Homologous expression of a bacterial phytochrome. The cyanobacterium Fremyella diplosiphon incorporates biliverdin as a genuine, functional chromophore.

Bacteriophytochromes constitute a light-sensing subgroup of sensory kinases with a chromophore-binding motif in the N-terminal half and a C-terminally located histidine kinase activity. The cyanobacterium Fremyella diplosiphon (also designated Calothrix sp.) expresses two sequentially very similar bacteriophytochromes, cyanobacterial phytochrome A (CphA) and cyanobacterial phytochrome B (CphB). Cyanobacterial phytochrome A has the canonical cysteine residue, by which covalent chromophore attachment is accomplished in the same manner as in plant phytochromes; however, its paralog cyanobacterial phytochrome B carries a leucine residue at that position. On the basis of in vitro experiments that showed, for both cyanobacterial phytochrome A and cyanobacterial phytochrome B, light-induced autophosphorylation and phosphate transfer to their cognate response regulator proteins RcpA and RcpB [Hübschmann T, Jorissen HJMM, Börner T, Gärtner W & deMarsac NT (2001) Eur J Biochem268, 3383-3389], we aimed at the identification of a chromophore that is incorporated in vivo into cyanobacterial phytochrome B within the cyanobacterial cell. The approach was based on the introduction of a copy of cphB into the cyanobacterium via triparental conjugation. The His-tagged purified, recombinant protein (CphBcy) showed photoreversible absorption bands similar to those of plant and bacterial phytochromes, but with remarkably red-shifted maxima [lambda(max) 700 and 748 nm, red-absorbing (P(r)) and far red-absorbing (P(fr)) forms of phytochrome, respectively]. A comparison of the absorption maxima with those of the heterologously generated apoprotein, assembled with phycocyanobilin (lambda(max) 686 and 734 nm) or with biliverdin IXalpha (lambda(max) 700 and 750 +/- 2 nm), shows biliverdin IXalpha to be a genuine chromophore. The kinase activity of CphBcy and phosphotransfer to its cognate response regulator was found to be strictly P(r)-dependent. As an N-terminally located cysteine was found as an alternative covalent binding site for several bacteriophytochrome photoreceptors that bind biliverdin and lack the canonical cysteine residue (e.g. Agrobacterium tumefaciens and Deinococcus radiodurans), this corresponding residue in heterologously expressed cyanobacterial phytochrome B was mutated into a serine (C24S); however, there was no change in its spectral properties. On the other hand, the mutation of His267, which is located directly after the canonical cysteine, into alanine (H267A), caused complete loss of the capability of cyanobacterial phytochrome B to form a chromoprotein.

A probable link between the DedA protein and resistance to selenite.

To understand the molecular events involved in the reduction of selenite to non-toxic elemental selenium, 4000 clones of Ralstonia metallidurans CH34 were produced by random Tn5 transposon integration mutagenesis. Eight mutants were able to resist up to 15 mM selenite while the MIC for the wild-type strain was estimated as 4-6 mM selenite. The identification of the disrupted genes was carried out by Southern blot analysis and inverse PCR. The three resistant mutants containing only one insertion were further characterized. Tn5 disrupted a gene that encoded a protein which was closely related to proteins of the DedA family. This family represents a group of integral membrane proteins with completely unknown functions. Phenotypic characterization of the dedA mutants and selenite consumption experiments strongly suggest that DedA is involved in the uptake of selenite.

Chromophore selectivity in bacterial phytochromes: dissecting the process of chromophore attachment.

Bacterial phytochromes (Bphs) are ancestors of the well characterized plant photoreceptors. Whereas plant phytochromes perform their photoisomerization exclusively via a covalently bound bilin chromophore, Bphs are variable in their chromophore selection. This is demonstrated in the cyanobacterium Calothrix PCC7601 that expresses two Bphs, CphA and CphB. CphA binds phycocyanobilin (PCB) covalently, whereas CphB, lacking the covalently binding cysteine of the plant phytochromes, carries biliverdin IXalpha (BV) as the chromophore. Our experiments elucidate the different modes of chromophore-protein interaction in CphA and CphB and offer a rationale for their chromophore selectivity. The tight binding of BV by CphB prevents PCB from competing for the binding cavity. Even when the chromophore-binding cysteine has been inserted (CphB-mutant L266C), PCB replaces BV very slowly, indicating the tight, but not irreversible binding of BV. The mutant CphB L266C showed a redox-sensitivity with respect to its PCB binding mode: under reducing conditions, the chromoprotein assembly leads to spectra indicative for a covalent binding, whereas absence of dithiothreitol or its removal prior to assembly causes spectra indicative for noncovalent binding. Regarding the CphB-type Bphs lacking the covalently binding cysteine, our results support the involvement of the succeeding histidine residue in chromophore fixation via a Schiff base-like bond between the bilin A-ring carbonyl and the histidine imidazole group. The assembly process and the stability of the holo-proteins were strongly influenced by the concentration of added imidazole (mimicking the histidine side-chain), making the attachment of the chromophore via the histidine more likely than via another cysteine of the protein.

Listening to the blue: the time-resolved thermodynamics of the bacterial blue-light receptor YtvA and its isolated LOV domain.

YtvA is a bacterial flavo-protein related to plant phototropin. The photochemistry of YtvA and of its isolated LOV domain (YtvA-LOV) has been characterized by optical, mass spectrometric and photocalorimetric methods. The energy content (E390) of the FMN-C4a-thiol photoadduct (YtvA390 and YtvA-LOV390) and its structural volume change (deltaV390), with respect to the parent state, have been determined by means of Laser Induced Optoacoustic Spectroscopy (LIOAS). The high value of E390, 136 and 115 kJ mol(-1), respectively, ensures a large driving force for the dark recovery to the unphotolyzed state and points to a strained conformation of the protein or/and the chromophore in the photoadduct. The value of deltaV390 is significantly different for the two proteins, deltaV390 = -12.5 ml mol(-1) in YtvA and -17.2 ml mol(-1) in YtvA-LOV. The kinetics of the dark recovery reaction for YtvA-LOV is slower than for full-length YtvA, with taurec = 3900 and 2600 s at 25 degrees C, respectively, and shows a different temperature dependence. A similarly slow kinetics can be induced in YtvA by high ionic strength. Minor differences are observed in the fluorescence and photoadduct formation quantum yield. The overall stability is higher for YtvA than for YtvA-LOV. The data as a whole are indicative of an interaction between the two domains of YtvA, most probably mediated by electrostatic interactions that renders the full-length protein a compact and more rigid unit. The results reported here support the idea that the formation of the photoadduct changes the dynamics of the protein, depending on the conformational flexibility of the parent state. Flashing of the photoadduct induces a negligible deltaV, with 96% of the excitation energy dissipated as heat in <20 ns, indicating that the photoadduct does not undergo a photocycle on the LIOAS time scale, or that the photoinduced reactions occur with very low yield.

Two independent, light-sensing two-component systems in a filamentous cyanobacterium.

Two ORFs, cphA and cphB, encoding proteins CphA and CphB with strong similarities to plant phytochromes and to the cyanobacterial phytochrome Cph1 of Synechocystis sp. PCC 6803 have been identified in the filamentous cyanobacterium Calothrix sp. PCC7601. While CphA carries a cysteine within a highly conserved amino-acid sequence motif, to which the chromophore phytochromobilin is covalently bound in plant phytochromes, in CphB this position is changed into a leucine. Both ORFs are followed by rcpA and rcpB genes encoding response regulator proteins similar to those known from the bacterial two-component signal transduction. In Calothrix, all four genes are expressed under white light irradiation conditions, albeit in low amounts. For heterologous expression and convenient purification, the cloned genes were furnished with His-tag encoding sequences at their 3' end and expressed in Escherichia coli. The two recombinant apoproteins CphA and CphB bound the chromophore phycocyanobilin (PCB) in a covalent and a noncovalent manner, respectively, and underwent photochromic absorption changes reminiscent of the P(r) and P(fr) forms (red and far-red absorbing forms, respectively) of the plant phytochromes and Cph1. A red shift in the absorption maxima of the CphB/PCB complex (lambda(max) = 685 and 735 nm for P(r) and P(fr), respectively) is indicative for a noncovalent incorporation of the chromophore (lambda(max) of P(r), P(fr) of CphA: 663, 700 nm). A CphB mutant generated at the chromophore-binding position (Leu246-->Cys) bound the chromophore covalently and showed absorption spectra very similar to its paralog CphA, indicating the noncovalent binding to be the only cause for the unexpected absorption properties of CphB. The kinetics of the light-induced P(fr) formation of the CphA-PCB chromoprotein, though similar to that of its ortholog from Synechocystis, showed differences in the kinetics of the P(fr) formation. The kinetics were not influenced by ATP (probing for autophosphorylation) or by the response regulator. In contrast, the light-induced kinetics of the CphB-PCB complex was markedly different, clearly due to the noncovalently bound chromophore.

Phytochromes with noncovalently bound chromophores: the ability of apophytochromes to direct tetrapyrrole photoisomerization.

Chromophore-apoprotein interactions were studied with recombinant apoproteins, oat phytochrome (phyA) and CphB of the cyanobacterium Calothrix PCC7601, which were both incubated with the bilin compounds biliverdin (BV) IXalpha, phycocyanobilin (PCB) and the 3'-methoxy derivative of PCB. Previously it was shown that CphB and its homolog in Calothrix, CphA, show strong sequence similarities with each other and with the phytochromes of higher and lower plants, despite the fact that CphB carries a leucine instead of a cysteine at the chromophore attachment position and thus holds the chromophore only noncovalently. CphA binds tetrapyrrole chromophores in a covalent, phytochrome-like manner. For both eyanobacterial phytochromes, red and far-red light-induced photochemistry has been reported. Thus, the role of the binding site of CphB in directing the photochemistry of noncovalently bound tetrapyrroles was analyzed in comparison with the apoprotein from phyA phytochrome. Both the aforementioned compounds, which were used as chromophores, are not able to form covalent bonds with a phytochrome-type apoprotein because of their chemical structure (vinyl group at position 3 or methoxy group at position 3'). The BV adducts of both apoproteins showed phytochrome-like photochemistry (formation of red and far-red-absorbing forms of phytochrome [P(r) and P(fr) forms]). However, incubation of the oat apophytochrome with BV primarily yields a 700 nm form from which the P(r)-P(fr) photochemistry can be initiated and to which the system relaxes in the dark after illumination. The results for CphB were compared with a CphB mutant where the chromophore-binding cysteine had been introduced, which, upon incubation with PCB, shows spectral properties nearly identical with its (covalently binding) CphA homolog. A comparison of the spectral properties (P(r) and P(fr) forms) of all the PCB- and BV-containing chromoproteins reveals that the binding site of the cyanobacterial apoprotein is better suited than the plant (oat) phytochrome to noncovalently incorporate the chromophore and to regulate its photochemistry. Our findings support the proposal that the recently identified phytochrome-like prokaryotic photoreceptors, which do not contain a covalently bound chromophore, may trigger a light-induced physiological response.

First evidence for phototropin-related blue-light receptors in prokaryotes.

A prokaryotic protein, YtvA from Bacillus subtilis, was found to possess a light, oxygen, voltage (LOV) domain sharing high homology with the photoactive, flavin mononucleotide (FMN)-binding LOV domains of phototropins (phot), blue-light photoreceptors for phototropism in higher plants. Computer-based three-dimensional modeling suggests that YtvA-LOV binds FMN in a similar pocket as phot-LOVs. Recombinant YtvA indeed exhibits the same spectroscopical features and blue-light-induced photochemistry as phot-LOVs, with the reversible formation of a blue-shifted photoproduct, assigned to an FMN-cysteine thiol adduct (Thio383). By means of laser-flash photolysis and time-resolved optoacoustic experiments, we measured the quantum yield of formation for Thio383, Phi(Thio) = 0.49, and the enthalpy change, DeltaH(Thio) = 135 kJ/mol, with respect to the parent state. The formation of Thio383 is accompanied by a considerable volume contraction, DeltaV(Thio) = -13.5 ml/mol. Similar to phot-LOVs, Thio383 is formed from the decay of a red-shifted transient species, T650, within 2 micros. In both YtvA and free FMN, this transient has an enthalpy content of approximately 200 kJ/mol, and its formation is accompanied by a small contraction, DeltaV(T) approximately -1.5 ml/mol, supporting the assignment of T650 to the FMN triplet state, as suggested by spectroscopical evidences. These are the first studies indicating that phototropin-related, blue-light receptors may exist also in prokaryotes, besides constituting a steadily growing family in plants.