consists of two BLUF-cyclase motifs arranged in tandem. They function in a PAC-α/PAC-β heterodimeric complex as BL-regulated adenylate cyclases, responsible for step-up (but not step-down) photophobic responses as well as both positive and negative phototaxis (197,198). PAC proteins are conserved among euglenoids (199), but to our knowledge, BLUF proteins are not present in other eukaryotic phyla.

Also for bacteria, functional information for BLUF proteins is still sparse, with the notable exception of AppA from R. sphaeroides and of YcgF from E. coli. AppA is a light and redox regulator for the expression of photosynthesis genes, built of a light-sensing BLUF module (87,200) and a redox-sensing C-terminal domain, shown to bind heme (180,201). This light-dependent regulatory function of AppA is accomplished only at intermediate oxygen concentration, when BL excitation induces the release of the transcription factor PpsR (179). Interestingly, a BLUF domain from a Euglena PAC protein fused to the C-terminus of AppA is fully functional in regulating light-dependent gene expression in R. sphaeroides (202). This finding implies that a BLUF domain can convey signals to completely different output domains and that a eukaryotic light-sensing module can fully replace its homolog in a prokaryotic cell, although the sequence similarity between PAC-BLUF and AppA-BLUF is lower than 30%.

Recently it was suggested that BL-induction of photosynthesis pigments in the marine gammaproteobacterium Congregibacter litoralis might be under the control of BLUF proteins, indeed well represented in this organism (see Data S2) (203).

YcgF from E. coli carries out BL-regulation of biofilm formation (132). The EAL domain in YcgF neither hydrolyzes c-di-GMP nor binds it, but is rather active in protein–protein interaction. The molecular pathway leading to light-induced up-regulation of a biofilm matrix component (colanic acid) and acid resistance genes and to the down-regulation of adhesive curli fimbriae has been illustrated in the section above. The Slr1694 short-BLUF protein from Synechocystis sp. PCC 6803 has been identified to be involved in phototaxis (204,205), but its action appears to be integrated with that of other photoreceptors and its precise function still remains elusive (206).

Blue light, an ancient signal of danger?

At first glance it seems surprising to find a large number of positive hits for BL photoreceptor genes from Genbank surveys. In fact ca 13% of fully sequenced bacteria possess at least one LOV or BLUF protein (10) (Data S1 and S2). Obviously, blue light represents an important environmental factor that penetrates deepest into a water column, compared to all other spectral ranges (15). It also induces potentially harmful photochemical reactions which have to be efficiently counteracted, making the initiation of physiological responses upon the detection of BL essential for survival. This assumption also explains the variation in signaling domains for one and the same detector domain in several microorganisms (e.g. Erythrobacter litoralis exhibits three histidine kinases fused to LOV domains and one HTH—helix-turn-helix—motif), allowing a wider set of BL-regulated physiological responses (9,10,51,195). Alternatively, microorganisms are found where several stress factors are integrated in a “stressosome” to initiate a more stereotypical response (well studied in B. subtilis), e.g. activation of the σB regulon (176,177). BL is an ambivalent environmental factor: on the one hand, it represents an attracting light quality allowing activation of the photosynthetic apparatus, and also it is essential for the function of light-activated DNA-repairing enzymes (6). On the other hand, porphyrinic compounds that are ubiquitous, e.g. as cofactors in cytochromes, exhibit a strong absorption (εmaxca 105 m−1 cm−1) in the blue region of the visible spectrum (207). These compounds usually function via an iron atom bound in the center of the tetrapyrrole ring structure. In case, however, the central metal ion is absent, the porphyrins exhibit a high tendency to form triplets from their excited singlet state (16,207). Triplet state porphyrins, in turn, convert with high yield the ever present triplet oxygen into its singlet state (16), which is outstandingly reactive and readily causes deleterious damage in its direct surrounding, eventually even causing cell death by photooxidative stress (207). This photosensitization effect is presently being exploited in the photodynamic therapy of cancer (207), and as a potential killing device for bacteria: it has been demonstrated that the antibiotic-resistant Staphylococcus aureus can be killed, in vitro, with 470 nm light (208). Interestingly, one finds in several cases genes encoding BL photoreceptors in vicinity to genes of the iron metabolism (e.g. encoding Fe-chelatases and iron scavengers) (G. Pathak, personal communication). Such an increase in the biosynthesis of iron scavengers (pyoverdins), which is up-regulated selectively by BL, has recently been demonstrated for P. putida (209).

In an aerobic environment the deleterious oxidative effects rely on the same BL bioabsorbers that optimize both respiration and photosynthesis, i.e. porphyrins and flavins, but living organisms have learnt how to take advantage of O2 and BL, at the same time minimizing photodamages. One of the organisms that has best exploited this interplay between O2 and BL is R. sphaeroides, a member of the cosmopolitan α-proteobacteria family. Rhodobacter sphaeroides can switch between photosynthesis and aerobic respiration via a complex regulation mechanism that includes the AppA protein (179). At high pO2 photosynthesis genes are not expressed, no matter if BL is present: it would be useless, costly and potentially dangerous. The contrary is true under oxygen-limited or anaerobic conditions. At low pO2, it is BL that promotes the transcription of photosynthesis genes, by triggering the AppA photocycle (210).

New blue light trends

  1. Top of page
  2. Abstract
  3. Introduction
  4. From photochemistry to signaling
  5. LOV and BLUF-mediated physiological responses in prokaryots
  6. New blue light trends
  7. Author Biographies
  8. References
  9. Supporting Information

BL sensors as tools for cellular studies: reporters and optogenetics tools

The employment of the ubiquitous and photochemically versatile flavins as chromophores in BL photoreceptors distinguishes these chromoproteins as promising tools for biotechnological applications, particularly in the exciting field of optogenetics, where light-gated proteins originally designed by nature are exploited as tools to photomodulate activities even in cells of higher organisms (79,211).

The high fluorescence quantum yield of LOV domains (ca 0.25 in the dark state [56]) allows applications such as those now common for the various GFP derivatives (212). As fluorescence is lost upon the photochemical formation of the photoadduct state, this process can be annihilated by exchanging the covalent bond forming a cysteine residue for an alanine or serine, yielding a permanently fluorescent molecule (213). In fact, compared to GFPs, where the fluorescent chromophore forms via an intramolecular condensation of three amino acids, requiring the presence of oxygen, the LOV domains are advantageous for selected applications in anaerobic or microaerobic environments or during viral infections of plants, where they even outperform GFP (213,214).

Beyond the inherent properties of the LOV domains, their combination with various signaling domains, as found by genome surveys (10,108), has seen the emergence of an entirely new field of biomedical applications. The possibility to regulate intracellular physiological processes simply by an external irradiation is definitely an intriguing aspect and has caused an explosion in applications. Initially, retinal (vitamin A aldehyde)-based photoreceptors have opened this research field (“channelrhodopsins”) (215–218), however, the above discussed PAC from E. gracilis (carrying as functional elements a combination of a BLUF domain and an AMP-cyclase) has initiated optogenetic research with flavin-based receptors (219,220). This function is based on the light-regulated cAMP-mediated activation of certain neurons upon functional expression of PAC, causing light-induced changes in behavior via cAMP formation by the BLUF-domain activation of the cyclase (219,220). The fast increase in cAMP in turn regulates, via a phosphorylation cascade, gene expression and other processes in eukaryotic cells. Additional applications are expected, especially after the characterization of a bacterial BLUF adenylyl cyclase from the gammaproteobacterium Beggiatoa sp. PS, denominated bPAC (221) or BlaC (222). The small (350 aa) bacterial protein appears to be more efficient and versatile than the Euglena PAC in eliciting cAMP-regulated activities triggered by light once integrated in a given cell system (e.g. pyramidal neurons, Xenopus oocytes, cAMP-deficient E. coli) (221). BlaC can also be converted into a cGMP cyclase by suitable mutations, extending its range of applications (222).

One step further in the optogenetic direction has been made by the combination of BL-sensing domains (LOV and BLUF) with enzymatic or regulatory functions from other proteins. Early experiments have shown that the kinase domain of phot2 is constitutively active toward the heterologous substrate casein (223). Adding in vitro the phot2-LOV2 domain enables a light-control of this kinase activity, even in the absence of a covalent LOV2–kinase interaction (223). A direct light-regulated DNA binding was accomplished by a hybrid protein consisting of a LOV2 domain from A. sativa phot1 and the E. coli Trp repressor protein (TrpR) (224), based on the light-induced undocking of the Jα-helix from the LOV core that activates the effector domain (118). Employing the building principle of many LOV domain proteins being fused to histidine kinases, the LOV domain of YtvA was fused to a histidine kinase from the oxygen-sensing protein FixL (126). The light-induced dampening of kinase activity, paralleling the inhibition promoted by the natural oxygen substrate, relies in this case most probably on a “torque” effect carried out by helical linker regions connecting the sensing and effector domains, arranged in dimeric units (79). Coupling the LOV domain to the original heme-binding PAS-B domain of FixL, the system becomes a double sensor, albeit with low activity (225).

Recently, exciting results have been published, whereby Rho-like GTPases, Rac and Cdc42, caged with a LOV domain have been engineered: the proteins are activated upon irradiation, repeatedly and reversibly, in living cells to produce protrusions, filopodia, etc. and probe signaling pathways (226).

These works certainly pioneered the use of LOV domains in the design and engineering of photofunctional LOV proteins, but researchers became soon aware that the design of tools for optogenetics must be optimized for practical applications (120,126). The point is thoroughly made in a dedicated review (79). We just recall here the impressive improvement obtained by Strickland et al. (120): in the LOV2-TrpR hybrid protein mentioned above, the degree of light activation is modest and the overall DNA-binding affinity of LOV2-TrpR is about two orders of magnitude lower than that of the wildtype TrpR (224). This is, at least partially, linked to the fact that in the presence of the effector domain (i.e. the TrpR module here) the docked-undocked equilibrium of Jα-helix to the LOV core is shifted toward the undocked state, i.e. the protein is functionally mostly active even in the dark state (vide supra) (120). Nevertheless, using site-directed mutagenesis, the regulatory effect of light on DNA binding by LOV-TrpR could be improved to ca 64-fold, by shifting the equilibrium toward a preferential docked Jα-helix in the dark state, thus largely extending the light sensitivity of the enginereed system (120).

From the laboratory to the real world: metagenomics and the blue-light world

The broad distribution of LOV domains in many prokaryotic genomes, documented by ongoing GenBank surveys (10), has initiated a novel activity to investigate even uncharacterized material for the presence of BL photoreceptors (14,227), assuming that the presence of BL photoreceptors would be similarly high as in genome-classified organisms. The rationale for such an approach is the expectation that searching exotic or extreme environments, proteins with novel and unusual properties might be found. In addition, the amount of sequence material from such “metagenomic” approaches is already nowadays higher by at least a factor of 10 compared to the genome sequences deposited in the databases. Such studies are readily performed on a sequence-based setup employing DNA microarrays: assuming a distribution of LOV domains in metagenome material similar as in sequenced genomes, a set of oligonucleotides, encoding representative LOV domain regions within a highly conserved, canonical sequence region, is covalently attached to a solid support material (a glass or plastic slide or an electronic chip). This DNA microarray serves as bait for hybridization with fluorescence-labeled, unknown DNA (potentially cloned into BAC, phasmids or cosmids). It will give rise to a positive fluorescent signal at positions where hybridization to an oligonucleotide with known sequence took place. Moving out from the known sequence motif back into the unknown DNA then allows “harvesting” the full-length gene encoding a novel protein. The intermediate cloning step of uncharacterized DNA even allows, after identification of the full-length gene, a heterologous expression and a functional characterization of the gene product (14), and in addition identifies gene neighborhood relations and operon structures of simultaneously regulated genes. Such an approach reveals only sequences with a relatively high degree of similarity to the oligonucleotides used as bait. A function-based investigation, however, would enable us to identify a protein function irrespective of the sequence. In case of LOV domain proteins, the recovery of the fluorescent parent state (LOV445) could readily be followed in a high-throughput heterologous-expression approach.

Also entirely in silico-based investigations have recently been perfomed for both BLUF- and LOV domains (227) (G. Pathak, A. Losi, and W. Gärtner, unpublished). These studies, again based on sequence comparisons, revealed the presence of BLUF and also LOV domains in practically any environment, deriving from many locations in various oceans (Sargasso and Global Ocean Sampling Project), or from waste water-treatment plants or mining drainage (Pathak et al., unpublished).

The last decade has seen an enormous growth in our understanding of the function of BL photoreceptors, both with respect to their inherent photochemical reactivity and also related to their role in regulating physiological functions. This knowledge is now starting to be extended by investigations into the role of (blue) light as an important and regulatory function in environmental research and microbial communities, including screening databases and metagenomes. Based on these developments, one can expect in the near future that novel research directions, e.g. the regulation of physiological processes by BL domains in naturally occurring photoreceptors or as parts of hybrid proteins, will become a rapidly growing research area with outstanding potential in biomedical applications.

Author Biographies

  1. Top of page
  2. Abstract
  3. Introduction
  4. From photochemistry to signaling
  5. LOV and BLUF-mediated physiological responses in prokaryots
  6. New blue light trends
  7. Author Biographies
  8. References
  9. Supporting Information
  • image

[ Aba Losi ]

Aba Losi received her PhD in Biophysics in 1997 at the University Parma, Italy. In 1998 she was a Marie Curie postdoc fellow at the MPI for Radiation Chemistry, presently MPI for Bio-Inorganic Chemistry in Mülheim (Germany), where she performed physicochemical studies and time-resolved thermodynamics of photosensors. In 2002 she became a permanent researcher at U. Parma and published her first work dealing with prokaryotic blue–light-sensing proteins of the LOV superfamily. Since then she has published more that 30 papers in this field and organized several symposia within international congresses.

  • image

[ Wolfgang Gärtner ]

Wolfgang Gärtner studied chemistry at the Universities of Göttingen and Würzburg (Germany) and received his PhD in 1982. He presented his thesis for habilitation for Bioorganic Chemistry in 1993 at the University of Duisburg and was nominated Professor adjunct to the same university in 1999. Being a staff member and P.I. at the MPI for Bio-Inorganic Chemistry in Mülheim, he teaches biochemistry at the University of Düsseldorf. His research topics are biological photosensors and hydrogenases.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. From photochemistry to signaling
  5. LOV and BLUF-mediated physiological responses in prokaryots
  6. New blue light trends
  7. Author Biographies
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. From photochemistry to signaling
  5. LOV and BLUF-mediated physiological responses in prokaryots
  6. New blue light trends
  7. Author Biographies
  8. References
  9. Supporting Information

Data  S1. LOV proteins in prokaryotes.

Data  S2. BLUF proteins in prokaryotes.

FilenameFormatSizeDescription
PHP_913_sm_Gartner-SUPPL1.doc676KSupporting info item
PHP_913_sm_Gartner-SUPPL2.doc552KSupporting info item

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