Genome-Wide Mutagenesis of Xanthomonas axonopodis pv. citri Reveals Novel Genetic Determinants and Regulation Mechanisms of Biofilm Formation

Abstract

Xanthomonas axonopodis pv. citri (Xac) causes citrus canker disease, a major threat to citrus production worldwide. Accumulating evidence suggests that the formation of biofilms on citrus leaves plays an important role in the epiphytic survival of this pathogen prior to the development of canker disease. However, the process of Xac biofilm formation is poorly understood. Here, we report a genome-scale study of Xac biofilm formation in which we identified 92 genes, including 33 novel genes involved in biofilm formation and 7 previously characterized genes, colR, fhaB, fliC, galU, gumD, wxacO, and rbfC, known to be important for Xac biofilm formation. In addition, 52 other genes with defined or putative functions in biofilm formation were identified, even though they had not previously reported been to be associated with biofilm formation. The 92 genes were isolated from 292 biofilm-defective mutants following a screen of a transposon insertion library containing 22,000 Xac strain 306 mutants. Further analyses indicated that 16 of the novel genes are involved in the production of extracellular polysaccharide (EPS) and/or lipopolysaccharide (LPS), 7 genes are involved in signaling and regulatory pathways, and 5 genes have unknown roles in biofilm formation. Furthermore, two novel genes, XAC0482, encoding a haloacid dehalogenase-like phosphatase, and XAC0494 (designated as rbfS), encoding a two-component sensor protein, were confirmed to be biofilm-related genes through complementation assays. Our data demonstrate that the formation of mature biofilm requires EPS, LPS, both flagellum-dependent and flagellum-independent cell motility, secreted proteins and extracellular DNA. Additionally, multiple signaling pathways are involved in Xac biofilm formation. This work is the first report on a genome-wide scale of the genetic processes of biofilm formation in plant pathogenic bacteria. The report provides significant new information about the genetic determinants and regulatory mechanism of biofilm formation.

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

“…Surprisingly, XAC2583/gumD showed not only impairment in sliding motility, but also that its ability to swim was affected. In a previous biofilm screening (Li & Wang, 2011b), this mutant was also found to have shortcomings in both types of motilities, confirming our results. We observed that the production of EPS is affected in XAC1260, XAC0533 and XAC2301 (data not shown), although these genes are not known to be related with xanthan synthesis.…”

“…These genes are involved in xanthan production (gumB, galU and gumD) (Guo et al, 2010;Li & Wang, 2011b;Rigano et al, 2007), in amino acid metabolism (sahH) (Li & Wang, 2011b) and in periplasmic glucan biosynthesis (hrpM) (Malamud et al, 2012).…”

“…So far, no relationship between chemotaxis and biofilm development has been demonstrated in Xcc. In a previous biofilm screening, four different genes involved in bacterial chemotaxis were found, but the authors did not characterize them (Li & Wang, 2011b). In order to establish a relationship between biofilm development and chemotaxis in Xcc, we performed a chemotaxis assay consisting of the measurement of bacteria migration ability towards grapefruit leaf extract.…”

“…A direct relationship between this system and biofilm development in Xcc has not been described yet. However, a former mutant screening also found a hypothetical protein related to biofilm formation that interacts with VirD4 (Li & Wang, 2011b). Both proteins share a conserved domain of approximately 120 amino acids that is present in all the VirD4-associated proteins (Alegria et al, 2005).…”

Identification of New Genes Related to Virulence of <i>Xanthomonas axonopodis</i> Pv. <i>Citri</i> during Citrus Host Interactions

“…A previous study based on the in planta analysis of Xac pathotype A mutants has led to the identification of 44 mutated genes in strains that show total or partial inability to induce the Xac virulence phenotype in the host plant [14]. Another study involving the same organism described 292 mutants showing a massive reduction in virulence phenotypes [15]. More recently, seven new genes associated with virulence in X. citri subsp.…”