Functional characterization of two proteins belonging to toxin-antitoxin operon and a LysR-type transcription factor from Xylella fastidiosa

Author(s)

André da Silva Santiago

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2015-09-25

Examining board members

Anete Pereira de Souza; Ljubica Tasic; Cristina Elisa Alvarez Martinez; Nelson Arno Wulff; Cleide Marques Ferreira

• Advisor: Alessandra Alves de Souza; Clelton Aparecido dos Santos; Anete Pereira de Souza

Abstract

After the genome sequencing of the Xylella fastidiosa greater knowledge about the bacterial physiology and pathogenicity have become available, however many proteins still do not have predicted functions and are classified as hypothetical proteins. Characterization of these proteins can bring new insights about its role within bacteria physiology. Therefore, our work aimed to characterize three proteins termed XfYcjZ (orf Xf1480), which possesses high identity with the LysR-type transcriptional regulator (LTTR) family; XfMqsR (orf Xf2162) and XfMqsA (orf Xf2163) which possess identity with the toxin-antitoxin operon MqsR and MqsA from Escherichia coli. These genes were reported to be involved in cell cycle, quorum sensing, biofilm formation, virulence and oxidative stress response. The functional characterization of these proteins was based on circular dichroism (CD), analytical size-exclusion chromatography, analytical ultracentrifugation and western blottings, which demonstrated the protein XfYcjZ is present in tetrameric form in solution and is globular, as shown by size exclusion chromatography and analytical ultracentrifugation. Our analyses also showed that XfYcjZ is differentially induced in the X. fastidiosa growth phases as well as its expression changed to cupper-induced oxidative stress, which is a indicative that XfYcjZ is involved in oxidative stress. The characterization of the orfs Xf2162 and Xf2163 was based on the cloning, expression and purification of the heterologous proteins and they were analysed as for the capacity of interaction between the proteins XfMqsR and XfMqsA and between XfMqsA and promoter of the operon. We have shown that XfMqsA is present as a dimer and the XfMqsR in monomer in solution and the interaction occurs in molar ratio 1:1 and the complex XfMqsA and XfMqsR is thermodynamically favorable and more stable that the isolated proteins. XfMqsR possesses RNAse activity and XfMqsA was able to inhibit it in fluorimetric assay. Moreover, it was demonstrated by MS/MS and western blotting that the XfMqsA is secreted by X. fastidiosa via outer membrane vesicles. We hope these results can contribute to better understand the involvement of these proteins in the X. fastidiosa lifecycle and the possible involvement in its pathogenicity (AU)