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

CONFORTE, VALERIA; ANTELO, GIULIANO; SIRIGU, SERENA; GOLDBAUM, FERNANDO A.; OTERO, LISANDRO H.; RINALDI, JIMENA; CHAVAS, LEONARDO; MALAMUD, FLORENCIA; TOUM, LAILA; KLINKE, SEBASTIÁN; VOJNOV, ADRIAN; BONOMI, HERNAN R.

La Plata

Congreso; V Reunión Grupo Argentino de Fotobiología; 2020

Grupo Argentino de Fotobiologia

Redand far-red light sensing bacteriophytochrome photoreceptor (BphP)and blue light sensing LOV domain proteins have been showed to playkey roles in bacterial physiology and in virulence factors (VFs)modulation. We have previously shown that Xanthomonas campestris pv.campestris (Xcc), the causal agent of black rot disease, has in itsgenome a sequence that encodes for a single bathy type BphP (XccBphP)that negatively regulates its virulence.These bilin-binding proteinshave the capacity to photoswitch between two states, Pr (redabsorbing) and Pfr (far-red absorbing), by the isomerization of thebilin chromophore and generating structural changes that result inthe transduction of the light signal into biochemical signaling.Here, we designed and constructed three different site-directedmutations that affect XccBphP photocycle favoring its Pr state:D199A, L193Q and L193N. The mutant recombinant proteins have beenproduced in vitro and assayed by UV-Vis spectroscopy, showing that Pris their preferred state. As expected, D199A locked the photoreceptorin a Pr-like state, however, L193Q and L193N transformed XccBphP froma bathy-type phytochrome into a canonical one, exhibiting a Prthermal ground state. The X-ray structures for all the mutants wereobtained in the Pr conformation, identical to the onefrom thewild-type previously determined, showing no significant differencesin the quaternary, tertiary or secondary structures. Finally, wetested the in vivo implications of manipulating the XccBphPphotocycle using xanthan production and stomata aperture as thebiological responses of its signaling output. The null mutantcomplementation experiments show that locking XccBphP in a Pr-likestate (using D199A) or, converting it into a Pr-stabilizedphytochrome (using L193N or L193Q) decreases bacterialexopolysaccharide production in dark condition when compared to nullmutant. This is reverted when the complemented strains were grownunder red light.p { margin-bottom: 0.25cm; line-height: 115% }