The antitoxin protein of a toxin-antitoxin system from Xylella fastidiosa is secreted via outer membrane vesicles.
The Xylella fastidiosa subsp pauca strain 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 limit...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2016-12-01
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| Series: | Frontiers in Microbiology |
| Subjects: | |
| Online Access: | http://journal.frontiersin.org/Journal/10.3389/fmicb.2016.02030/full |
| _version_ | 1819073357030621184 |
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| author | André Santiago Juliano Sales Mendes Clelton A Santos Marcelo AS Toledo Lilian L Beloti Aline Crucello Maria AC Horta Marianna Favaro Duber Marcel Murillo Munar Alessandra A Souza Mônica Alonso Cotta Mônica Alonso Cotta Anete Pereira De Souza |
| author_facet | André Santiago Juliano Sales Mendes Clelton A Santos Marcelo AS Toledo Lilian L Beloti Aline Crucello Maria AC Horta Marianna Favaro Duber Marcel Murillo Munar Alessandra A Souza Mônica Alonso Cotta Mônica Alonso Cotta Anete Pereira De Souza |
| author_sort | André Santiago |
| collection | DOAJ |
| description | The Xylella fastidiosa subsp pauca strain 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, from X. fastidiosa subsp pauca strain 9a5c. These proteins display a high similarity to their homologues in X. fastidiosa strain Temecula and a predicted tridimensional structure that is similar to MqsR-YgiT from Escherichia coli. The characterization was performed using in vitro assays 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 that X. fastidiosa secretes 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 from X. fastidiosa strain 9a5c, and we also discuss the possible influence of wild-type XfYgiT in the cell. |
| first_indexed | 2024-12-21T17:52:20Z |
| format | Article |
| id | doaj.art-c5b50757ae0d4547a7171c1bfa43d2a1 |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-12-21T17:52:20Z |
| publishDate | 2016-12-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-c5b50757ae0d4547a7171c1bfa43d2a12022-12-21T18:55:19ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2016-12-01710.3389/fmicb.2016.02030203225The antitoxin protein of a toxin-antitoxin system from Xylella fastidiosa is secreted via outer membrane vesicles.André Santiago0Juliano Sales Mendes1Clelton A Santos2Marcelo AS Toledo3Lilian L Beloti4Aline Crucello5Maria AC Horta6Marianna Favaro7Duber Marcel Murillo Munar8Alessandra A Souza9Mônica Alonso Cotta10Mônica Alonso Cotta11Anete Pereira De Souza12University of CampinasUniversity of CampinasUniversity of CampinasUniversity of CampinasUniversity of CampinasUniversity of CampinasUniversity of CampinasUniversity of CampinasUniversity of CampinasInstituto Agronômico de CampinasUniversity of CampinasUniversity of CampinasUniversity of CampinasThe Xylella fastidiosa subsp pauca strain 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, from X. fastidiosa subsp pauca strain 9a5c. These proteins display a high similarity to their homologues in X. fastidiosa strain Temecula and a predicted tridimensional structure that is similar to MqsR-YgiT from Escherichia coli. The characterization was performed using in vitro assays 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 that X. fastidiosa secretes 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 from X. fastidiosa strain 9a5c, and we also discuss the possible influence of wild-type XfYgiT in the cell.http://journal.frontiersin.org/Journal/10.3389/fmicb.2016.02030/fullBacterial Pathogenesisprotein characterizationXylella fastidiosaOMVstoxin-antitoxin system |
| spellingShingle | André Santiago Juliano Sales Mendes Clelton A Santos Marcelo AS Toledo Lilian L Beloti Aline Crucello Maria AC Horta Marianna Favaro Duber Marcel Murillo Munar Alessandra A Souza Mônica Alonso Cotta Mônica Alonso Cotta Anete Pereira De Souza The antitoxin protein of a toxin-antitoxin system from Xylella fastidiosa is secreted via outer membrane vesicles. Frontiers in Microbiology Bacterial Pathogenesis protein characterization Xylella fastidiosa OMVs toxin-antitoxin system |
| title | The antitoxin protein of a toxin-antitoxin system from Xylella fastidiosa is secreted via outer membrane vesicles. |
| title_full | The antitoxin protein of a toxin-antitoxin system from Xylella fastidiosa is secreted via outer membrane vesicles. |
| title_fullStr | The antitoxin protein of a toxin-antitoxin system from Xylella fastidiosa is secreted via outer membrane vesicles. |
| title_full_unstemmed | The antitoxin protein of a toxin-antitoxin system from Xylella fastidiosa is secreted via outer membrane vesicles. |
| title_short | The antitoxin protein of a toxin-antitoxin system from Xylella fastidiosa is secreted via outer membrane vesicles. |
| title_sort | antitoxin protein of a toxin antitoxin system from xylella fastidiosa is secreted via outer membrane vesicles |
| topic | Bacterial Pathogenesis protein characterization Xylella fastidiosa OMVs toxin-antitoxin system |
| url | http://journal.frontiersin.org/Journal/10.3389/fmicb.2016.02030/full |
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