title: The Antitoxin Protein of a Toxin-Antitoxin System fromXylella fastidiosaIs Secreted via Outer Membrane Vesicles.
abstract: TheXylella fastidiosasubsppaucastrain 9a5c is a Gram-negative, xylem-limited bacterium that is able to form a biofilm and affects citrus crops in Brazil. Some genes are considered to be involved in biofilm formation, but the specific mechanisms involved in this process remain unknown. This limited understanding of how some bacteria form biofilms is a major barrier to our comprehension of the progression of diseases caused by biofilm-producing bacteria. Several investigations have shown that the toxin-antitoxin (TA) operon is related to biofilm formation. This operon is composed of a toxin with RNAse activity and its cognate antitoxin. Previous reports have indicated that the antitoxin is able to inhibit toxin activity and modulate the expression of the operon as well as other target genes involved in oxidative stress and mobility. In this study, we characterize a toxin-antitoxin system consisting of XfMqsR and XfYgiT, respectively, fromX. fastidiosasubsp.paucastrain 9a5c. These proteins display a high similarity to their homologs inX. fastidiosastrain Temecula and a predicted tridimensional structure that is similar to MqsR-YgiT fromEscherichia coli. The characterization was performed usingin vitroassays such as analytical ultracentrifugation (AUC), size exclusion chromatography, isothermal titration calorimetry, and Western blotting. Using a fluorometric assay to detect RNAses, we demonstrated that XfMqsR is thermostable and can degrade RNA. XfMqsR is inhibited by XfYgiT, which interacts with its own promoter. XfYgiT is known to be localized in the intracellular compartment; however, we provide strong evidence thatX. fastidiosasecretes wild-type XfYgiT into the extracellular environment via outer membrane vesicles, as confirmed by Western blotting and specific immunofluorescence labeling visualized by fluorescence microscopy. Taken together, our results characterize the TA system fromX. fastidiosastrain 9a5c, and we also discuss the possible influence of wild-type XfYgiT in the cell.
authors.affiliation: Centro de Biologia Molecular e Engenharia Genética, Instituto de Biologia, Universidade Estadual de Campinas Campinas, Brazil.
authors.forename: André da Silva
authors.initials: AD
authors.lastname: Santiago
authors.affiliation: Centro de Biologia Molecular e Engenharia Genética, Instituto de Biologia, Universidade Estadual de Campinas Campinas, Brazil.
authors.forename: Juliano S
authors.initials: JS
authors.lastname: Mendes
authors.affiliation: Centro de Biologia Molecular e Engenharia Genética, Instituto de Biologia, Universidade Estadual de Campinas Campinas, Brazil.
authors.forename: Clelton A
authors.initials: CA
authors.lastname: Dos Santos
authors.affiliation: Centro de Biologia Molecular e Engenharia Genética, Instituto de Biologia, Universidade Estadual de Campinas Campinas, Brazil.
authors.forename: Marcelo A S
authors.initials: MA
authors.lastname: de Toledo
authors.affiliation: Centro de Biologia Molecular e Engenharia Genética, Instituto de Biologia, Universidade Estadual de Campinas Campinas, Brazil.
authors.forename: Lilian L
authors.initials: LL
authors.lastname: Beloti
authors.affiliation: Centro de Biologia Molecular e Engenharia Genética, Instituto de Biologia, Universidade Estadual de Campinas Campinas, Brazil.
authors.forename: Aline
authors.initials: A
authors.lastname: Crucello
authors.affiliation: Centro de Biologia Molecular e Engenharia Genética, Instituto de Biologia, Universidade Estadual de Campinas Campinas, Brazil.
authors.forename: Maria A C
authors.initials: MA
authors.lastname: Horta
authors.affiliation: Centro de Biologia Molecular e Engenharia Genética, Instituto de Biologia, Universidade Estadual de Campinas Campinas, Brazil.
authors.forename: Marianna T de Pinho
authors.initials: MT
authors.lastname: Favaro
authors.affiliation: Departamento de Física Aplicada, Instituto de Física Gleb Wataghin, Universidade Estadual de Campinas Campinas, Brazil.
authors.forename: Duber M M
authors.initials: DM
authors.lastname: Munar
authors.affiliation: Instituto Agronômico de Campinas, IAC-APTA Citrus Cordeirópolis, Brazil.
authors.forename: Alessandra A
authors.initials: AA
authors.lastname: de Souza
authors.affiliation: Departamento de Física Aplicada, Instituto de Física Gleb Wataghin, Universidade Estadual de Campinas Campinas, Brazil.
authors.forename: Mônica A
authors.initials: MA
authors.lastname: Cotta
authors.affiliation: Centro de Biologia Molecular e Engenharia Genética, Instituto de Biologia, Universidade Estadual de CampinasCampinas, Brazil; Departamento de Biologia Vegetal, Instituto de Biologia, Universidade Estadual de CampinasCampinas, Brazil.
authors.forename: Anete P
authors.initials: AP
authors.lastname: de Souza
keywords: OMV; Xylella fastidiosa; biofilm; protein characterization; toxin-antitoxin system