Characterization of the mreB gene of Xantomonas citri subsp. citri coding for a bacterial actin

Author(s)

Camila Malvessi Pereira

• Document type: Master's Dissertation
• Press: Rio Claro. 2019-04-17.
• Institution: Universidade Estadual Paulista (Unesp). Instituto de Biociências. Rio Claro
• Defense date: 2019-02-26
• Advisor: Henrique Ferreira; Paula Maria Moreira Martins

Abstract

Xanthomonas citri subsp. citri (X. citri) is the etiological agent of Citrus canker, a widespread plant disease that attacks all varieties of commercial citrus plants, causing economic loss worldwide. In endemic areas, citrus canker is controlled mainly by the application of cupric bactericides. Alternative compounds to control this pathogen are desirable, since copper based bactericides have negative environmental impact and induce resistance in X. citri. An important step for the discovery and evaluation of new compounds to control X. citri is the characterization of genes/proteins that are potential targets. The mreB gene codes for a protein that is a bacterial actin homologue with a key role in rod shape morphogenesis. This gene was characterized in some bacteria, in which it was shown to be essential. There are no reports of characterization of mreB in X. citri. MreB and actin have a weak sequence similarity, despite being functional homologues. These characteristics make MreB an interesting target for new antibacterial chemical compounds. The objectives of this work were to study the function and subcellular localization of MreB in X. citri. To study the function of MreB in X. citri, two strategies were used to delete the mreB gene. However, neither strategy produced deleted mutants. This result confirmed that mreB is essential to X. citri and showed that the complementation method used was not adequate to ensure the viability of X. citri. To investigate MreB subcellular localization, a sandwich fusion of MreB with GFP was developed, and cells expressing the fusion were imaged with fluorescence and phase contrast microscopy. The fluorescence and phase contrast microscopy images obtained of X. citri expressing the sandwich fusion showed rod shaped cells with spots of fluorescence predominantly at the periphery of the cells. These images indicated that the sandwich fusion was successfully expressed in X. citri with no detectable negative effects for the cells. These images also revealed for the first time that the subcellular distribution of MreB in X. citri is very similar to that seen in other bacteria. The results of this work expanded the knowledge of the biology of X. citri and will also be useful to future evaluations of chemical compounds to control citrus canker. (AU)

• FAPESP's process: 17/08856-2 - Characterization of the bacterial actin homologue mreB in Xanthomonas citri subsp. citri
• Grantee: Camila Malvessi Pereira
• Support Opportunities: Scholarships in Brazil - Master