The bacterial counterparts of plant phototropins

Aba LosiDepartment of Physics, University of Parma, Istituto Nazionale per la Fisica della Materia,Parco Area delle Scienze 7/A, 43100, Parma, Italy. E-mail: losia@fis.unipr.it

Received 15th January 2004, Accepted 24th February 2004First published as an Advance Article on the web 10th March 2004

We review and analyze the growing family of bacterialproteins carrying the LOV (light oxygen voltage) motif, aflavin-binding photoactive domain first characterized inplant blue-light receptors, the phototropins. A total of 29sequences encoding LOV-proteins can be detected in thegenomes of 24 bacterial species. In the bacterial LOVdomains, the majority of the amino acids known to inter-act with the flavin mononucleotide (FMN) chromophore inphototropin LOVs are conserved, supporting the sugges-tion of their possible role as blue-light sensors. The Bacillussubtilis protein YtvA has been the first bacterial LOV-protein shown to bind FMN and to undergo the same light-induced reactions as plant phototropins. The photocycleinvolves the reversible formation of a covalent adductbetween FMN and a conserved cysteine. In this work wereport preliminary results on a Caulobacter crescentusLOV-kinase, that undergoes the same photochemistry asYtvA. The bacterial LOV-proteins exhibit a variety ofeffector domains associated to the light-responsive LOV-domain, e.g. histidine kinase, transcriptional regulators,putative phosphodiesterases and regulators of stressfactors, pointing to their physiological role as sensing andsignalling proteins.

IntroductionGenome sequencing and annotation projects are revealing thatbacteria are equipped with a range of putative photosensorproteins, whose physiological significance remains, in mostcases, poorly characterized. The analysis of the different lightsensing/light responsive protein modules and the elucidationof their light-triggered reactions, is more and more pointing toa scenario where given photosensing paradigms (e.g. retinal-binding proteins, phytochromes, flavin-based photoreceptors)are shared between eukaryotes and prokaryotes.1–4

In this paper we will focus on bacterial proteins related to

Aba Losi

Aba Losi studied Biology atthe University of Parma (Italy),where she also received herPhD in Biophysics in 1997. Shewas the recipient of a MarieCurie EU post-doctoral fellow-ship, which she completed in thelaboratory of Prof. Silvia E.Braslavsky in Muelheim an derRuhr (Germany). She has beenactively studying the thermo-dynamics of light inducedreactions in biological photo-receptors, by means of time-resolved optical and photo-calorimetric techniques. Her

interests include the developing field of flavin-based blue-lightphotoreceptors in bacteria. Aba Losi presently holds a researchposition at the Faculty of Science of Parma University.

the plant blue-light receptors phototropins (phot). This classforms a family in bacteria about which knowledge is rapidlygrowing,3,5 albeit their possible significance as photosensors isstill to be assessed and their molecular properties are largerlyuncharacterized. Phot are membrane associated kinases thatundergo UVA/blue light-induced autophosphorylation 6 andrepresent the main photoreceptors for phototropism, chloro-plast relocation and stomatal opening in higher plants.7

Chlamydomonas reinhardtii phot is instead involved in bluelight-mediated gametogenesis.8 Phot proteins possess two N-terminal photoactive LOV (light, oxygen and voltage) domains(LOV1 and LOV2), a subset of the PAS (PerArntSim) super-family,9 and a C-terminal serine/threonine kinase domain.Phot-LOV1 and LOV2 bind oxidized flavin mono-nucleotide(FMN) as chromophore and absorb maximally at ca. 450 nm(LOV447).

10 Blue-light illumination of phot-LOV domainstriggers a photocycle involving the formation of a blue-shiftedFMN-cysteine C(4a)-thiol adduct (LOV390) that slowly revertsto LOV447 in the dark.10–12 In the following we will only discuss asubset of LOV domains that contain the conserved cysteine andare likely to undergo phot-like photochemical reactions. Thephotocycle of LOV domains comprises a red-shifted transientspecies, LOV660 (appearing on the fs time-scale 13), assigned tothe FMN triplet excited state and decaying on the short micro-second time-scale into LOV390.

14,15 Detailed structural inform-ation both in the dark (LOV447) and in the photoactivated state(LOV390), are available for the LOV2 domain of phy3 16,17

(a phytochrome-phot hybrid photoreceptor from the fernAdiantum capillus-veneris) and for C. reinhardtii phot-LOV1.18

The two structures appear very similar, exhibiting a typicalα/β PAS fold and have allowed to identify the residues thatinteract with FMN and the structural changes occurring uponlight activation.

Phot proteins are the first plant blue-light receptors for whichidentity, function and photochemistry have been elucidated.Their photocycle has established a new paradigm in the fieldof photosensory biology, in contrast to chromophore cis–trans isomerization.19 The phot light-sensing mechanism hasapparently been conserved across distant phyla. In fact, theBacillus subtilis protein YtvA, a 261 aa protein that possesses aphot-like LOV domain, was found to bind FMN and undergophot-like light-induced reactions.5 A Caulobacter crescentusLOV protein with a kinase C-terminal domain is shown here toundergo the same photochemical reactions, although problemsin protein expression and purification still impair detailedcharacterization. This system is of high interest given that it isrelated to both phot and to the bacterial two-component signaltransduction paradigm (vide infra).20

On the basis of sequence and domain analysis it is pre-dictable that other bacterial proteins exhibit similar features,3

coupling a LOV light sensing module to diverse effectorsdomains, such as kinases (similar to phot), phosphodiesterases,response regulators, DNA-binding transcription factors andregulators of stress sigma factors. These effector modules indi-cate that bacterial LOV-proteins are part of the cell signalingmachinery, although a light-driven regulation of their activityremains to be demonstrated, as well as their light dependentphysiological role.D

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Table 1 Bacteria whose genome contain sequences encoding LOV-proteins, with protein accession numbers and aa interval of the phot-likeLOV domain

Species Accession Nr. (LOV-protein) Length (aa number) LOV domain (aa interval)

Bacillus subtilis a O34627 r 261 25–126Oceanobacillus iheyensis b Q8ESN8 r 264 21–124Listeria monocytogenes c P58724 r 253 19–121Listeria innocua c Q92DM1 r 253 19–121Rhodopirellula baltica d Q7USG5 r 1637 998–1101Pseudomonas syringae pv. syringae e ZP_00124092 s 534 33–136Pseudomonas syringae pv. tomato f Q881J7 r 534 33–136Xanthomonas campestris g Q8P827 r 540 39–142Xanthomonas axonopodis g Q8PJH6 r 540 39–142Brucella melitensis h Q8YC53 r 489 32–135Brucella suis i Q8FW73 r 463 6–109Magnetospirillum magnetotacticum e ZP_00051334 (1) s

ZP_00052303 (2) s454365

70–17341–144

Caulobacter crescentus l Q9ABE3 r 449 114–217Novosphingobium aromaticivorans e sZP_00095689 (1)

sZP_00093141 (2)473244

164–27060–163

Thermosynechococcus elongatus m Q8DJE3 r 1353 328–431Nostoc punctiforme (1) e ZP_00105980 s 1403 240–343Nostoc sp. PCC 7120 (1) n Q8YT51 r 1817 449–552Nostoc punctiforme (2) e ZP_00111211 s 1043 239–342Nostoc sp. PCC 7120 (2) n Q8YSB9 r 1021 213–316Synechocystis sp. PCC 6803 o Q55576 r 1244 307–413Ralstonia solanacearum p Q8XT61 r 1178 635–738Burkholderia fungorum e ZP_00034273 s 1036 486–589Rhodobacter sphaeroides e ZP_00007036 s 176 18–121Chloroflexus aurantiacus e ZP_00020459 s 242 43–147Pseudomonas fluorescens e ZP_00084650 s 158 16–121Pseudomonas putida (KT2440) q Q88E39 (1) r

Q88JB0 (2) r142151

16–11919–122

a Ref. 65. b Ref. 66. c Ref. 67. d Ref. 68. e Genome annotation in progress at the NCBI. f Ref. 69. g Ref. 70. h Ref. 71. i Ref. 72. l Ref. 73. m Ref. 74.n Ref. 67. o Ref. 24. p Ref. 75. q Ref. 76. r Swiss-Prot/TrEMBL databank. s NCBI databank.

In the following, we review the bacterial LOV-proteins thatare presently detectable through genome digging and sequencecomparison, keeping in mind that the scenario is prone torapidly evolve thanks to ongoing genome projects. The presentinformation on the photochemistry of bacterial LOV-proteinsis summarized. We also group the bacterial LOV-proteins inprotein families according to their putative molecular function,in view of their similarity to well characterized systems. Finallywe discuss the possible molecular mechanisms of light-driveninterdomain communication on the basis of current hypothesesproposed for phot and for other PAS-based sensing proteins.

MethodsBacterial phot-like LOV domains were searched by usingthe BLAST 21 network service at the Swiss Institute of Bio-informatics (SIB) and the National Center for BiotechnologyInformation (NCBI), over all annotated genome sequences(completed or in progress). The expectation value (E ) thresholdwas set at 0.0001 and sequences were filtered for low-complexityregions. Only sequences with E ≤ 10�10 were accepted andfurther visually inspected. Sequence alignments and phylo-genetic trees were computed at the ClustalW network service ofthe European Bioinformatics Institute (EBI), using defaultparameters.22 The phylogenetic tree was drawn using the Phylipoutput at the ClustalW network service.22 Protein domainswere detected by using the InterProScan network service atthe EBI.23 Secondary sequence prediction was performed withPSIPRED.24 Prediction of transmembrane helices was donewith the TMHMM-2.0 network service at the Center forBiological Sequences Analysis (CBS).25

Bacterial phot-like LOV domainsThe first evidence for the occurrence of LOV-proteins inbacteria was presented by Huala et al., who compared the

sequences of phot-LOV domains with those from Bacillussubtilis YtvA (O34627, SwissProt-TrEMBL accession number)and Synechocystis PCC 6803 Q55576 (gene slr0359).6 Recently,Crosson et al. reported a sequence alignment including addi-tional bacterial LOV domains from Xanthomonas campestris,Xanthomonas axonopodis, Caulobacter crescentus, Brucellamelitensis, Nostoc sp. PCC 7120, Listeria monocytogenes andListeria innocua. 3

A BLAST 21 search through public databases, allowspresently to detect a total of 29 sequences encoding LOV-proteins, in the genome of 24 bacterial species (Table 1 andFig. 1).

Sequence comparison with Avena sativa and Arabidopsisthaliana phot1, C. reinhardtii phot and Adiantum phy3, showsthat bacterial LOV domains present intermediate charac-teristics between LOV1 and LOV2. In some cases the similaritywith LOV2 is markedly higher, with an identity of about 49%,against ca. 41% with LOV1 (e.g. in B. subtilis, P. syringae, X.axonopodis, X. campestris and N. aromaticivorans ZP_00093141proteins). Interestingly, the LOV domains from photosyntheticbacteria, are among the proteins with the lowest identity degreewith phot-LOVs (<ca. 40%, last 13 sequences in Fig. 1).

Secondary structure prediction of bacterial LOV domainsdoes not show any appreciable deviation from LOV1 or LOV2(data not shown), as expected given the high sequence similarityand the existence of only few gaps in the alignment. Neverthe-less, in some cases, aa substitutions that could impair FMNbinding are present. In the following we will refer to the aanumbering and protein structure of C. reinhardtii phot-LOV1.18

The reactive C57 is found in most bacterial LOV domainswithin the conserved GXNCRFLQ motif. In LOV crystal struc-tures, N56, R58 and Q61 are part of the hydrogen bond (HB)network that stabilizes the FMN chromophore (Fig. 2).16,18 TheQ61L substitution in the Synechocystis sp. 6803 Q55576 andNostoc sp. 7120 Q8YT51 proteins appears particularly critical,given that leucine is not polar and cannot form the same HB

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Fig. 1 Sequence alignment of bacterial LOV domains (see Table 1 for the primary protein accession numbers) with phot-LOV domains of knownstructure. The order from top to bottom is the same as in Table 1. Computation was performed at the ClustalW 22 network service of the EBI. Thesecondary elements in the crystal structure of C. reinhardtii phot-LOV1 18 and Adiantum phy3-LOV2 16 are shown above the alignment. The residuesinteracting with FMN are indicated as follows: ¡: interactions with the ribityl chain; ↓ hydrophobic pocket around the dimethyl benzyl ring of FMN;↓: interactions with the isoalloxazine ring; *: reactive cysteine. With the arrows ⇓ we have indicated the conserved E and K residues, forming a saltbridge at the surface of LOV domains.3 In green: residues conserved in all phot-LOV domains; in red: residues conserved in all phot-LOV2 domains;in light blue: residues conserved in all plant phot-LOV1 domains [from Crosson et al.16 (note that some residues are changed in C. reinhardtii phot-LOV1)]. In dark green: residues interacting with FMN and not conserved (neither with any LOV1 nor LOV2) in the bacterial LOV domains.Underlined are the two bacterial LOV-proteins for which phot-like photochemistry has been demonstrated.

as the lateral chain of glutamine. Other residues part of theFMN-centered HB network are N89, that is conserved in allLOV domains, Q120, changed for I in the B. fungorumZP_00034273 protein and N99, changed for S in C. aurantiacusZP_00020459. The pattern of residues that build the hydro-phobic pocket around the dimethyl benzyl ring of FMN isinstead conserved in all bacterial LOV domains. In the βDstrand, L101 in C. reinhardtii phot-LOV1 corresponds to F1010in phy3-LOV2, that is stacked on the re face of the FMNisoalloxazine (opposite to the reactive cysteine).16 An F residuein this position is characteristic of LOV2 and the distance withFMN-C4a becomes considerably larger upon formation of thephotoadduct (from 4.1 to 5.2 Å),17 whereas in LOV1 themovement of L101 is less pronounced (the distance increasesfrom 3.4 to 4.1 Å).18 As all phot-LOV1, the large majority ofthe bacterial LOV domains present an L in this position.

A peculiarity of the bacterial LOV-proteins is the presence ofN in place of S38, the latter conserved in all phot-LOVs andshown to be essential for FMN binding (suppressed upon S38Fmutation).11 The S38N substitution does not impair FMNbinding in B. subtilis YtvA,5 but could partially account forthe lower stability of the isolated YtvA-LOV with respect tofull length YtvA and to phot-LOVs.26 The S38N substitutionoccurs also in the FMN-binding LOV domain of the A. thali-

Fig. 2 Residues that form hydrogen bonds with FMN in the crystalstructure of C. reinhardtii phot-LOV1 18 and that are changed in someof the bacterial LOV domains. The picture was created from the PDBdatabank coordinates 1N9l using the beta-version of Deep View/Swiss-PDB viewer 3.7.77

ana photoreceptor FKF1.27 F41, also essential for FMNbinding,11 is conserved in all bacterial LOV domains. Both S38and F41 are not part of the FMN binding pocket, but could beimportant for correct protein folding

The few short gaps appearing in the sequence alignment ofFig. 1 are confined to the loops between secondary structureelements. The longest insertion is found in the Synechocystis sp.Q55576 protein and localized within the α�A–αC loop. Inser-tions in the same region, albeit longer, occur in the LOVdomains of the Neurospora crassa VIVID and WC-1, and A.thaliana FKF1 flavin-based photoreceptors.28 This extensionhas been suggested to be important for accommodating a largerchromophore than FMN, given that WC-1 is associated withFAD (flavin-adenin dinucleotide) 29 and purified VIVID canbind both FMN and FAD.30 The LOV domain of FKF1 hasbeen nevertheless shown to bind FMN and undergo phot-likephotochemical reactions, albeit the formation of the photo-product is irreversible.27 The extra insertion in the α�AxynαCloop, could therefore have a regulatory role.

In summary, the majority of the bacterial LOV domains arelikely to bind FMN and undergo phot-like photochemistry. Aphylogenetic tree, built on the basis of the sequence alignment,shows that the LOV domains cluster into two groups after thefirst root (Fig. 3).

LOV domains from photosynthetic bacteria (bottom partof the tree) are related to Adiantum phy3-LOV1 and to proteo-bacteria proteins showing the lowest similarity with phot-LOV1 and LOV2 (in P. putida, P. fluorescens, B. fungorum,R.solanacearum). Plant phot-LOV domains and the highlyhomologous proteins from the remaining proteobacteria,appear to derive from the LOV domains of gram-positivebacteria (e.g. B. subtilis YtvA-LOV). The LOV-domains fromproteobacteria falling in this cluster are associated with a kinasefunction in the full length proteins. The only exception is theN. aromaticivorans ZP_00093141, a putative DNA-bindingtranscriptional regulator (see the Protein families section). Onemust keep in mind that the phylogenetic analysis presentedhere is extremely simple and relies solely on the LOV domainsalignment of Fig. 1. Alternative alignments are possible and amore sophisticated phylogenetic analysis should take intoaccount the associated domains.

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Gene sequences encoding for phot-related LOV proteinswere not found in Archaea. The closest match was found in thebacterio-opsin activator (Bat), whose expression is triggeredunder low oxygen tension.31 In Bat the majority of the FMNinteracting residues is conserved but the photoreactive cysteineis substituted for a histidine. It is probable that Bat binds aflavin within its PAS domain, that could function as a redox-sensitive switch. The same features characterize in fact theredox-sensing, FAD-based aerotaxis receptor Aer 32 and NifL,a modulator of nitrogen-fixation genes transcription.33

Photochemistry of bacterial LOV proteinsAlbeit the sequence analysis presented above strongly suggeststhat bacterial LOV proteins can undergo phot-like photo-chemical reactions, this feature has been demonstrated only intwo cases: the B. subtilis σB regulator YtvA (O34627) 5 and theCaulobacter crescentus LOV-kinase Q9ABE3 (this work).

The B. subtilis YtvA protein

YtvA is 261 aa protein, that acts as a positive regulator of thegeneral stress transcription factor σB and was first suggested tobe a flavo-protein by Akbar et al.34 The N-terminal LOVdomain is followed in YtvA by a STAS (Sulfate transporter/Anti-Sigma-factor antagonist), carrying a putative nucleosidetriphosphate (NTP) binding site.35 When YtvA is expressed inE. coli, the protein is readily purified with a bound FMNchromophore and shows, in the dark, an absorption spectrumsimilar to phot-LOV domains (YtvA447, Fig. 4A).5 The darkstate YtvA447 emits a green fluorescence, with quantum yield ΦF

= 0.22 at room temperature. Upon light activation YtvA447

undergoes phot-like reactions, eventually leading to the form-ation of the blue-shifted, non fluorescent covalent adduct(YtvA390) via the 2 µs decay of the FMN triplet state (YtvA660)

5

Fig. 3 Phylogenetic tree calculated on the basis of Fig. 1, extendedto Avena sativa and Arabidopsis thaliana phot1-LOV1 and LOV2,Adiantum phy3-LOV1, C. reinhardtii phot-LOV2 (alignment notshown).

The quantum yield of formation for YtvA660 and YtvA390 havebeen measured by means of laser flash photolysis and photo-thermal methods to be Φ660 = 0.62 and Φ390 = 0.49, respectively.5

In the dark, YtvA390 slowly recovers to YtvA447 with lifetime τrec

= 2600 s at 25 �C (Fig. 4A).26 The isolated YtvA-LOV domainshows similar features but, contrary to the case of phot,exhibits a lower stability than the full-length protein and alonger τrec = 3900 s at 25 �C.26 YtvA-LOV also shows somehowdifferent photophysical parameters (ΦF = 0.16, Φ660 = 0.69 andΦ390 = 0.55). The data indicate that the conformation of theN-terminal LOV-domain is affected by the remaining partof the protein, a feature also confirmed by photothermalexperiments.26

The τrec = 3900 s for YtvA-LOV is very slow compared tophot-LOV domains, although in the latter the range ofmeasured τrec is quite large, namely between 5 and 330 s at roomtemperature.10,17 There is no difference in the primary sequencethat can easily account for this large variation in τrec. However,even tiny modifications in the chromophore cavity and/or inthe protein folding, can affect the high activation energy,11,26

thus dramatically changing the recovery kinetics at roomtemperature. We have recently shown, by means of photo-thermal experiments, that the formation of the photoadductin LOV-proteins is invariably accompanied by a volume con-traction (∆V390 < 0) with respect to the unphotolyzed state,indicative for a protein structural change.5,26,36 Mutationalanalysis in C. reinhardtii phot-LOV1 has revealed that themagnitude of ∆V390 is proportional to τrec, whereas there is norelation between τrec and the energy content of the photo-adduct.36 This effect is general and has been observed also inYtvA and YtvA-LOV,26 pointing to a structural barrier asmajor rate-determining factor for the dark recovery reaction.Furthermore the results obtained with C. reinhardtii phot-LOV1 indicate a key role of the HB network centred on theFMN phosphate group, in determining τrec (vide infra).

The Caulobacter crescentus LOV-kinase

The Caulobacter crescentus Q9ABE3 protein contains anN-terminal LOV domain, associated to a C-terminal kinasefunction, therefore it is intrinsically similar to phot. Uponexpression and purification of the C. crescentus Q9ABE3kinase in E. coli by using a similar protocol as for YtvA,5

we obtained only a small fraction of protein associated withFMN and undergoing phot-like photochemistry (Fig. 4B). The

Fig. 4 (A) Photochemistry of B. subtilis full-length YtvA, expressed inE. coli and purified as previously described.5 In the inset: photocycle ofYtvA at 25 �C. (B) Photo-induced spectral changes of the C. crescentusLOV-kinase Q9ABE3, expressed in E. coli. Inset: light � darkdifference spectrum of the C. crescentus LOV-kinase.

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Fig. 5 Domain architecture of selected bacterial LOV proteins and plant phot. The proteins are named after the bacterium of origin. Domainanalysis was performed at the EBI using the InterproScan service.23 Proportions are respected (for groups of proteins, the structure for the organismin bold is shown). TM: transmembrane helices; STAS: Sulfate transporter/anti-sigma factor antagonist domain; HisKIN: histidine kinase;RR: response regulator, receiver domain; HTH: HelixLoopHelix transcriptional regulator domain; Hpt = Histidine phosphotransfer domain;CheB: CheB methylesterase domain; CheR: CheR methyltransferase domain; GGDEF, EAL = domains named after their conserved aa motifs,found in diguanylate cyclases and phosphodiesterases; HAMP = domain found in Histidine kinases, Adenylyl cyclases, Methyl binding proteins,Phosphatases. See also Crosson et al.3 and the InterproScan service documentation.23

protein is also unstable, preventing detailed characterization.Similar features were observed with the isolated LOV domain,appearing even more unstable than the full length protein. Amore sophisticated experimental approach is apparently neededin order to fully characterize this system and to test its putativelight-regulated kinase activity.

Protein familiesThe bacterial LOV-proteins can be grouped in families, basedon the inferred function of the associated effector domains(Fig. 5).

In some cases high similarity in the primary sequenceand domain architecture is observed within the same family,resembling the situation observed among plant phot.37 Theproteins forming the most homologous groups are (1) theYtvA-like putative σB regulators and (2a) the hybrid kinase-response regulators from γ-proteobacteria.

1. Putative �B regulators

The LOV proteins from the four gram-positive bacteria (B.subtilis, O. iheyensis, L. monocytogenes and L. innocua), presentthe YtvA domain structure (ca. 260 aa), with a STAS domainC-terminal to LOV and form a highly homologous family.STAS domains have been detected in prokaryotic and eukary-otic proteins, and have been suggested to possess a general NTP(Nucleoside Triphosphate) binding role.35 The binding of aNTP to YtvA, albeit predictable on the basis of sequenceanalysis,5 could not be demonstrated up to now. YtvA wasrecently included in a family of novel regulators of the generalstress transcription factor σB (albeit with a relatively modesteffect) during salt and ethanol stress conditions.34 Differentfrom the other members of the regulators family, YtvApossesses a sensing domain (LOV), enhances the activity ofσB and cannot be phosphorylated by the RsbT environmentalsignaling kinase.34 The mechanism by which YtvA regulates σB

probably involves its interaction with two other regulators,RsbR and YqhA.34 It is intriguing to speculate that the inter-action might be regulated by light-activation, that is UVA/blue-light may represent a stress factor sensed by the YtvA photo-sensing domain, although this remains to be experimentallyassessed.

2. Kinases

A considerably large number (14) of the bacterial LOV proteinsare members of the histidine protein kinase (HPK) kinase

superfamily.38 They are found in P. syringae pv. syringae, P.syringae pv. tomato, X. axonopodis, X. campestris, C. crescentus,B. suis, B. melitensis, N. aromaticivorans (ZP_00095689), M.magnetotacticum (ZP_00051334 and ZP_00052303), R. baltica,Nostoc sp. PCC 7120 (Q8YT51), N. punctiforme (ZP_00105980)and T. elongatus (Q8DJE3). In bacteria, signal transducingHPKs, together with phoshoaspartyl response regulators(RR), are the key elements of two-component signal trans-duction systems.20 The HPKs generally contain an N-terminalsensing domain (e.g. LOV) and a C- terminal kinase core, butother domains may be present. The kinase core of HPKfeatures the phospho-accepting histidine box (H-box) withinthe homodimerization domain and, downstream of it, thehighly conserved homology boxes of the nucleotide-binding,catalytic domain (N-, D-, F- and G-boxes).38 In response to asignal, HPKs autophosphorylate the H-box histidine residue,from which the phosphoryl group is transferred to a conservedaspartic acid residue in the receiver domain of a RR.20

Generally RRs contain one or more output domains down-stream of the receiver domain, in some only the receiverdomain is present, often fused with the cognate HPK (hybridHPK-RR). HPKs can be further divided into subfamiliesaccording to their sequence similarity.38

2a. The LOV proteins from P. syringae pv. syringae, P. syrin-gae pv. tomato, X. axonopodis and X. campestris are highlyhomologous hybrid HPK-RRs (Fig. 5). The kinase domain ischaracteristic of the HPK4 class: besides a typical H-box, theseproteins exhibit the PF-TTK signature in the F-box (Fig. 6).38

These proteins are very similar to the rhizobial, PAS-based,heme O2 sensor-kinase FixL.39 Furthermore, their LOVdomains are among the most homologous ones to phot-LOVs(Fig. 1 and 3). Given these features, namely the association ofan ‘ideal’ phot-like LOV domain with prototypical kinase andRR motives, they are good candidates to test the molecularproperties of this novel, putative blue light-driven two-com-ponent signalling system in bacteria.

2b. The C. crescentus, B. suis, B. melitensis and N. aromati-civorans ZP_00095689 LOV-kinases show no canonical H-box,rather they are most similar to the HpK11 methanobacterialkinases.24 The two M. magnetotacticum LOV-kinases do not fallin any of the described group. In these six proteins the F-andG2-box appear to be missing or very reduced (Fig. 6). Thisregion forms a variable, mobile loop in the structure of diverseHPK, located near the ATP binding site (ATP lid) 20 and itsshortening could affect ATP binding or catalytic activity.

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2c. The R. baltica, Nostoc Q8YT51, N. punctiformeZP_00105980 and T. elongatus Q8DJE3 multidomain/hybridLOV-kinases, belong to class HPK1, the largest kinase sub-family. The KFT motif in the N-box is typical of the HPK1b

subgroup.38 The R. baltica LOV-kinase has a unique domainstructure, with a CheB methylesterase and CheR methyl-transferase domain in the N-terminus. In motile bacteria CheBand CheR control the level of methylation of glutamate resi-dues in methyl-accepting chemotaxis proteins.40 The functionof these two catalytic domains fused on the same protein hasnot been described. The three cyanobacterial kinases possessadditional GAF (cGMP phosphodiesterase, Adenylate cyclase,FhlA) domains, present also in phytochrome and cGMP-specific phosphodiesterases.41 The GAF domains do not showthe extra-insertion responsible for the binding of a tetrapyrrolechromophore in phytochromes.41 The kinases from Nostoc sp.and N. punctiforme contain a C-terminal Hpt (histidinephosphotransfer) domain, downstream of the two RR units.This feature has been previously characterised in the so-calledunorthodox, multistep His–Asp–His–Asp phosphorelaysystems where the HPt domain serves as a histidine-phos-phorylated intermediate during phosphoryl transfer betweentwo RR domains.42 Accordingly the genes for Q8YT51 andZP_00105980 are coupled with sequences encoding foradditional RRs. As a whole Q8YT51 and ZP_00105980 showstriking similarity, in the domain architecture, with E. coli ArcB(aerobic respiration control sensor protein), one of the bestcharacterised multi-step phosphorelay sensors.43

3. Transcriptional regulators

This family is formed by a single member, the transcriptionalregulator of N. aromaticivorans (ZP_00093141) that carries atypical helix turn helix (HTH) DNA-binding motif. HLH/PASproteins are well documented in plants and animals.44,45 Theytend to be ubiquitous, latent transcription factors whoseactivity is signal regulated, but the molecular mechanism bywhich they control gene activity is not well understood.45

4. Single LOV-domains

The small proteins in P. putida (Q88E39, Q88JB0), P. fluores-cens, R. sphaeroides and C. aurantiacus exhibit a single LOVdomain with variable N- and C-terminal caps. This featureis reminiscent of N. crassa VIVID 30 and of the photoactiveyellow protein (PYP), a small bacterial blue-light photo-receptor that binds 4-hydroxycinnamic acid as chromophore.46

In the P. putida genome, the sequences encoding for the twoLOV proteins are associated with putative DNA-binding

Fig. 6 Sequence alignment of conserved boxes in thephosphoacceptor/dimerization domain (H-box, the site ofautophosphorylation in bold) and in the ATP-binding catalytic domain(N-, D-, F- and G-boxes) of representative LOV-kinases. Shadowedresidues characterize the different boxes.38,78 For comparison, thecorresponding sequences of the E. coli Mg2� sensor PhoQ 78 andosmolarity sensor EnvZ, are reported.79 Ec: E. coli, Rb: R. baltica, Mm:M. magnetotacticum, Cc: C. crescentus, Xc: X. campestris. For proteinaccession numbers, see Table 1. Dots indicate segments not shown inthe alignment. The asterisks show residues that interact with a non–hydrolyzable ATP analogs and a Mg2� cation in the structure of thecatalytic PhoQ domain.78

transcriptional regulators, which could represent theirmolecular partners.

5. Putative nucleotide cyclases and phosphodiesterases

The last family encompasses five LOV bacterial proteins(in Nostoc sp. (Q8YSB9), N. punctiforme (ZP_00111211),Synechocystis sp., B. fungorum and R. solanacearum) containingthe GGDEF domain associated with EAL (GGDEF and EALare conserved aa sequences), a well documented tandemmotif.47 Although the function of most of the GGDEF proteinshas not been characterized, some of them show catalyticactivity dependent on a novel effector molecule, cyclic diguanyl-ate (c-di-GMP, bis(3�,5�)-cyclic diguanylinic acid).48,49 Twoenzymes involved in cellulose biosynthesis, DGC1 (diguanylatecyclase 1) and PDEA1 (phosphodiesterase 1), have both theGGDEF-EAL module.50 The GGDEF domain was indicatedas a probable cyclase, while EAL is a good candidate as diguan-ylate phosphodiesterase site.47 The presence of the two domainsassociated on the same protein, is therefore indicative of a c-di-GMP dependent phosphodiesterase (and/or cyclase) activity.Should this catalytic role be confirmed for the LOV-proteins ofNostoc, Ralstonia solanacearum, Synechocystis and Nostocpunctiforme (ZP_00111211), this finding would constitute anunprecedented mechanism in bacterial light-signal trans-duction chains. A blue-light driven adenylyl cyclase fromthe unicellular flagellate Euglena gracilis, has been recentlycharacterized.51

Light-driven activation and interdomaincommunicationDespite availability of detailed information on the structure,photochemistry and photophysics of isolated phot-LOVdomains, the molecular mechanisms of light-to-signal con-version are largely unknown, also because structural andfunctional studies on full-length phot are hampered by theirlow solubility.10 In LOV proteins light activation must changethe interaction of the chromophore bearing parts withmolecular partners, in order to trigger the subsequent physio-logical responses. Alternatively, light-induced conformationalchanges can be transmitted to partner domains via rearrange-ments of secondary structure elements (vide infra).

Evidence that light-driven conformational changes occur inoat phot1-LOV2 have been obtained by means of one-dimen-sional nuclear magnetic spectroscopy (NMR) spectroscopy 12

and circular dichroism.52 X-ray crystallography and FourierTransform Infrared (FTIR) spectroscopy of plant and algalphot-LOV domains, show that the conformational changes areminor and restricted to the vicinity of FMN.17,18,53,54 Photo-acoustic calorimetry experiments and mutational analysis withC. reinhardtii phot-LOV1, have shown that the volume contrac-tion accompanying the formation of LOV390 receives large con-tributions from the rearrangements of the HB network centeredon Arg58 (adjacent to the reactive Cys57) and involving theFMN phosphate group.36 It is possible that this protein regionacts as regulator for the LOV domain itself (e.g. by affectingstability of the photoproduct and kinetics of the recoveryreaction), rather than constituting the interaction site withpartner domains. In summary the data collected on phot-LOVdomains, indicate that the light-driven conformational changesare relatively small and mainly originate in the vicinity of thechromophore.

At variance with these results, FTIR experiments with aphy3-LOV2 extended construct (including ca. 20 residuesupstream and ca. 40 downstream of the LOV2 core), suggestthe existence of conformational substates of the photoadductthat can be cryotrapped.55 The relevance of these substates inthe room temperature photocycle remains to be clarified, aswell as the contribution of the extra residues in the construct.Recently, Harper et al. used NMR spectroscopy to characterize

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the light-dependent conformational changes of a A. sativaphot1-LOV2 extended construct, comprising 40 aa downstreamof the LOV core.56 Their results showed the existence of a ca. 20aa long amphipatic helix (Jα), C-terminal to the PAS core andconnected to βE by a flexible loop (10 aa). The Jα helix affectsthe LOV domain structure in the dark, but the constraint isremoved after light activation and the helix becomes dis-ordered. A low resolution structure, obtained upon combining3D-NOEs (Nuclear Overhauser Effects) spectra and a model ofthe LOV2 core, shows that in the dark the Jα helix lies on thesolvent-exposed face of the central β-sheet (formed by the βC,βD and βE strands).56 The authors suggest a general modelfor kinase activation (in phot), in which the extended LOV2domain is converted from a ‘closed’ (dark) to an ‘open’ (lit)state.56 It remains to be demonstrated if the interaction of theJα helix in the construct is the same as in the full-length proteinand what role the light-induced destabilization of the Jα helixplays in kinase activation. Secondary structure prediction ofbacterial LOV proteins, indicates the occurrence of similaramphipatic α-helices downstream of the LOV core, but withconnecting loops of different length, sometimes much shorterthan in oat phot1-LOV2 (e.g. 1–2 aa in YtvA, data notshown). The sequence of YtvA, both within the LOV core andimmediately downstream of it, shares large similarity with theB. japonicum FixL O2 sensor.57 In the crystal structure of FixLa rigid α-helix follows, without loops, the βE strand and pro-trudes away from the PAS core.58 Albeit the latter feature couldbe an artifact of crystal packing, it shows that the helix canactually assume different orientations. Recently the structureof the ligand-binding PAS-core domain of CitA, an integralmembrane kinase that regulates transport and metabolismof citrate in K. pneumoniae, was solved.59 CitA-PAS is flankedby two transmembrane helices (N- and C-terminal to the PAScore) and is spatially separated by the cytoplasmic effectorkinase domain, constituting the first example of PAS mediatedtransmembrane signalling. The authors suggest that kinaseactivation is mediated by rearrangements among the trans-membrane helices and the PAS β-sheet, induced by ligandbinding, a mechanism that does not require direct contactbetween the PAS core and the kinase domain.59 Interestinglythis model, as the one proposed by Harper et al. for phot,56

implies rearrangements among the PAS β-sheet and a down-stream α-helix as key factors for signal transduction.

Crosson and Moffat have proposed that LOV domains mightcommunicate with domain partners by means of a conservedsalt bridge between a glutamic acid and a lysine residue (seeFig. 1), located at the end of a structural motif that connectsthe FMN binding pocket to the protein surface.3 The salt bridgemay break or destabilize upon formation of LOV390. Recently,we have demonstrated that the LOV domain of YtvA interactswith the remaining part of the protein and that the interactionis mediated by electrostatic effects.26 According to the structuralmodelling of YtvA-LOV,5 in the vicinity of the conservedsalt-bridge (Fig. 7), two additional glutamic acid residue builda cluster of negative charges. The cluster could be importantin intradomain communication also given the high numberof lysine residues present on the STAS domain. Site specificmutagenesis studies as well as the determination of the YtvAcrystal structure will help clarify this point.

In summary three possible regulatory or protein-proteininteraction sites (not necessarily coinciding) have been identi-fied in LOV domains: the solvent exposed face of the βC, βDand βE strands,56 the conserved E–K salt bridge 3 and theprotein region binding the FMN phosphate group.36

An additional regulatory role could be played by loops con-necting secondary structure elements. In PYP and Aer, the co-factor binding and the supposed active site for signalling arecentered around the α�A–αC loop and the PAS core.60,61 In con-trast the αC–βC loop has been suggested to have a regulatoryrole in the PAS domain of FixL 58 and in the N-terminal PAS1

domain of a mammalian kinase.62 In the latter protein, anexceptionally flexible αC–βC loop serves also as interactionsite with the kinase domain. In summary, the literature dataon PAS-mediated signalling indicate that PAS domains haveconserved folding but different cofactors introduce significantvariations in the signalling/regulatory pathways. These vari-ations originate in the protein regions that are most variableand define cofactor specificity.

Concluding remarksDespite the fact that photoreactivity of bacterial phot-relatedproteins has been proven, we still have no knowledge abouttheir physiological relevance, although some speculations canbe made. Blue light is potentially harmful for all bacteria,because it is efficiently absorbed by porphyrins, ubiquitousphotosensitisers for singlet oxygen (see Ghetti et al., and refer-ences therein 63). All bacteria synthesize porphyrins as hemeor chlorophyll precursors, and it would not be surprising thatboth photosynthetic and non-photosynthetic microorganismsthat experience a changing environment during their life coursehave evolved blue-light sensors able to elicit a motile response.UVA/blue-light driven photoavoidance responses have beendescribed for some of the bacteria listed in Table 1 (seeArmitage and Hellingwerf for a review 64); however, they havenot yet been linked to a LOV-domain protein. Genetic studiesare needed in order to clarify this point.

The discovery of LOV proteins in eubacteria opens a newresearch area in the field of bacterial blue-light sensing andsignificance, likely to give in the near future substantial contri-bution in our understanding of how microorganisms ‘see’ theirworld. Their photoreactivity constitutes a new mechanism ofblue-light sensing in bacteria, besides the well known photo-active yellow protein 46 and the emerging AppA paradigm.2

The fact that they can utilize as signal partners catalytic func-tions located on the same sensing molecule, suggest possiblemechanisms of light-to-signal transduction. The light sensingLOV domain can be easily triggered by blue-light, undergoingsubstantial conformational changes which are communicatedto the output domains. The modularity of the bacterial LOVproteins can thus constitute a powerful tool (and a potentialmodel for protein engineering) in understanding the modalitiesof interdomain communication in sensor proteins.

Abbreviationsphot, phototropin; FMN, flavin mononucleotide; LOV, LightOxygen Voltage domain; aa, amino acid(s); PAS, PerArntSimdomain; FAD, flavin-adenin dinucleotide; STAS, Sulfate trans-porter/anti-sigma factor antagonist domain; NTP, NucleosideTriphosphate; HAMP, domain found in Histidine kinases,Adenylyl cyclases, Methyl binding proteins, Phosphatases; Hpt,Histidine phosphotransfer domain; GGDEF, EAL, domains

Fig. 7 3-D homology modeling of YtvA-LOV domain.5 Structuralelements known or predicted to be involved in interdomaincommunication or to play regulatory roles in LOV (bold) and otherPAS domains are indicated. The three arrows indicate the central β-sheet, composed of the three βC-, βD- and βE-strands.

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named after their conserved aa motifs; GAF, domain present inphytochrome and cGMP-specific phosphodiesterases; HPK,Histidine Protein Kinase; HB, hydrogen bond; HPK, histidineprotein kinase; RR, response regulators.

Acknowledgements

I thank Benjamin Quest for cloning the gene of the C. crescen-tus LOV-kinase and Helene Steffen for technical help. I amindebted to Wolfgang Gärtner for critical reading of the manu-script and to Silvia E. Braslavsky for the use of spectroscopyfacilities and her support.

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