Subcellular localization of proteins labeled with GFP in Xanthomonas citri ssp. citri: targeting the division septum

Abstract

Xanthomonas citri ssp. citri (Xac) is the causal agent of citrus canker, an economically important disease that affects citrus worldwide. To initiate the characterization of essential biological processes of Xac, we constructed integrative plasmids for the ectopic expression of green fluorescent protein (GFP)-labeled proteins within this bacterium. Here, we show that the disruption of the alpha-amylase gene (amy), the site of plasmid integration into the bacterial chromosome, does not alter its pathogenesis while abolishing completely the ability of Xac to degrade starch. Furthermore, our GFP expression system was used to characterize ORF XAC3408, a hypothetical protein encoded by Xac that shares significant homology to the FtsZ-stabilizing factor ZapA from Bacillus subtilis (ZapA(Bsu)). GFP-XAC3408 expressed in Xac exhibited a septal localization pattern typical of GFP-ZapA(Bsu), which indicates that XAC3408 is the Xac orthologue of the cell division protein ZapA(Bsu). 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.

Antibacterial Activity of Alkyl Gallates against Xanthomonas citri subsp. citri

“…citri mutant strains were used for these analyses: one was the X. citri subsp. citri amy::pPM2a-zapA mutant expressing extra copies of ZapA as GFP fusions (GFP-ZapA) (27),…”

“…The X. citri subsp. citri 306 mutant strains expressing green fluorescent protein (GFP)-ZapA (27) and ParB-GFP (A. P. Ucci and H. Ferreira, unpublished data) were X. citri subsp. citri amy::pPM2a-zapA and X. citri subsp.…”

“…Cells of wild-type and mutant strains of X. citri subsp. citri were treated with the alkyl gallates for 6 h at 30°C and immobilized on agarose-covered slides for observations by microscopy, as previously described (27). Cells were visualized by using an Olympus BX-61 microscope and documented with a monochromatic XM-10 camera.…”

Xanthomonas citri MinC Oscillates from Pole to Pole to Ensure Proper Cell Division and Shape

“…To construct pGCD21, gfp was removed from pPM7g (Martins et al, 2010) using the restriction enzyme Bam HI/ Xba I; the isolated gfp cassette was ligated to the backbone of pHF5Ca digested with the same enzymes, giving rise to pGCD19 (GenBank KJ619486). Next, the arabinose repressor and promoter ( araC-para ) were amplified by PCR using pEB304 (Gully et al, 2003) as a template and the primer pair pARAF/pARAR.…”

“…Since then, some studies have expanded upon the knowledge we have about biological processes in Xac, mostly concerning pathogenicity mechanisms (Alegria et al, 2004; Casabuono et al, 2011; Li and Wang, 2012; Huang et al, 2013; Alexandrino et al, 2016). Recently, new tools for protein expression in Xac have been developed (Martins et al, 2010; Ucci et al, 2014; Lacerda et al, 2017), enabling studies on chromosome segregation and cell division. These cellular systems represent potential targets for antimicrobials, as the proteins involved in such processes share little homology with eukaryotic equivalents (Pan et al, 2006; Vollmer, 2006; Sass and Brötz-Oesterhelt, 2013; Broughton et al, 2016).…”