The Xanthomonas axonopodis pv. citri flagellum is required for mature biofilm and canker development

Abstract

The Xanthomonas axonopodis pv. citri flagellum is required for mature biofilm and canker development Xanthomonas axonopodis pv. citri (Xac) is the causative agent of citrus canker. This bacterium develops a characteristic biofilm on both biotic and abiotic surfaces. To evaluate the participation of the single flagellum of Xac in biofilm formation, mutants in the fliC (flagellin) and the flgE (hook) genes were generated. Swimming motility, assessed on 0.25 % agar plates, was markedly reduced in fliC and flgE mutants. However, the fliC and flgE mutants exhibited a flagellarindependent surface translocation on 0.5 % agar plates. Mutation of either the rpfF or the rpfC gene, which both encode proteins involved in cell-cell signalling mediated by diffusible signal factor (DSF), led to a reduction in both flagellar-dependent and flagellar-independent surface translocation, indicating a regulatory role for DSF in both types of motility. Confocal laser scanning microscopy of biofilms produced in static culture demonstrated that the flagellum is also involved in the formation of mushroom-shaped structures and water channels, and in the dispersion of biofilms. The presence of the flagellum was required for mature biofilm development on lemon leaf surfaces. The absence of flagellin produced a slight reduction in Xac pathogenicity and this reduction was more severe when the complete flagellum structure was absent.

Identification and characterization of biofilm formation-defective mutants of Xanthomonas citri subsp. citri

“…WT Xcc strain 306 and mutant strains were grown at 28 uC in PYM medium or in a minimal medium (YMM) as described by Malamud et al (2011). Escherichia coli strains were cultured at 37 uC in Luria-Bertani medium.…”

“…Briefly, bacterial strains were grown overnight in PYM nutrient medium and then inoculated into YMM. Aliquots of 150 ml were used to fill different wells of a microtitre plate and then incubated at 28 uC as previously described (Malamud et al, 2011); the assay was performed in triplicate. These first candidates were subsequently subjected to the same assay three more times independently with eight replicates each time.…”

“…The assay was performed by growing cells at 28 uC for 24 h without agitation in YMM minimal medium as previously described (Malamud et al, 2011;Rigano et al, 2007).…”

“…Previous studies have revealed that Xcc forms biofilms on both abiotic and biotic surfaces (Malamud et al, 2011;Rigano et al, 2007). Bacterial biofilm development is a temporal process involving transition through different stages of multicellular organization (Monds & O'Toole, 2009).…”

“…Some features of biofilm, known to be required in other micro-organisms, were also found necessary for the biofilm establishment of Xcc. Xanthan production (Rigano et al, 2007), quorum sensing (Siciliano et al, 2006), a filamentous haemagglutinin-like adhesin (Gottig et al, 2009), flagellum synthesis (Malamud et al, 2011), a UTPglucose-1-phosphate uridylyltransferase (Guo et al, 2010), LPS biosynthesis (Li & Wang, 2011a; and IP: 13.66.222.141…”