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

Abstract

Red and far-red light sensing bacteriophytochrome photoreceptor (BphP) and blue light sensing LOV domain 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 causal agent 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 (redabsorbing) and Pfr (far-red absorbing), by the isomerization of the bilin chromophore and generating structural changes that result inthe transduction of the light signal into biochemical signaling. Here, we designed and constructed three different site-directed mutations 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 photoreceptor in a Pr-like state, however, L193Q and L193N transformed XccBphP froma bathy-type phytochrome into a canonical one, exhibiting a Pr thermal ground state. The X-ray structures for all the mutants were obtained in the Pr conformation, identical to the one from the wild-type previously determined, showing no significant differences in 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 mutant complementation experiments show that locking XccBphP in a Pr-likestate (using D199A) or, converting it into a Pr-stabilized phytochrome (using L193N or L193Q) decreases bacterial exopolysaccharide production in dark condition when compared to null mutant. This is reverted when the complemented strains were grown under red light.