STRUCTURAL AND FUNCTIONAL ANALYSES OF Pr-INDUCED VARIANTS OF XccBphP BACTERIOPHYTOCHROME FROM XANTHOMONAS CAMPESTRIS

VALERIA CONFORTE; OTERO LISANDRO; LAILA TOUM; SIRIGU S; ANTELO GT; RINALDI JIMENA; KLINKE S; CHAVAS L; VOJNOV A.A.; GOLDBAUM FA; MALAMUD F; BONOMI HR

VIRTUAL

Congreso; Reunion conjiunta SAIB SAMIGE; 2020

Photoreceptors are able to detect light and transduce that signal generating a cellular response. Among them are the red/far red light sensing bacteriophytochromes (BphP). The genome of Xanthomonas campestris pv. campestris (Xcc), the causative agent of black rot in crucifers, codesfor a functional BphP (XccBphP) which has been described in previous works (Bonomi et al., 2016; Otero et al., 2016). This is a cytoplasmic protein that displays four conserved domains: Period/Arndt/Single minded (PAS2 family), cGMP phosphodiesterase/ adenyl cyclase/FhlA (GAF),phytochrome-associated (PHY), and output/effector module (PAS9 family). The PAS2?GAF?PHY triad represents the photosensory module which binds the chromophore billiverdin-IXa (BV).Phytochromes exhibit two conformational states: Pr and Pfr, that absorb red and far-red light, respectively. A Pr ground state and a dark conversion from Pfr to Pr are characteristic of canonical phytochromes. Bathy-phytochromes present a Pfr ground state (Rottwinkel et al., 2010).XccBphP is a bathy-like BphP, with an incomplete Pfr conversion in the dark (~85%).We have previously reported that XccBphP exerts a regulatory effect in Xcc physiology affecting virulence mechanisms (i.e. xanthan exopolysaccharide production, extracellular hydrolytic enzymes secretion, biofilm formation) and impacting in the plant defense mechanisms (i.e. callosedeposition and stomata regulation) (Bonomi et al., 2016). We proposed that XccBphP functions as a negative regulator for virulence through transcriptional regulation.The purpose of this work was to investigate the Pr photostate of XccBphP. To this aim, we generated specific site-directed mutants coding variants with altered photocycles, either by locking or stabilizing the Pr conformation. Two conserved residues located in the photosensory domain,which are crucial for photochemical conversion, were selected, Leu193 and Asp199, generating three mutant variants: L193N, L193Q and D199A. We solved their structures by X-ray crystallography and explored the effects of altering the photocycle in bacterial physiology.