Identification and Regulation of the N-Acetylglucosamine Utilization Pathway of the Plant Pathogenic Bacterium Xanthomonas campestris pv. campestris ▿ †

Xanthomonas campestris pv. campestris, the causal agent of black rot disease of brassicas, is known for its ability to catabolize a wide range of plant compounds. This ability is correlated with the presence of specific carbohydrate utilization loci containing TonB-dependent transporters (CUT loci) devoted to scavenging specific carbohydrates. In this study, we demonstrate that there is an X. campestris pv. campestris CUT system involved in the import and catabolism of N-acetylglucosamine (GlcNAc). Expression of genes belonging to this GlcNAc CUT system is under the control of GlcNAc via the LacI family NagR and GntR family NagQ regulators. Analysis of the NagR and NagQ regulons confirmed that GlcNAc utilization involves NagA and NagB-II enzymes responsible for the conversion of GlcNAc-6-phosphate to fructose-6-phosphate. Mutants with mutations in the corresponding genes are sensitive to GlcNAc, as previously reported for Escherichia coli. This GlcNAc sensitivity and analysis of the NagQ and NagR regulons were used to dissect the X. campestris pv. campestris GlcNAc utilization pathway. This analysis revealed specific features, including the fact that uptake of GlcNAc through the inner membrane occurs via a major facilitator superfamily transporter and the fact that this amino sugar is phosphorylated by two proteins belonging to the glucokinase family, NagK-IIA and NagK-IIB. However, NagK-IIA seems to play a more important role in GlcNAc utilization than NagK-IIB under our experimental conditions. The X. campestris pv. campestris GlcNAc NagR regulon includes four genes encoding TonB-dependent active transporters (TBDTs). However, the results of transport experiments suggest that GlcNAc passively diffuses through the bacterial envelope, an observation that calls into question whether GlcNAc is a natural substrate for these TBDTs and consequently is the source of GlcNAc for this nonchitinolytic plant-associated bacterium

The plant pathogen Xanthomonas campestris pv. campestris exploits N-acetylglucosamine during infection.

N-Acetylglucosamine (GlcNAc), the main component of chitin and a major constituent of bacterial peptidoglycan, is present only in trace amounts in plants, in contrast to the huge amount of various sugars that compose the polysaccharides of the plant cell wall. Thus, GlcNAc has not previously been considered a substrate exploited by phytopathogenic bacteria during plant infection. Xanthomonas campestris pv. campestris, the causal agent of black rot disease of Brassica plants, expresses a carbohydrate utilization system devoted to GlcNAc exploitation. In addition to genes involved in GlcNAc catabolism, this system codes for four TonB-dependent outer membrane transporters (TBDTs) and eight glycoside hydrolases. Expression of all these genes is under the control of GlcNAc. In vitro experiments showed that X. campestris pv. campestris exploits chitooligosaccharides, and there is indirect evidence that during the early stationary phase, X. campestris pv. campestris recycles bacterium-derived peptidoglycan/muropeptides. Results obtained also suggest that during plant infection and during growth in cabbage xylem sap, X. campestris pv. campestris encounters and metabolizes plant-derived GlcNAc-containing molecules. Specific TBDTs seem to be preferentially involved in the consumption of all these plant-, fungus- and bacterium-derived GlcNAc-containing molecules. This is the first evidence of GlcNAc consumption during infection by a phytopathogenic bacterium. Interestingly, N-glycans from plant N-glycosylated proteins are proposed to be substrates for glycoside hydrolases belonging to the X. campestris pv. campestris GlcNAc exploitation system. This observation extends the range of sources of GlcNAc metabolized by phytopathogenic bacteria during their life cycle.Despite the central role of N-acetylglucosamine (GlcNAc) in nature, there is no evidence that phytopathogenic bacteria metabolize this compound during plant infection. Results obtained here suggest that Xanthomonas campestris pv. campestris, the causal agent of black rot disease on Brassica, encounters and metabolizes GlcNAc in planta and in vitro. Active and specific outer membrane transporters belonging to the TonB-dependent transporters family are proposed to import GlcNAc-containing complex molecules from the host, from the bacterium, and/or from the environment, and bacterial glycoside hydrolases induced by GlcNAc participate in their degradation. Our results extend the range of sources of GlcNAc metabolized by this phytopathogenic bacterium during its life cycle to include chitooligosaccharides that could originate from fungi or insects present in the plant environment, muropeptides leached during peptidoglycan recycling and bacterial lysis, and N-glycans from plant N-glycosylated proteins present in the plant cell wall as well as in xylem sap

The N-Glycan Cluster from Xanthomonas campestris pv. campestris: A toolbox for sequential plant n-glycan processing

N-Glycans are widely distributed in living organisms but represent only a small fraction of the carbohydrates found in plants. This probably explains why they have not previously been considered as substrates exploited by phytopathogenic bacteria during plant infection. Xanthomonas campestris pv. campestris, the causal agent of black rot disease of Brassica plants, possesses a specific system for GlcNAc utilization expressed during host plant infection. This system encompasses a cluster of eight genes (nixE to nixL) encoding glycoside hydrolases (GHs). In this paper, we have characterized the enzymatic activities of these GHs and demonstrated their involvement in sequential degradation of a plant N-glycan using a N-glycopeptide containing two GlcNAcs, three mannoses, one fucose, and one xylose (N2M3FX) as a substrate. The removal of the α-1,3-mannose by the α-mannosidase NixK (GH92) is a prerequisite for the subsequent action of the β-xylosidase NixI (GH3), which is involved in the cleavage of the β-1,2-xylose, followed by the α-mannosidase NixJ (GH125), which removes the α-1,6-mannose. These data, combined to the subcellular localization of the enzymes, allowed us to propose a model of N-glycopeptide processing by X. campestris pv. campestris. This study constitutes the first evidence suggesting N-glycan degradation by a plant pathogen, a feature shared with human pathogenic bacteria. Plant N-glycans should therefore be included in the repertoire of molecules putatively metabolized by phytopathogenic bacteria during their life cycle

EffectorK, a comprehensive resource to mine for Ralstonia, Xanthomonas, and other published effector interactors in the Arabidopsis proteome

Pathogens deploy effector proteins that interact with host proteins to manipulate the host physiology to the pathogen's own benefit. However, effectors can also be recognized by host immune proteins, leading to the activation of defence responses. Effectors are thus essential components in determining the outcome of plant-pathogen interactions. Despite major efforts to decipher effector functions, our current knowledge on effector biology is scattered and often limited. In this study, we conducted two systematic large-scale yeast two-hybrid screenings to detect interactions betweenArabidopsis thalianaproteins and effectors from two vascular bacterial pathogens:Ralstonia pseudosolanacearumandXanthomonas campestris. We then constructed an interactomic network focused onArabidopsisand effector proteins from a wide variety of bacterial, oomycete, fungal, and invertebrate pathogens. This network contains our experimental data and protein-protein interactions from 2,035 peer-reviewed publications (48,200Arabidopsis-Arabidopsisand 1,300Arabidopsis-effector protein interactions). Our results show that effectors from different species interact with both common and specificArabidopsisinteractors, suggesting dual roles as modulators of generic and adaptive host processes. Network analyses revealed that effector interactors, particularly "effector hubs" and bacterial core effector interactors, occupy important positions for network organization, as shown by their larger number of protein interactions and centrality. These interactomic data were incorporated in EffectorK, a new graph-oriented knowledge database that allows users to navigate the network, search for homology, or find possible paths between host and/or effector proteins. EffectorK is available at and allows users to submit their own interactomic data

The xylan utilization system of the plant pathogen Xanthomonas campestris pv campestris controls epiphytic life and reveals common features with oligotrophic bacteria and animal gut symbionts

Xylan is a major structural component of plant cell wall and the second most abundant plant polysaccharide in nature. Here, by combining genomic and functional analyses, we provide a comprehensive picture of xylan utilization by Xanthomonas campestris pv campestris (Xcc) and highlight its role in the adaptation of this epiphytic phytopathogen to the phyllosphere. The xylanolytic activity of Xcc depends on xylan-deconstruction enzymes but also on transporters, including two TonB-dependent outer membrane transporters (TBDTs) which belong to operons necessary for efficient growth in the presence of xylo-oligosaccharides and for optimal survival on plant leaves. Genes of this xylan utilization system are specifically induced by xylo-oligosaccharides and repressed by a LacI-family regulator named XylR. Part of the xylanolytic machinery of Xcc, including TBDT genes, displays a high degree of conservation with the xylose-regulon of the oligotrophic aquatic bacterium Caulobacter crescentus. Moreover, it shares common features, including the presence of TBDTs, with the xylan utilization systems of Bacteroides ovatus and Prevotella bryantii, two gut symbionts. These similarities and our results support an important role for TBDTs and xylan utilization systems for bacterial adaptation in the phyllosphere, oligotrophic environments and animal guts

EffectorK, a comprehensive resource to mine for Ralstonia, Xanthomonas,

International audiencePathogens deploy effector proteins that interact with host proteins to manipulate the host physiology to the pathogen's own benefit. However, effectors can also be recognized by host immune proteins, leading to the activation of defence responses. Effectors are thus essential components in determining the outcome of plant-pathogen interactions. Despite major efforts to decipher effector functions, our current knowledge on effector biology is scattered and often limited. In this study, we conducted two systematic large-scale yeast two-hybrid screenings to detect interactions betweenArabidopsis thalianaproteins and effectors from two vascular bacterial pathogens:Ralstonia pseudosolanacearumandXanthomonas campestris. We then constructed an interactomic network focused onArabidopsisand effector proteins from a wide variety of bacterial, oomycete, fungal, and invertebrate pathogens. This network contains our experimental data and protein-protein interactions from 2,035 peer-reviewed publications (48,200Arabidopsis-Arabidopsisand 1,300Arabidopsis-effector protein interactions). Our results show that effectors from different species interact with both common and specificArabidopsisinteractors, suggesting dual roles as modulators of generic and adaptive host processes. Network analyses revealed that effector interactors, particularly "effector hubs" and bacterial core effector interactors, occupy important positions for network organization, as shown by their larger number of protein interactions and centrality. These interactomic data were incorporated in EffectorK, a new graph-oriented knowledge database that allows users to navigate the network, search for homology, or find possible paths between host and/or effector proteins. EffectorK is available at and allows users to submit their own interactomic data