GLUCAN BIOSYNTHESIS IN Xanthomonas citri subsp. citri REQUIRES OF THE GLUCOSYLTRANSFERASES HrpM AND NdvB AS CONCERTED COMPLEX AND GalU, AN UTP-GLUCOSE-1-PHOSPHATE URIDYLTRANSFERASE

DE PINO, VERÓNICA; PABLO SEBASTIÁN TORRES; CONFORTE, VALERIA P.; FLORENCIA MALAMUD; CHAZARRETA, CRISTIAN N.; PABLO M. YARYURA; MODENUTTI CARLOS; MARTÍ, MARCELO; VOJNOV, ADRIAN ALBERTO

Buenos Aires

Congreso; SAIB; 2017

Xanthomonas citri subsp. Citri (Xcc) is the causal agent of citruscanker disease in citrus plant. One of the virulence factor producesby the bacteria is a β-1, 2 cyclic glucans. In previous work of ourlaboratory, two Xcc mutants impaired in glucan biosynthesis wereobtained one of them by marker exchange mutagenesis (ndvB) andthe other (hrpM) was identified in an Xcc EZ-Tn5 Tnp transposon library. Also, from the library, a tn5 insertion in galU,a gene encoding an enzyme that is supposed to be involved in theformation of uridine diphosphate (UDP)-glucose from UTP and glucose-1-phosphate, was identified. Previous study showed that galUis required for biosynthesis of extracellular polysaccharides xanthangum. Our aim is to study the synthesis of cyclic glucans as a possibletarget for the control of citrus canker. We show here, for the firsttime, that galU mutant is also affected in the cyclic β-1,2 glucan invivo. Instead, in vitro, using total membrane preparation from Xccand incubated with UDP-[14C]Glucose and in the presence of Cl2Mg,the galU mutant was able to synthesis β-1,2 glucan, demonstratingthat GalU is a UTP-glucose-1-phosphate uridylyltransferase inwhich product is required for β-1,2 glucan synthesis as a precursor.On the other hand, while the membrane preparations from the ndvBand hrpM Xcc mutants were unable of β-1,2 glucan production bothin vivo and in vitro, a mixture of membranes of both mutants restoredthe glucan synthesis if they were previously sonicated and preincubatedin buffer Tris-HCl pH8 in the presence of Cl2Mg and then andUDP (14C)-Glc, suggesting an interaction between NdvB and HrpMcould be essential in the process. An in silico model, by homology,shows a putative complex which includes HrpM as an integral innermembrane protein and NdvB as a peripheral inner membraneprotein facing the periplasmic space. These results present new insightsin the biosynthesis mechanism of β-1, 2 cyclic glucan in Xcc.