The (6-4) photolyases use blue light to reverse UV-induced (6-4) photoproducts in DNA. This (6-4) photorepair was thought to be restricted to eukaryotes. Here we report a prokaryotic (6-4) photolyase, PhrB from Agrobacterium tumefaciens, and propose that (6-4) photolyases are broadly distributed in prokaryotes. The crystal structure of photolyase related protein B (PhrB) at 1.45 Å resolution suggests a DNA binding mode different from that of the eukaryotic counterparts. A His-His-X-X-Arg motif is located within the proposed DNA lesion contact site of PhrB. This motif is structurally conserved in eukaryotic (6-4) photolyases for which the second His is essential for the (6-4) photolyase function. The PhrB structure contains 6,7-dimethyl-8-ribityllumazine as an antenna chromophore and a [4Fe-4S] cluster bound to the catalytic domain. A significant part of the Fe-S fold strikingly resembles that of the large subunit of eukaryotic and archaeal primases, suggesting that the PhrB-like photolyases branched at the base of the evolution of the cryptochrome/photolyase family. Our study presents a unique prokaryotic (6-4) photolyase and proposes that the prokaryotic (6-4) photolyases are the ancestors of the cryptochrome/photolyase family.DNA repair | UV light | energy transfer

Phytochromes are biliprotein photoreceptors that are found in plants, bacteria, and fungi. Prototypical phytochromes have a Pr ground state that absorbs in the red spectral range and is converted by light into the Pfr form, which absorbs longer-wavelength, far-red light. Recently, some bacterial phytochromes have been described that undergo dark conversion of Pr to Pfr and thus have a Pfr ground state. We show here that such so-called bathy phytochromes are widely distributed among bacteria that belong to the order Rhizobiales. We measured in vivo spectral properties and the direction of dark conversion for species which have either one or two phytochrome genes. Agrobacterium tumefaciens C58 contains one bathy phytochrome and a second phytochrome which undergoes dark conversion of Pfr to Pr in vivo. The related species Agrobacterium vitis S4 contains also one bathy phytochrome and another phytochrome with novel spectral properties. Rhizobium leguminosarum 3841, Rhizobium etli CIAT652, and Azorhizobium caulinodans ORS571 contain a single phytochrome of the bathy type, whereas Xanthobacter autotrophicus Py2 contains a single phytochrome with dark conversion of Pfr to Pr. We propose that bathy phytochromes are adaptations to the light regime in the soil. Most bacterial phytochromes are light-regulated histidine kinases, some of which have a C-terminal response regulator subunit on the same protein. According to our phylogenetic studies, the group of phytochromes with this domain arrangement has evolved from a bathy phytochrome progenitor.

Photolyases and cryptochromes are evolutionarily related flavoproteins with distinct functions. While photolyases can repair UV-induced DNA lesions in a light-dependent manner, cryptochromes regulate growth, development and the circadian clock in plants and animals. Here we report about two photolyase-related proteins, named PhrA and PhrB, found in the phytopathogen Agrobacterium tumefaciens. PhrA belongs to the class III cyclobutane pyrimidine dimer (CPD) photolyases, the sister class of plant cryptochromes, while PhrB belongs to a new class represented in at least 350 bacterial organisms. Both proteins contain flavin adenine dinucleotide (FAD) as a primary catalytic cofactor, which is photoreduceable by blue light. Spectral analysis of PhrA confirmed the presence of 5,10-methenyltetrahydrofolate (MTHF) as antenna cofactor. PhrB comprises also an additional chromophore, absorbing in the short wavelength region but its spectrum is distinct from known antenna cofactors in other photolyases. Homology modeling suggests that PhrB contains an Fe-S cluster as cofactor which was confirmed by elemental analysis and EPR spectroscopy. According to protein sequence alignments the classical tryptophan photoreduction pathway is present in PhrA but absent in PhrB. Although PhrB is clearly distinguished from other photolyases including PhrA it is, like PhrA, required for in vivo photoreactivation. Moreover, PhrA can repair UV-induced DNA lesions in vitro. Thus, A. tumefaciens contains two photolyase homologs of which PhrB represents the first member of the cryptochrome/photolyase family (CPF) that contains an iron-sulfur cluster.

Response to changes in light conditions involves a variety of receptors that can modulate gene expression, enzyme activity and/or motility. For the study of light-regulated effects of Agrobacterium tumefaciens, we used a global analysis approach - proteomics - and compared the protein patterns of dark- and light-grown bacteria. These analyses revealed a significant reduction of FlaA and FlaB - proteins of the flagellum - when the cells were grown in light. The light effect was confirmed by SDS-PAGE with isolated flagella. Quantitative PCR experiments showed a 10-fold increase of the transcription level of flaA, flaB and flaC within 20 min after the transfer from light to darkness. Electron microscopy revealed that these molecular events result in a light-induced reduction of the number of flagella per cell. These changes have major physiological consequences regarding motility, which is considerably reduced with exposure to light. The inhibitory effect of light on the motility is not unique to A. tumefaciens and was also seen in other species of the Rhizobiaceae. Previous studies suggested that the flagella function is significant for bacteria-plant interactions and bacterial virulence. In our studies, light reduced the attachment of A. tumefaciens to tomato roots and the virulence of the bacteria in a cucumber infection assay.

Background: Photolyases use light energy to repair UV-damaged DNA. Results: The crystal structure of a "class III CPD" photolyase reveals a new binding pocket for an "5,10-methenyltetrahydrofolate" antenna chromophore. Conclusion: Related plant cryptochromes might use the same antenna chromophore binding pocket. Significance: The photolyase crystal structure allows a better understanding of the evolutionary transition of photolyases to plant cryptochromes.