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Competition assays are a simple phenotyping strategy that confront two bacterial strains to evaluate their relative fitness. Because they are more accurate than single-strain growth assays, competition assays can be used to highlight slight differences that would not otherwise be detectable. In the frame of host-pathogens interactions, they can be very useful to study the contribution of individual bacterial genes to bacterial fitness and lead to the identification of new adaptive traits. Here, we describe how to perform such competition assays by taking the example of the model phytopathogenic bacterium Xanthomonas campestris pv. campestris during infection of the mesophyll of its cauliflower host. This phenotypic assay is based on the use of a Competitive Index (CI) that compares the relative abundance of co-inoculated strains before and after inoculation. Since multiplication is a direct proxy for bacterial fitness, the evolution of the ratio between both strains in the mixed population is a direct way to assess differences in fitness in a given environment. In this protocol, we exploit the blue staining of GUS-expressing bacteria to count blue vs. white colonies on plates and estimate the competitiveness of the strains of interest in plant mesophyll.

International audience; Plant diseases are an important threat to food production. While major pathogenicity determinants required for disease have been extensively studied, less is known on how pathogens thrive during host colonization, especially at early infection stages. Here, we used randomly barcoded-transposon insertion site sequencing (RB-TnSeq) to perform a genome-wide screen and identify key bacterial fitness determinants of the vascular pathogen Xanthomonas campestris pv campestris (Xcc) during infection of the cauliflower host plant (Brassica oleracea). This high-throughput analysis was conducted in hydathodes, the natural entry site of Xcc, in xylem sap and in synthetic media. Xcc did not face a strong bottleneck during hydathode infection. In total, 181 genes important for fitness were identified in plant-associated environments with functional enrichment in genes involved in metabolism but only few genes previously known to be involved in virulence. The biological relevance of 12 genes was independently confirmed by phenotyping single mutants. Notably, we show that XC_3388, a protein with no known function (DUF1631), plays a key role in the adaptation and virulence of Xcc possibly through c-di-GMP-mediated regulation. This study revealed yet unsuspected social behaviors adopted by Xcc individuals when confined inside hydathodes at early infection stages.

Plant diseases are an important threat to food production. While major pathogenicity determinants required for disease have been extensively studied, less is known on how pathogens thrive during host colonization especially at early infection stages. Here, we used randomly barcoded-transposon insertion site sequencing (RB-TnSeq) to perform a genome-wide screen and identify key bacterial fitness determinants of the vascular pathogenXanthomonas campestrispv.campestris(Xcc) during infection of the cauliflower host plant (Brassica oleracea). This high-throughput analysis was conducted in hydathodes, the natural entry site ofXcc, in xylem sap and in synthetic media.Xccdid not face a strong bottleneck during hydathode infection. 183 genes important for fitness were identified in plant-associated environments with functional enrichment in genes involved in metabolism when only few genes known to be involved in virulence were found to be affected. The biological relevance of 13 genes was independently confirmed by phenotyping single mutants. Notably, we show that the XC_3388, a protein with no known function (DUF1631), plays a key role in the adaptation and virulence ofXccpossibly through c-di-GMP-mediated regulation. This study thus revealed yet unsuspected social behaviors adopted byXccindividuals when confined inside hydathodes at early infection stages.

International audience; DNA footprinting is a classic technique to investigate protein-DNA interactions. However, traditional footprinting protocols can be unsuccessful or difficult to interpret if the binding of the protein to the DNA is weak, the protein has a fast off-rate, or if several different protein-DNA complexes are formed. Our protocol differs from traditional footprinting protocols, because it provides a method to isolate the protein-DNA complex from a native gel after treatment with the footprinting agent, thus removing the bound DNA from the free DNA or other protein-DNA complexes. The DNA is then extracted from the isolated complex before electrophoresis on a sequencing gel to determine the footprinting pattern. This analysis provides a possible solution for those who have been unable to use traditional footprinting methods to determine protein-DNA contacts.

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.

The Bvg-regulated promoters for the fimbrial subunit genes fim2 and fim3 of Bordetella pertussis behave differently from each other both in vivo and in vitro. In vivo Pfim2 is significantly stronger than Pfim3 , even though predictions based on the DNA sequences of BvgA-binding motifs and core promoter elements would indicate the opposite. In vitro Pfim3 demonstrated robust BvgA∼P-dependent transcriptional activation, while none was seen with Pfim2 . This apparent contradiction was investigated further. By swapping sequence elements we created a number of hybrid promoters and assayed their strength in vivo. We found that, while Pfim3 promoter elements upstream of the +1 transcriptional start site do indeed direct Bvg-activated transcription more efficiently than those of Pfim2 , the overall promoter strength of Pfim3 in vivo is reduced due to sequences downstream of +1 that inhibit transcription more than 250-fold. This element, the DRE (downstream repressive element), was mapped to the 15 bp immediately downstream of the Pfim3 +1. Placing the DRE in different promoter contexts indicated that its activity was not specific to fim promoters, or even to Bvg-regulated promoters. However it does appear to be specific to Bordetella species in that it did not function in Escherichia coli.

Summary We have used protein electrophoresis through poly- acrylamide gels derivatized with the proprietary ligand Phos-tag™ to separate the response regulator BvgA from its phosphorylated counterpart BvgA~P. This approach has allowed us to readily ascertain the degree of phosphorylation of BvgA in in vitro reac- tions, or in crude lysates ofBordetellapertussis grown under varying laboratory conditions. We have used this technique to examine the kinetics of BvgA phos- phorylation after shift of B. pertussis cultures from non-permissive to permissive conditions, or of its dephosphorylation following a shift from permissive to non-permissive conditions. Our results provide the first direct evidence that levels of BvgA~P in vivo correspond temporally to the expression of early and late BvgA-regulated virulence genes. We have also examined a number of other aspects of BvgA function predicted from previous studies and by analogy with other two-component response regulators. These include the site of BvgA phosphorylation, the exclu- sive role of the cognate BvgS sensor kinase in its phosphorylation inBordetellapertussis, and the effect of the T194M mutation on phosphorylation. We also detected the phosphorylation of a small but consist- ent fraction of BvgA purified after expression in Escherichia coli.

Summary 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.

Protein phosphorylation plays a central role in signal transduction in bacteria. However, separation and detection of the phosphorylated protein from its nonphosphorylated form remain challenging. Here we describe a method to detect phosphorylation of the Bordetella pertussis response regulator BvgA, which is phosphorylated at an aspartate residue (Boulanger et al., 2013). This method is based on the proprietary adduct, Phos-tag™, a dinuclear metal complex, which together with Zn2+ or Mn2+, forms a complex with a phosphomonoesterdianion, such as the phosphorylated aspartate of a response regulator (Barbieri and Stock, 2008; Kinoshita and Kinoshita-Kikuta, 2011). For in vivo detection, B. pertussis cells are lysed in mild formic acid at 4 °C to minimize the disruption of the phospho-aspartate bond, and the phosphorylated BvgA is separated from its nonphosphorylated form by electrophoresis (SDS-PAGE) containing Phos-tag™. Both forms of BvgA are subsequently detected by Western Blot analysis. Quantification of the level of phosphorylated BvgA formed after treatment with acetyl phosphate in vitro is also easily accomplished. Thus, this technique allows one to readily assess the levels of BvgA phosphorylation in B. pertussis and in E. coli under different laboratory conditions in vivo or after phosphorylation under varying reaction conditions in vitro (this research was supported in part by the Intramural Research Program of the NIH, NIDDK).

Crop diseases impact both yield and product quality and result in important health, economic, environmental, and societal issues worldwide (Scholthof, 2003; Strange and Scott, 2005). These diseases are caused by microorganisms like viruses, fungi, and bacteria. Among phytopathogenic bacteria, the genus Xanthomonas comprises 20 species which together infect more than 400 plant species, among which several important crops such as rice, cassava, cabbages, citrus, or tomato (Mansfield et al., 2012). In contrast, individual Xanthomonas species have a narrow host range usually restricted to a specific plant genus or species. The genetic and molecular bases for host range determination in Xanthomonas are essentially unknown at this date (Jacques et al., 2016): It likely involves bacterial factors enhancing the physiological adaptation to the plant environment (e.g., tropism, attachment, nutrition, degradation of plant compounds) or limiting the elicitation of plant immunity (Buttner and Bonas, 2010). Elicitors of plant immunity include both pathogen-associated molecular patterns (PAMP) and type III effectors (T3E) (Jones and Dangl, 2006). PAMP-triggered immunity (PTI) is the first layer of plant's innate immunity and strongly restricts host range for most bacteria. Key to pathogenicity and host range expansion is pathogen's capacity to suppress PTI. This is the prime function of most T3E known to date. T3E proteins are directly injected inside plant cells using a molecular syringe known as the type III secretion system. As a countermeasure, given T3E might specifically elicit potent immune responses known as effector-triggered immunity (ETI) in a set of plant species resulting in a reduced host range. It is thus key to determine pathogen T3E repertoires. Large scale sequencing of collections of phytopathogenic bacteria at the inter- and intra-specific level has revealed a large diversity of T3E repertoires leading to the definition of core vs. accessory T3E (Potnis et al., 2011; Bart et al., 2012; Roux et al., 2015). The paper by Schwartz et al. (2015) perfectly illustrates the power of comparative genomics to understand pathogenicity and host specificity of this important genus of pathogens. The authors focussed on bacterial spot disease of tomato and pepper caused by three distinct Xanthomonas species [gardneri (Xg), perforans (Xp), and euvesicatoria (Xe)]. Draft genome sequences were determined for 67 strains sampled in the southeastern and midwestern United States between 1994 and 2013 and subjected to comparative analysis. Importantly, core genome analysis supported the recent reclassification of these strains in three distinct bacterial species (Jones et al., 2004) and evidenced further intraspecific organization of Xp strains. Identification, comparison, and analysis of T3E repertoires allowed the definition of surprisingly large core effectomes (24/29 T3E in Xp; 26/31 in Xe; 19/25 in Xg). Thirteen T3E are conserved among all three species. Identification of these core T3E and their sequence conservation should prove instrumental to design durable disease resistance strategies against bacterial spot disease in tomato and pepper. Such large interspecific core T3E repertoire might highlight the need for a future larger worldwide sampling of Xanthomonas infecting tomato and pepper. Beyond presence-absence of T3E, the conservation of T3E genes or proteins was also investigated in Xe, Xp, and Xg revealing the evolution and dynamics of these pathogenicity determinants. Overall, a lot more diversity is observed in T3E gene repertoires than in core genome analyses, suggesting a central role of horizontal gene transfer and distinct evolutionary forces applying to T3E genes. In Xe, the authors witnessed the evolution of T3E gene profiles over a 20 years period which could have suggested that such effect was driven by plant breeding and the introduction of novel tomato or pepper varieties. However, changes in prevalent bacterial species on tomato observed the last decade in different states (Florida, Ohio, Michigan) is not associated with strong changes in tomato cultivars used in commercial production fields (Potnis et al., 2015). Abiotic conditions could drive diversity and prevalent strains in diseased tomato fields. For instance, Xg is generally prevalent in cooler regions as observed for Pseudomonas syringae pv. tomato, another bacterial spot agent (Potnis et al., 2015). These two pathogens share several T3Es that are not present in other strains of Xanthomonas associated with bacterial spot. Horizontal gene transfer between different pathogens found in similar regions could be a strong source of diversity. The second part of the analysis reports on host range determination in Xp by two T3E, AvrBsT and XopQ. Indeed, the authors demonstrate that the loss of avrBsT alone explains Xp pathogenicity on pepper while the double loss of avrBsT and xopQ renders Xp pathogenic on Nicotiana benthamiana. This result illustrates how plant immunity might restrict host range by ETI activation. The manuscript by Schwartz and colleagues thus illustrates the potential of large-scale pathogenomics in understanding pathogen evolution and disease emergence and offers perspectives to design durable resistance against bacterial spot in tomato and pepper. Such approaches would be further reinforced by an improved sampling design, an increased number of sequenced genomes, and the use of complete genomes now accessible by SMRT sequencing (Pacific Biosciences). Such bioinformatics analyses should now be extended to pan genomes, plasmids, InDels, SNPs, recombinations, and use the pioneering approach developed by Guttman and colleagues (McCann et al., 2012) which followed evolutionary forces applied to individual P. syringae genes resulting in the identification of several novel PAMP elicitors.

International audience; How pathogens coevolve with and adapt to their hosts are critical to understanding how host jumps and/or acquisition of novel traits can lead to new disease emergences. The Xanthomonas genus includes Gram-negative plant-pathogenic bacteria that collectively infect a broad range of crops and wild plant species. However, individual Xanthomonas strains usually cause disease on only a few plant species and are highly adapted to their hosts, making them pertinent models to study host specificity. This review summarizes our current understanding of the molecular basis of host specificity in the Xanthomonas genus, with a particular focus on the ecology, physiology, and pathogenicity of the bacterium. Despite our limited understanding of the basis of host specificity, type III effectors, microbe-associated molecular patterns, lipopolysaccharides, transcriptional regulators, and chemotactic sensors emerge as key determinants for shaping host specificity.

Two-component systems [sensor kinase/response regulator (RR)] are major tools used by microorganisms to adapt to environmental conditions. RR phosphorylation is typically required for gene activation, but few studies have addressed how and if phosphorylation affects specific steps during transcription initiation. We characterized transcription complexes made with RNA polymerase and the Bordetella pertussis RR, BvgA, in its nonphosphorylated or phosphorylated (BvgA∼P) state at P(fim3), the promoter for the virulence gene fim3 (fimbrial subunit), using gel retardation, potassium permanganate and DNase I footprinting, cleavage reactions with protein conjugated with iron bromoacetamidobenzyl-EDTA, and in vitro transcription. Previous work has shown that the level of nonphosphorylated BvgA remains high in vivo under conditions in which BvgA is phosphorylated. Our results here indicate that surprisingly both BvgA and BvgA∼P form open and initiating complexes with RNA polymerase at P(fim3). However, phosphorylation of BvgA is needed to generate the correct conformation that can transition to competent elongation. Footprints obtained with the complexes made with nonphosphorylated BvgA are atypical; while the initiating complex with BvgA synthesizes short RNA, it does not generate full-length transcripts. Extended incubation of the BvgA/RNA polymerase initiated complex in the presence of heparin generates a stable, but defective species that depends on the initial transcribed sequence of fim3. We suggest that the presence of nonphosphorylated BvgA down-regulates P(fim3) activity when phosphorylated BvgA is present and may allow the bacterium to quickly adapt to the loss of inducing conditions by rapidly eliminating P(fim3) activation once the signal for BvgA phosphorylation is removed.

Transcription of the Escherichia coli osmB gene is induced by several stress conditions. osmB is expressed from two promoters, osmBp1 and osmBp2 . The downstream promoter, osmBp2 , is induced after osmotic shock or upon entry into stationary phase in a σ S -dependent manner. The upstream promoter, osmBp1 , is independent of σ S and is activated by RcsB, the response regulator of the His-Asp phosphorelay signal transduction system RcsCDB. RcsB is responsible for the induction of osmBp1 following treatment with chlorpromazine. Activation of osmBp1 by RcsB requires a sequence upstream of its −35 element similar to the RcsB binding site consensus, suggesting a direct regulatory role. osmB appears as another example of a multistress-responsive gene whose transcription involves both a σ S -dependent promoter and a second one independent of σ S but controlled by stress-specific transcription factors.

The Bvg-regulated promoters for the fimbrial subunit genes fim2 and fim3 of Bordetella pertussis behave differently from each other both in vivo and in vitro. In vivo Pfim2 is significantly stronger than Pfim3 , even though predictions based on the DNA sequences of BvgA-binding motifs and core promoter elements would indicate the opposite. In vitro Pfim3 demonstrated robust BvgA∼P-dependent transcriptional activation, while none was seen with Pfim2 . This apparent contradiction was investigated further. By swapping sequence elements we created a number of hybrid promoters and assayed their strength in vivo. We found that, while Pfim3 promoter elements upstream of the +1 transcriptional start site do indeed direct Bvg-activated transcription more efficiently than those of Pfim2 , the overall promoter strength of Pfim3 in vivo is reduced due to sequences downstream of +1 that inhibit transcription more than 250-fold. This element, the DRE (downstream repressive element), was mapped to the 15 bp immediately downstream of the Pfim3 +1. Placing the DRE in different promoter contexts indicated that its activity was not specific to fim promoters, or even to Bvg-regulated promoters. However it does appear to be specific to Bordetella species in that it did not function in Escherichia coli.

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.

Bacteria often encounter diverse and rapidly changing environments. To overcome harmful situations, they must be capable of sensing external changes and transmitting this information across biological membranes into the cell, which results in the appropriate redirection of gene expression to prevent or repair cellular damages caused by stress. Extracytoplasmic function (ECF) σ factors provide one common means of bacterial signal transduction to regulate gene expression in response to various extracellular changes (65). ECF σ factors represent the largest and most diverse subfamily of σ70 proteins. They generally recognize a −35 box with a clear bias toward a GAAC in their target promoters, while the −10 region tends to be highly variable between ECF subfamily members (65). One of the best-studied ECF σ factors is the key regulator of the extracytoplasmic stress response factor σE from Escherichia coli, encoded by the rpoE gene (56). ECF proteins were recently divided into 43 major phylogenetically distinct groups named ECF01 to ECF43 (65). RpoE-like ECF σ factors are part of one predominant subgroup found in most bacterial phyla and comprise ECF01 to -04 proteins. RpoE-like ECF σ factors are autoregulated and are required for a wide range of functions. For instance, the E. coli σE factor is essential for growth and promotes the expression of factors that help to preserve and/or restore cell envelope integrity (2). Salmonella enterica serovar Typhimurium σE is required for protection against reactive oxygen species and antimicrobial peptides and for stationary-phase survival (20, 67). Bacillus subtilis σW seems to constitute an antibiosis regulon acting against cell envelope stress (34). S. Typhimurium σE, Pseudomonas aeruginosa AlgU, and Vibrio cholerae σE are required for virulence (5). ECFs can thus be considered models to understand how bacteria sense and respond to their environment both during their interaction with their host and in their free-living state. RpoE-like ECF σ factors are tightly regulated in order to coordinate their activation with the appropriate environmental cues. In most cases, the σE factor is cotranscribed with a cognate transmembrane anti-σ factor possessing an extracytoplasmic domain and an intracellular σ-binding domain. In the absence of stimulus, the membrane-bound anti-σ binds tightly to the σ factor, thereby keeping it inactive (33). Upon receiving a proper signal, the anti-σ factor is inactivated by regulated intramembrane proteolysis (RIP), resulting in the release and subsequent activation of the σE factor. This mechanism has been well studied for the anti-σ factors RseA, MucA, and RsiW, regulating the activity of E. coli σE, P. aeruginosa AlgU, or B. subtilis σW, respectively (1, 32, 75). In E. coli, the accumulation of C-terminal domains of unfolded porins is the activating signal of the RpoE response by triggering the activation of the inner-membrane-anchored protease, DegS (site 1 protease), and the subsequent cleavage of RseA within its periplasmic domain by DegS. The resulting truncated anti-σ factor is then a suitable substrate for a second inner-membrane protease, RseP/YaeL (site 2 protease), which cleaves RseA near the cytoplasmic face of the inner membrane, releasing an RseAcyto-σE complex into the cytoplasm, where the remaining RseA fragment is degraded by cytoplasmic proteases, resulting in the active σE (1). Another important mediator of the extracytoplasmic stress response is the periplasmic protease DegP, also known as HtrA and DO in E. coli or MucD in P. aeruginosa (22, 55). DegP binds to and degrades misfolded proteins and acts as a chaperone to direct the proper folding of some envelope proteins (66). As such, this family of proteases regulates the σE stress response system by removing misfolded proteins in the periplasm that could activate the degradation pathway of the anti-σE factor, even in the absence of stress (27). The Gram-negative phytopathogenic bacterium Xanthomonas campestris pv. campestris is an epiphytic bacterium that can become a vascular pathogen, causing black rot disease of crucifers (52). The bacterium produces a large amount of extracellular polysaccharide (EPS) that plays an important role during bacterial infection, and X. campestris pv. campestris has been used as a model organism for investigating the mechanism of bacterial pathogenesis. X. campestris pv. campestris flourishes in and adapts to a wide range of habitats: during epiphytic life, X. campestris pv. campestris is exposed to harsh stresses, such as oligotrophic conditions, desiccation, or large changes in temperature. Upon entry into plant tissues, X. campestris pv. campestris cells must face defense reactions of the host, including oxidative conditions. Finally, the natural life cycle of X. campestris pv. campestris includes long periods of survival on seeds or plant scraps or in the soil, where again it must survive a variety of stressful conditions before it can infect a new host plant. Its ability to manage variable and often lethal external conditions can be partly attributed to its large repertoire of alternative σ factors. Of the 4,179 open reading frames (ORFs) comprising the large 5.1-Mb X. campestris pv. campestris strain ATCC 33913 genome, 15 ORFs encode characterized or putative σ factors, 10 of which belong to the ECF subfamily (23). Little is known about which σ factors are required for the survival of X. campestris pv. campestris under stress and the contribution of these factors to virulence. The classification of ECF σ factors strongly suggested that the XCC1267 gene encodes the σE factor of X. campestris pv. campestris (65). Moreover, previous work by Cheng et al. (17) described the biochemical characterization of the σE factor of X. campestris pv. campestris strain 11 and suggested that it could have a role in the heat shock response. Therefore, we aimed at deciphering the roles and regulation mechanisms of the extracytoplasmic stress response regulator σE in X. campestris pv. campestris. In the present work we characterized the rpoE operon genes, rpoE, rseA, and mucD. Using primer extension and lacZ transcriptional reporter fusions, we show that rpoE transcription is autoregulated and that RseA and MucD are negative regulators of σE activity. We identified 45 putative members of the σE regulon by a transcriptome analysis, including the heat stress σ factor σH and a number of periplasmic or membrane proteins. We provided evidence that σE is an important regulator of stress responses in X. campestris pv. campestris, since it has a role in heat adaptation, resistance to cadmium, and stationary-phase survival. Furthermore, our results strongly suggest that σE is regulated by a RIP mechanism involving RseP (XCC1366) and DegS (XCC3898) putative proteases, as in many other bacteria. However, our data suggest that the RIP proteases RseP and DegS are not only dedicated to RseA cleavage and that the proteolytic cascade of RseA could involve other proteases.

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 c arbohydrate u tilization loci containing T onB-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.

TonB-dependent receptors (TBDRs) are outer membrane proteins mainly known for the active transport of iron siderophore complexes in Gram-negative bacteria. Analysis of the genome of the phytopathogenic bacterium Xanthomonas campestris pv. campestris (Xcc), predicts 72 TBDRs. Such an overrepresentation is common in Xanthomonas species but is limited to only a small number of bacteria. Here, we show that one Xcc TBDR transports sucrose with a very high affinity, suggesting that it might be a sucrose scavenger. This TBDR acts with an inner membrane transporter, an amylosucrase and a regulator to utilize sucrose, thus defining a new type of carbohydrate utilization locus, named CUT locus, involving a TBDR for the transport of substrate across the outer membrane. This sucrose CUT locus is required for full pathogenicity on Arabidopsis, showing its importance for the adaptation to host plants. A systematic analysis of Xcc TBDR genes and a genome context survey suggested that several Xcc TBDRs belong to other CUT loci involved in the utilization of various plant carbohydrates. Interestingly, several Xcc TBDRs and CUT loci are conserved in aquatic bacteria such as Caulobacter crescentus, Colwellia psychrerythraea, Saccharophagus degradans, Shewanella spp., Sphingomonas spp. or Pseudoalteromonas spp., which share the ability to degrade a wide variety of complex carbohydrates and display TBDR overrepresentation. We therefore propose that TBDR overrepresentation and the presence of CUT loci designate the ability to scavenge carbohydrates. Thus CUT loci, which seem to participate to the adaptation of phytopathogenic bacteria to their host plants, might also play a very important role in the biogeochemical cycling of plant-derived nutrients in marine environments. Moreover, the TBDRs and CUT loci identified in this study are clearly different from those characterized in the human gut symbiont Bacteroides thetaiotaomicron, which allow glycan foraging, suggesting a convergent evolution of TBDRs in Proteobacteria and Bacteroidetes.