Phisiologycal characterization of a glycosyltransferase-deficient mutant of Ralstonia solanacearum and analysis of its interaction with host and non-host plants

TONDO, M. LAURA; SENSOLINI, RAQUEL; ORELLANO, ELENA G.

Rosario

Congreso; IX Congreso Argentino de Microbiología General (SAMIGE); 2013

Adherence to eukaryotic cells is an important early step during the infection of hosts by many bacterial pathogens. One of the most widespread adherence structures among Gram-negative bacteria are type IV pili (Tfp), filamentous appendages primarily composed of the pilin protein, that extend from the bacterial cell surface. In addition to their role in adherence, Tfp also mediate a form of flagella-independent surface translocation called twitching motility, and can be important for autoaggregation, biofilm formation and pathogenesis. In diverse animal-pathogenic bacteria pilins are glycosylated by oligosaccharyltransferases (OTases), integral inner membrane proteins that transfer short preassembled oligosaccharides to selected residues of the pilin. This posttranslational modification is thought to contribute to Tfp adhesiveness and/or to serve a protective role allowing glycosylated strains to evade immune responses and colonize host tissues. Ralstonia solanacearum is a Gram-negative soil-borne bacterium that causes disease in more than 200 plant species around the world, including economically important crops such as tomato, potato, banana and peanut. Analysis of the genomic DNA sequence of R. solanacearum GMI1000 identified the pilA structural gene, coding for type IV pilin, and an open reading frame (RSc0559) next to the pilA gene, with high homology to OTases involved in pilin glycosylation. The presence of this ORF raises the possibility that the pilin of R. solanacearum is glycosylated by a mechanism similar to the described in animal pathogens. To address this question, in this work we have generated a R. solanacearum RSc0559 mutant strain by insertional mutagenesis of a local isolation and we have characterized it both physiologically and in the interaction with host and non host plants. We found that RSc0559 mutation does not affect bacterial viability; however the mutant strain exhibited significant differences in colony morphology when grown on solid BG medium. In addition, the OTase mutant was enable to develop swimming and twitching motilities and lacked the typical polar flagellum observed in wild-type cells. During the interaction with tomato (host) plants the mutant strain exhibited a drastic reduction of virulence, with no development of typical wilting disease symptoms at 35 days post-infection. On the other hand, the OTase mutant did not elicit a hypersensitive response when inoculated in tobacco (non-host) plants. Some of these phenotypes were previously reported for pilin mutants of R. solanacearum, which suggest that Tfp function might be altered in the RSc0559 mutant. Altogether our results indicate that the putative OTase RSc0559 would play an important role for bacterial physiology and pathogenesis in R. solanacearum.