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| Author(s): |
Maxuel de Oliveira Andrade
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ) |
| Defense date: | 2006-07-06 |
| Examining board members: |
Shaker Chuck Farah;
Regina Lúcia Baldini;
Marcos Antonio Machado
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| Advisor: | Shaker Chuck Farah |
| Abstract | |
In Xanthomonas a group of genes named regulators of the pathogenicity factors (rpf) control the synthesis of virulence factors as a function of cellular density in a process termed quorum sensing. Alegria (2002) cloned the rpf genes from Xanthomonas axonopodis pv citri (Xac) in the pOBD vector and used them as baits in two hybrid assays against a Xac prey library cloned in the pOAD vector and showed that RpfC interacts with RpfG, RpfF and CMF. Homologous of the cmf gene are found only in amoebas such as Dictyostelium, where plays a central function in the quorum sensing process. In this work, we expressed and purified RpfC and its domains, RpfG, RpfF and CMF and raised antibodies against these polypeptides. In vitro assays demonstrated the following interactions: RpfC-RpfF, RpfC-RpfG and RpfC-CMF. We show that RpfG and CMF interact with the response regulator domain of RpfC, and interactions RpfG and CMF also interact with the histidine phosphotransfer domain of RpfC. In addition, the recently characterized HD-GYP phosphodiesterase domain of RpfG was used as bait in the two hybrid assays. Interestingly, the majority of its preys were derived from a set of Xac proteins that possess GGDEF domains (diguanilate cyclase). In bacteria, many complex processes such virulence, motility and biofilm production are controlled by quorum sensing process and by levels of the second messenger cyclic diGMP. Our results demonstrate a direct link between quorum sensing and diGMP cyclic signaling pathways in the form of a direct physical interaction between the RpfG HD-GYP domain and GGDEF domains. Finally, studies with a Xac cmf-mutant show that CMF plays an important role in the quorum sensing process in Xanthomonas, including biofilm production, synthesis of xanthan gum and pathogenicity. (AU) | |