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, V; MALAMUD, F; MODENUTTI, C; CONFORTE, VP; YARYURA, PM; VOJNOV, AA; TORRES, PS; CHAZARRETA, CN; MARTÍ, M

Buenos Aires

Congreso; REUNIÓN CONJUNTA DE SOCIEDADES DE BIOCIENCIAS; 2017

Xanthomonas citri subsp. Citri (Xcc) is the causal agent of citruscanker disease in citrus plant. One of the virulence factor produces by the bacteria is a β-1, 2 cyclic glucans. In previous work of our laboratory, two Xcc mutants impaired in glucan biosynthesis were obtained one of them by marker exchange mutagenesis (ndvB) and the other (hrpM) was identified in an Xcc EZ-Tn5 <R6Kγori/KAN-2> Tnp transposon library. Also, from the library, a tn5 insertion in galU,a gene encoding an enzyme that is supposed to be involved in the formation of uridine diphosphate (UDP)-glucose from UTP and glucose-1-phosphate, was identified. Previous study showed that galU is required for biosynthesis of extracellular polysaccharides xanthan gum. Our aim is to study the synthesis of cyclic glucans as a possible target for the control of citrus canker. We show here, for the first time, that galU mutant is also affected in the cyclic β-1,2 glucan in vivo. Instead, in vitro, using total membrane preparation from Xcc and incubated with UDP-[14C]Glucose and in the presence of Cl2Mg, the galU mutant was able to synthesis β-1,2 glucan, demonstrating that GalU is a UTP-glucose-1-phosphate uridylyltransferase in which 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 both in vivo and in vitro, a mixture of membranes of both mutants restored the glucan synthesis if they were previously sonicated and preincubated in buffer Tris-HCl pH8 in the presence of Cl2Mg and then andUDP (14C)-Glc, suggesting an interaction between NdvB and HrpM could be essential in the process. An in silico model, by homology, shows a putative complex which includes HrpM as an integral inner membrane protein and NdvB as a peripheral inner membraneprotein facing the periplasmic space. These results present new insights in the biosynthesis mechanism of β-1, 2 cyclic glucan in Xcc.