Photosynthetic bacteria regulate photosystem synthesis in response to alterations in oxygen tension and light intensity. In this study we show that the PpsR repressor from Rhodobacter sphaeroides binds to DNA in a redox-dependent manner through the formation/breakage of an intramolecular disulfide bond. We also demonstrate that PpsR is antagonized by the flavin-containing antirepressor, AppA, that is capable of breaking the disulfide bond in oxidized PpsR as well as forming a stable AppA-PpsR(2) antirepressor-repressor complex. Blue light excitation of AppA induces a photocycle that is characterized by a long-lived red-shifted absorbance of the flavin. Light-excited AppA was found to be incapable of forming the AppA-PpsR(2) antirepressor complex. These results establish AppA as a transcription factor that controls both redox and blue light repression of photosystem gene expression by mediating DNA binding activity of PpsR.

The flavin-binding BLUF domain of AppA represents a new class of blue light photoreceptors that are present in a number of bacterial and algal species. The dark state X-ray structure of this domain was determined at 2.3 A resolution. The domain demonstrates a new function for the common ferredoxin-like fold; two long alpha-helices flank the flavin, which is bound with its isoalloxazine ring perpendicular to a five-stranded beta-sheet. The hydrogen bond network and the overall protein topology of the BLUF domain (but not its sequence) bear some resemblance to LOV domains, a subset of PAS domains widely involved in signaling. Nearly all residues conserved in BLUF domains surround the flavin chromophore, many of which are involved in an intricate hydrogen bond network. Photoactivation may induce a rearrangement in this network via reorientation of the Gln63 side chain to form a new hydrogen bond to the flavin O4 position. This shift would also break a hydrogen bond to the Trp104 side chain, which may be critical in induction of global structural change in AppA.

Crystal structures of the Synechocystis BLUF phototaxis photoreceptor Slr1694 have been determined in two crystal forms, a monoclinic form at 1.8 A resolution and an orthorhombic form at 2.1 A resolution. In both forms, the photoreceptor is comprised of two pentamer rings stacked face to face. Twenty total subunits in the two asymmetric units of these crystal forms display three distinct tertiary structures that differ in the length of the fifth beta-strand and in the orientation of Trp91, a conserved Trp residue near the FMN chromophore. Fluorescence spectroscopic analysis on Slr1694 in solution is consistent with motion of Trp91 from a hydrophobic environment in the dark state to a more hydrophilic environment in the light-excited state. Mutational analysis indicates that movement of Trp91 is dependent on the occupancy of the hydrophobic Trp binding pocket with a nearby Met. These different tertiary structures may be associated with absorption changes in the blue region of the spectrum.

The origin and evolution of photosynthesis have long remained enigmatic due to a lack of sequence information of photosynthesis genes across the entire photosynthetic domain. To probe early evolutionary history of photosynthesis, we obtained new sequence information of a number of photosynthesis genes from the green sulfur bacterium Chlorobium tepidum and the green nonsulfur bacterium Chloroflexus aurantiacus. A total of 31 open reading frames that encode enzymes involved in bacteriochlorophyll/porphyrin biosynthesis, carotenoid biosynthesis, and photosynthetic electron transfer were identified in about 100 kilobase pairs of genomic sequence. Phylogenetic analyses of multiple magnesium-tetrapyrrole biosynthesis genes using a combination of distance, maximum parsimony, and maximum likelihood methods indicate that heliobacteria are closest to the last common ancestor of all oxygenic photosynthetic lineages and that green sulfur bacteria and green nonsulfur bacteria are each other's closest relatives. Parsimony and distance analyses further identify purple bacteria as the earliest emerging photosynthetic lineage. These results challenge previous conclusions based on 16S ribosomal RNA and Hsp60/Hsp70 analyses that green nonsulfur bacteria or heliobacteria are the earliest phototrophs. The overall consensus of our phylogenetic analysis, that bacteriochlorophyll biosynthesis evolved before chlorophyll biosynthesis, also argues against the long-held Granick hypothesis.

The flavoprotein AppA is a blue-light photoreceptor that functions as an antirepressor of photosynthesis gene expression in the purple bacterium Rhodobacter sphaeroides. Heterologous expression studies show that FAD binds to a 156 amino acid N-terminal domain of AppA and that this domain is itself photoactive. A pulse of white light causes FAD absorption to be red shifted in a biphasic process with a fast phase occurring in <1 micros and a slow phase occurring at approximately 5 ms. The absorbance shift was spontaneously restored over a 30 min period, also in a biphasic process as assayed by fluorescence quenching and electronic absorption analyses. Site-directed replacement of Tyr21 with Leu or Phe abolished the photochemical reaction implicating involvement of Tyr21 in the photocycle. Nuclear magnetic resonance analysis of wild-type and mutant proteins also indicates that Tyr21 forms pi-pi stacking interactions with the isoalloxazine ring of FAD. We propose that photochemical excitation of the flavin results in strengthening of a hydrogen bond between the flavin and Tyr 21 leading to a stable local conformational change in AppA.

SUMMARY The Reg regulon from Rhodobacter capsulatus and Rhodobacter sphaeroides encodes proteins involved in numerous energy-generating and energy-utilizing processes such as photosynthesis, carbon fixation, nitrogen fixation, hydrogen utilization, aerobic and anaerobic respiration, denitrification, electron transport, and aerotaxis. The redox signal that is detected by the membrane-bound sensor kinase, RegB, appears to originate from the aerobic respiratory chain, given that mutations in cytochrome c oxidase result in constitutive RegB autophosphorylation. Regulation of RegB autophosphorylation also involves a redox-active cysteine that is present in the cytosolic region of RegB. Both phosphorylated and unphosphorylated forms of the cognate response regulator RegA are capable of activating or repressing a variety of genes in the regulon. Highly conserved homologues of RegB and RegA have been found in a wide number of photosynthetic and nonphotosynthetic bacteria, with evidence suggesting that RegB/RegA plays a fundamental role in the transcription of redox-regulated genes in many bacterial species.

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A phytochrome-like protein called Ppr was discovered in the purple photosynthetic bacterium Rhodospirillum centenum. Ppr has a photoactive yellow protein (PYP) amino-terminal domain, a central domain with similarity to phytochrome, and a carboxyl-terminal histidine kinase domain. Reconstitution experiments demonstrate that Ppr covalently attaches the blue light-absorbing chromophore p-hydroxycinnamic acid and that it has a photocycle that is spectrally similar to, but kinetically slower than, that of PYP. Ppr also regulates chalcone synthase gene expression in response to blue light with autophosphorylation inhibited in vitro by blue light. Phylogenetic analysis demonstrates that R. centenum Ppr may be ancestral to cyanobacterial and plant phytochromes.