Functional Analysis On Light-signaling Proteins Of Xanthomonas Campestris Pv.Campestris

Light is an energy source and an ever-present stimulus in environment, whichhas been revealed on most regions of wavelength in some researches on bacteria. Theseinfluences include light inhibition, phototaxis and photosynthesis, even virulence ofbacteria, etc. Xanthomonas campestris pathovar campestris （Xcc） is a cruciferousplant-infecting pathogen, which is the causal agent of black rot. PAS （Per–Arnt–Sim）domains are important signaling modules that monitor changes in light, redox potential,oxygen, small ligands, and overall energy level of a cell. Prokaryotic genome analysiswith bioinformatics methods revealed the presence of PAS proteins in¹5%of allsequenced genomes. PAS proteins participated in switching of lights as putativephotoreceptors according to more and more clues. Thirty-three PAS domain-containingproteins （thereafter PAS proteins） were found in Xcc genome and proteome with asystematic analysis via bioinformatics methods. These PAS proteins include8histidinekinases （HK）,10response regulators or hybrids （HK/RR hybrid）,8GGDEF familyproteins,3transcription regulators,2chemotaxis protein,1phytochrome-like proteinand1methyltransferase.The revealed PAS domain structures indicated that its general secondary structurewas very conservative, and cofactors frequently interact with helixes. Therefore,secondary structure topology （SST） of PAS domains were firstly predicted with threetools, and merged manually. Afterwards, a comparison alignment on SST of PASdomains was built, meanwhile, other16PAS domains that have been proven to functionas light, oxygen or voltage sensors, were added in the analysis. Three categories （light,oxygen and voltage） of known PAS domains were well clustered on the basis of SSTs,therefore, the six clusters of the full tree were compared with the results of the latermutants tests. The33mutants of PAS genes of Xcc, which include22in-frame deletionsand11insertional mutations, were detected in growth, motility and virulence assays, etc.Thirteen PAS proteins were detected to involve in light sensing or signaling in Xcc,which included4HK,5hybrid and4GGDEF family proteins. Four PAS proteins wereinvolved in virulence of Xcc with interacting with light signals. These results weremuch consistent with clustering analysis. The protein of XC₃829contains four predicted domains of two PAS, a PAC and aGGDEF, and the two PAS domains involved individually in deferent clusters. Fordetecting functions of PAS domain, the full length and three fragments of the proteinwere expressed as C-terminal His6-tagged fusion, and purified using nickel-affinitychromatography. The purified products were assayed with GTP in enzymatic reactions.The fragment of only GGDEF domain showed very low DGC activity. The DGCactivity of full protein was FMN dependent and was triggered in weak blue lightcondition. The PAS1domain interacted with FMN, and sensed blue light; the PAS2domain can sense light to inhibit the DGC activity. The interaction of the both PASdomains of XC₃829finely manipulates the DGC intensity of XC₃829.