The results demonstrate the potential of GFP labeling for protein functional characterizations in Xac, and, in addition, the Xac mutant strain labeled at the septum constitutes a biological model for the exploration of antibacterial compounds able to inhibit cell division in this plant pathogen. Xanthomonas
citri ssp. citri (Schaad et al., 2005, 2006) (also known as Xanthomonas axonopodis pv. citri or Xac) is a Gram-negative, plant-pathogenic bacterium that affects most citrus species and is the causal agent of citrus canker, a very economically important disease of citrus plants worldwide. An effective control for this disease is inexistent, and a more detailed understanding of the biology of the etiological agent may contribute substantially toward the development of strategies to prevent and control infection. A major effort to accomplish check details this task was the elucidation of the genome sequence of Xac (da Silva et al., 2002), which has stimulated a number of molecular studies using Xac as model microorganism,
and yet, little information is available regarding technical methods that could enhance its proteome exploration (Galvao-Botton et al., 2003; Mehta & Rosato, 2003; Alegria et al., 2004, 2005; Cernadas et al., 2008). Our main interest focused on the characterization of some essential biological processes of Xac, more specifically those GSK2118436 order involved with chromosome segregation and cell division. A common feature of such bacterial systems is that they are usually composed of proteins sharing little homology to their functional analogues in more derived eukaryotes; therefore, these proteins constitute ideal targets for antimicrobial drug development and pathogen control (e.g. Gitai et al., 2005; Pan et al., 2006; Haydon et al., PDK4 2008; Beuria et al., 2009; Kapoor & Panda, 2009). However, to undertake protein functional studies with/in Xac, we were limited
by the lack of biological tools developed and/or accessible for this purpose. Here, we describe a protein expression system dedicated to Xac, which can also be used for the subcellular localization of the green fluorescent protein (GFP)-labeled factors in this pathogen. We used the system to characterize a hypothetical protein of Xac that shares significant homology to the FtsZ-stabilizing factor ZapA, originally described in Bacillus subtilis (ZapABsu) (Gueiros-Filho & Losick, 2002). Furthermore, we show that the disruption of the α-amylase gene, the site of plasmid integration into the Xac chromosome, does not alter its pathogenesis. The Xanthomonas citri ssp. citri used was the sequenced strain (da Silva et al., 2002), formerly designated X. axonopodis pv. citri strain 306 (IBSBF 1594). Escherichia coli strain DH10B (Invitrogen) was used for cloning. Escherichia coli was cultivated at 37 °C in a Luria–Bertani (LB)/LB-agar medium (Sambrook et al.