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Functional characterization of the regulon of the alternative sigma factor ecfl of Xanthomonas citri subsp. citri

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Anthony Jhoao Fasabi Flores
Total Authors: 1
Document type: Master's Dissertation
Press: Campinas, SP.
Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
Defense date:
Examining board members:
Cristina Elisa Alvarez Martinez; Priscila Oliveira de Giuseppe; Germán Gustavo Sgro
Advisor: Cristina Elisa Alvarez Martinez

Citriculture is an important economic activity in Brazil, threatened by diseases that attack crops, such as citrus canker caused by the bacterium Xanthomonas citri, which leads to reduced yields and significant economic losses. It is therefore essential to carry out studies to understand the virulence mechanisms of X. citri, contributing to improve its detection and control. Bacterial sigma factors (?) are dissociable subunits of RNA polymerase responsible for the recognition of promoters and for promoting the initiation of transcription. Among them, the extracytoplasmic sigma factor (ECF) family regulates gene expression in response to signals detected in the bacterial envelope. Our research group has been studying the ECF factors encoded by the xac4128 (ecfK) and xac4129 (ecfL) genes. These are found in a gene cluster that encodes a type VI secretion system (SS6), a protein complex that crosses the bacterial envelope and injects proteins directly into prokaryotic and eukaryotic cells or into the extracellular medium. The ecfL gene has a typical genomic context of ECF factors involved in the response to iron deficiency, also called FecI (?FecI). Members of this group are encoded in an operon with the gene encoding an anti-sigma factor called FecR and in the vicinity of a gene encoding a TonB-dependent receptor (TBDR), called FecA, which acts on activation of the sigma factor in response to signals from the external environment. The transcriptome analysis of a strain with increased levels of ecfL led to the identification of the regulon of this ECF factor, which is composed of 3 genes, xac4131, xac4132, xac4133, located in the vicinity of ecfL in the genome of X. citri. Computer analysis of the proteins encoded by the EcfL regulon genes reveals that the xac4131 gene encodes a FecA homolog, suggesting that the protein acts in the activation of ?FecI and, possibly, in the uptake of nutrients or other small molecules, a function characterized for members of this family of outer membrane receptors. The xac4132 gene encodes a protein with a histidine-dependent acid phosphatase domain, this domain is found in phytases, enzymes that degrade phytic acid, the main source of phosphate stored in plants and soils. The xac4133 gene encodes a protein that combines a jacalin-like lectin domain, involved in carbohydrate binding, which suggests an interaction with the surface of eukaryotic cells, and an EEP (exonuclease-endonuclease-phosphatase) family domain, typical of enzymes that act in the cleavage of phosphodiester bonds, generally in DNA and phospholipids, which suggests an endonuclease or sphingomyelinase function. Bacterial sphingomyelinases are secreted enzymes that act to disrupt eukaryotic membranes, allowing, for example, phagosome escape and bacterial survival in the host. A possible role as toxins secreted by SS6 is therefore suggested by the domains found in Xac4132 and Xac4133. However, the phenotypic characterization of ?ecfL indicates that the gene is not necessary for resistance to predation and, similarly, the genes ecfL, xac4132 and xac4133 do not show expression induction during co-incubation with D. discoideum (Lima et al., submitted), a characteristic observed for the SS6 genes and its regulator EcfK (Bayer-Santos et al., 2018). The virulence reduction observed for the ?ecfL lineage may indicate that the Xac4132 and Xac4133 proteins act in the host cell during infection. Thus, despite knowing the ecfL regulon and having interesting phenotypic data for the ecfL mutant strain, further studies are needed to understand the role of this ECF sigma factor in the physiology of X. citri. For this reason, the objective of this work is the biochemical and functional characterization of the proteins encoded by the regulon members of the alternative sigma factor ecfL of X. citri. Thus, this study may lead to the identification of new important functions for the virulence of X. citri and for survival and adaptation to the environment (AU)

FAPESP's process: 19/03406-4 - Functional characterization of the Xanthomonas citri subsp citri EcfL regulon
Grantee:Anthony Jhoao Fasabi Flores
Support Opportunities: Scholarships in Brazil - Master