A study on Pr-induced variants of XccBphP bacteriophytochrome photoreceptor from Xanthomonas campestris

CONFORTE, VALERIA P.; OTERO, LISANDRO HORACIO; TOUM, LAILA; ANTELO, GIULIANO TOMÁS; RINALDI, JIMENA; KLINKE, SEBASTIÁN; SIRIGU, SERENA; CHAVAS, LEONARD MICHEL GABRIEL; VOJNOV, ADRIAN A.; GOLDBAUM, FERNANDO ALBERTO; MALAMUD, FLORENCIA; BONOMI, HERNÁN RUY

Rosario

Congreso; V Reunión GRAFOB 2020; 2020

GRAFOV

Red and far-red light sensing bacteriophytochrome photoreceptor (BphP) and blue light sensing LOVdomain proteins have been showed to play key roles in bacterial physiology and in virulence factors (VFs)modulation. We have previously shown that Xanthomonas campestris pv. campestris (Xcc), the causalagent of black rot disease, has in its genome a sequence that encodes for a single bathy type BphP(XccBphP) that negatively regulates its virulence.These bilin-binding proteins have the capacity to photoswitch between two states, Pr (red absorbing) andPfr (far-red absorbing), by the isomerization of the bilin chromophore and generating structural changesthat result in the transduction of the light signal into biochemical signaling. Here, we designed andconstructed three different site-directed mutations that affect XccBphP photocycle favoring its Pr state:D199A, L193Q and L193N. The mutant recombinant proteins have been produced in vitro and assayed byUV-Vis spectroscopy, showing that Pr is their preferred state. As expected, D199A locked the photoreceptorin a Pr-like state, however, L193Q and L193N transformed XccBphP from a bathy-type phytochrome into acanonical one, exhibiting a Pr thermal ground state. The X-ray structures for all the mutants were obtainedin the Pr conformation, identical to the one from the wild-type previously determined, showing nosignificant differences in the quaternary, tertiary or secondary structures. Finally, we tested the in vivoimplications of manipulating the XccBphP photocycle using xanthan production and stomata aperture asthe biological responses of its signaling output. The null mutant complementation experiments show thatlocking XccBphP in a Pr-like state (using D199A) or, converting it into a Pr-stabilized phytochrome (usingL193N or L193Q) decreases bacterial exopolysaccharide production in dark condition when compared tonull mutant. This is reverted when the complemented strains were grown under red light.