IBR   13079
INSTITUTO DE BIOLOGIA MOLECULAR Y CELULAR DE ROSARIO
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Functional analysis and transcriptional regulation of the o- oligosaccharyltransferase of Ralstonia solanacearum
Autor/es:
VICINO, PAULA; ORELLANO, ELENA G.; TONDO, M. LAURA
Lugar:
Córdoba
Reunión:
Congreso; XI Congreso Argentino de Microbiología General (SAMIGE); 2015
Resumen:
Protein O-glycosylation has become clearly established as a common posttranslational modification in bacteria. Glycans are often used to decorate several proteins at the bacterial surface such as the structural components of flagella and pili. Glycoproteins play diverse roles including adhesion, motility, immune evasion and host colonization. One of the proposed mechanisms of protein glycosylation in bacteria involves an oligosaccharyltransferase (OTase) that transfer short preassembled oligosaccharides to selected residues in the acceptor proteins. Although some OTases are specific to the pilin protein, others are classified as general OTases that target structurally and functionally diverse groups of membrane-associated proteins. Ralstonia solanacearum is a Gram-negative soil-borne alpha-proteobacterium which is the causal agent of bacterial wilting, one of the most devastating plant diseases in the world. The strong impact of this pathogen results from its worldwide geographical distribution and its wide host range; it affects more than 200 plant species including economically important food crops. In previous studies we have identified the product of the RSc0559 gene of R.solanacearum GMI1000 as a functional OTase, capable of transferring oligosaccharides to the pilin protein in an in vivo system in Escherichia coli cells. In the present work, an OTase deficient mutant generated by a clean deletion of the RSc0559 gene from the parental R. solanacearum GMI1000 strain was physiologically characterized. The OTase mutant exhibited similar growth kinetics than the wild-type strain in liquid BG medium and a normal colony morphology when grown on triphenyltetrazolium-supplemented agar plates. We found that the OTase mutant do not exhibit twitching motility and has an impaired ability to form biofilms on glass surfaces compared to the wild typestrain. However, both strains exhibited comparable swimming motilities in soft agar plates. In order to assess the significance of the OTase for bacterial virulence, the interaction with host and non-host plants was also analyzed. During the interaction with tomato (host) plants the mutant strain exhibited a drastic reduction of virulence, with no development of typical wilting symptoms at 20 days post-infection. On the other hand, the OTase mutant was able to elicit the typical hypersensitive response when inoculated in tobacco and cotton (non-host) plants. Finally, we constructed transcriptional fusions between the OTase promoter and the lacZ gene, and we measured the beta-galactosidase activity in mutant strains for different transcriptional regulators. By this approach we were able to determine that the expression of the OTase gene is controlled by main regulators of pathogenicity determinants of R. solanacearum.