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

VERÓNICA DE PINO; PABLO TORRES; VALERIA CONFORTE; FLORENCIA MALAMUD; CRISTIAN CHAZARRETA; PABLO YARYURA; CARLOS MODENUTTI; MARCELO MARTÍ; ADRIÁN VOJNOV

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencia; 2017

SAIB

Xanthomonas citri subsp. Citri (Xcc) is the causal agent of citrus canker 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 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 Cl2 Mg, 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 ndvB and 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 Cl2 Mg and then and UDP (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 membrane protein facing the periplasmic space. These results present new insights in the biosynthesis mechanism of β-1, 2 cyclic glucan in Xcc