Glucan biosynthesis in Xanthomonas citri subsp.citri requires of glycosyltransferases Hrpm and Ndvb as concerted complex and GalU, an UTP-glucose-1-phosphate uridyltransferase

VERONICA DE PINO; TORRES PABLO SEBASTIAN; MODENUTTI CARLOS; MARTI MARCELO; CONFORTE VALERIA; YARYURA PABLO; CHAZARRETA CRISTIAN; MALAMUD FLORENCIA; VOJNOV ADRIAN

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencia; 2017

Citrus canker is one of the most frequent citrus disease, which causes necrotic lesions in leaves, stems and fruits, its etiological agent is Xanthomonas citri subsp. Citri (Xanthomonas citri, Xanthomonas campestris pv. Citri, and Xanthomonas axonopodis pv. Citri), who enters the plant both through wound sites and stomata, persisting in the intercellular space of the spongy mesophyll. This bacterium evolved complicated mechanisms to circumvent the defense of the plant and acquire nutrients. One of this mechanism is the production of cyclic glucans, vital for virulence and interaction with the host. Glucans are synthesized into the inner membrane of the cell and then transported or secreted into the periplasm. The objective of this work is to study the synthesis of cyclic glucans as a possible target for the control of citrus canker, to do this we obtained a mutant strain for the galU gene that produces neither cyclic glucans nor xanthan. In this regard, GalU enzyme (EC 2.7.7.9) catalyzes the formation of uridine diphosphate (UDP) -glucose from UTP and glucose-1-phosphate. GalU strain lacks the characteristic mucosal phenotype of Xac, and does synthesize neither xanthan nor glucan, but the mutant strain complemented in trans recues the mutant phenotype. Glucan synthesis is restored at the wild-type level, using a system of total membranes incubated with UDP (14C) Glc in the presence of Cl2Mg. This fact is not occurring in other cyclic glucan mutant strains, such as HrpM and NdvB, suggesting not only that GalU is involved in the first step of glucan and xanthan biosynthesis, but also that HrpM and NdvB proceed downstream GalU. In the same line of research, it was found that the mixture of membranes of HrpM and NdvB, restored the cycic glucan synthesis if they were previously sonicated and preincubated in buffer Tris-HCl pH8 in the presence of Cl2Mg, and then incubated with UDP (14C)-Glc. This result would favor the hypothesis of an enzymatic complex responsible for the cyclic glucan synthesis. In this regard, the possible interaction between the two proteins involved in the biosynthesis downstream GalU, was modeled by homology, obtaining a putative complex which includes an integral inner membrane protein, hrpM and a peripheral inner membrane protein facing the periplasmic space, NdvB. This synthetic complex would be a good target to control the disease.