Blue light sensing by LOV protein in Xanthomonas axonopodis pv. citri

KRAISELBURD I, MONZON J, LOSI A, ORELLANO EG

La Plata

Encuentro; Primer Encuentro de Fotobiólogos Moleculares Argentinos; 2011

In living organisms perception of light is carried out by photoreceptor proteins that belong to one of six families: rhodopsins, phytochromes, xantopsins, cryptochromes, LOV (Light, Oxygen and Voltage)-proteins and BLUF (Blue-Light sensing Using Flavin). The first LOV proteins discovered were phototropins (phot), plant photoreceptors involved in phototropic bending, light-induced stomatal opening and chloroplast relocation. Phot are membrane-associated hydrophilic proteins with two LOV domains in their N-terminal half and a serine/threonine kinase domain in their C-terminal half. LOV domains contains FMN (flavin mononucleotide) attached noncovalently in the dark-adapted state. Blue-light absorption initiates a photocycle involving the reversible formation of a covalent bond between the C4a carbon atom of FMN and a cysteine residue, thus forming the signaling state, often referred to as photoadduct. Photoadduct formation leads to an enhancement of the kinase function. The sequencing of bacterial genomes has shown that bacteria encode a large number of photosensory proteins, chiefly LOV-proteins, whose physiological role are just beginning to be elucitated. Xanthomonas axonopodis pv. citri (Xac) is a gamma proteobacterium responsible for citrus canker, a disease damaging fruits, leaves and stems. Several studies demonstrated that an appropriate light environment is required to establish efficient resistance responses in several plant-pathogen interactions. On the other hand, the participation of light in regulation of bacterial virulence, via LOV- or BLUF-proteins, was demonstrated recently for important pathogens, such as Brucella abortus, Acinetobacter baumanni and Listeria monocytogenes. In silico analysis of Xac genome sequence showed the presence of a gene called fixL, which encodes a LOV protein containing a N-terminal LOV domain associated to a C-terminal histidine kinase and a response regulator (hybrid HK-RR). In order to study the photochemistry of Xac-FixL, we obtained the recombinant protein by expression in Escherichia coli and we performed a spectroscopic analysis of the purified protein. Studies of absorption and fluorescence spectroscopy of Xac-FixL showed a canonical LOV photochemistry, with formation of the photoadduct and loss of flavin fluorescence. Photoadduct formation also leads to conformational changes on the protein which were reflected on shifts on the fluorescence spectra of a conserved tryptophan residue. The photoadduct showed a very slow rate reversion to the ground state in the dark. We also constructed a knockout mutant of Xac by replacing the fixL gene with an antibiotic resistance cassette and we analyzed several physiological features of the bacteria, particularly those related with pathogen’s ability to colonize its host plant. We found significant differences in physiological features like motility, biofilm formation, exopolysaccharide production and bacterial adhesion between both strains which indicate that the LOV protein has a role in the regulation of these processes. Altogether these results suggest a light-dependent regulation of Xac virulence during host colonization.