Arabidopsis thaliana Early Foliar Proteome Response to Root Exposure to the Rhizobacterium Pseudomonas simiae WCS417.

Pseudomonas simiae WCS417 is a plant growth-promoting rhizobacterium that improves plant health and development. In this study, we investigate the early leaf responses of Arabidopsis thaliana to WCS417 exposure and the possible involvement of formate dehydrogenase (FDH) in such responses. In vitro-grown A. thaliana seedlings expressing an FDH::GUS reporter show a significant increase in FDH promoter activity in their roots and shoots after 7 days of indirect exposure (without contact) to WCS417. After root exposure to WCS417, the leaves of FDH::GUS plants grown in the soil also show an increased FDH promoter activity in hydathodes. To elucidate early foliar responses to WCS417 as well as FDH involvement, the roots of A. thaliana wild-type Col and atfdh1-5 knock-out mutant plants grown in soil were exposed to WCS417, and proteins from rosette leaves were subjected to proteomic analysis. The results reveal that chloroplasts, in particular several components of the photosystems PSI and PSII, as well as members of the glutathione S-transferase family, are among the early targets of the metabolic changes induced by WCS417. Taken together, the alterations in the foliar proteome, as observed in the atfdh1-5 mutant, especially after exposure to WCS417 and involving stress-responsive genes, suggest that FDH is a node in the early events triggered by the interactions between A. thaliana and the rhizobacterium WCS417. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Cruciferous Weed Isolates of Xanthomonas campestris Yield Insight into Pathovar Genomic Relationships and Genetic Determinants of Host and Tissue Specificity.

Pathovars of Xanthomonas campestris cause distinct diseases on different brassicaceous hosts. The genomic relationships among pathovars as well as the genetic determinants of host range and tissue specificity remain poorly understood despite decades of research. Here, leveraging advances in multiplexed long-read technology, we fully sequenced the genomes of a collection of X. campestris strains isolated from cruciferous crops and weeds in New York and California as well as strains from global collections, to investigate pathovar relationships and candidate genes for host- and tissue-specificity. Pathogenicity assays and genomic comparisons across this collection and publicly available X. campestris genomes revealed a correlation between pathovar and genomic relatedness and provide support for X. campestris pv. barbareae, the validity of which had been questioned. Linking strain host range with type III effector repertoires identified AvrAC (also 'XopAC') as a candidate host-range determinant, preventing infection of Matthiola incana, and this was confirmed experimentally. Furthermore, the presence of a copy of the cellobiosidase gene cbsA with coding sequence for a signal peptide was found to correlate with the ability to infect vascular tissues, in agreement with a previous study of diverse Xanthomonas species; however, heterologous expression in strains lacking the gene gave mixed results, indicating that factors in addition to cbsA influence tissue specificity of X. campestris pathovars. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Genome-wide identification of fitness determinants in the Xanthomonas campestris bacterial pathogen during early stages of plant infection.

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.

The Same against Many: AtCML8, a Ca2+ Sensor Acting as a Positive Regulator of Defense Responses against Several Plant Pathogens.

Calcium signals are crucial for the activation and coordination of signaling cascades leading to the establishment of plant defense mechanisms. Here, we studied the contribution of CML8, an Arabidopsis calmodulin-like protein in response to Ralstonia solanacearum and to pathogens with different lifestyles, such as Xanthomonas campestris pv. campestris and Phytophtora capsici. We used pathogenic infection assays, gene expression, RNA-seq approaches, and comparative analysis of public data on CML8 knockdown and overexpressing Arabidopsis lines to demonstrate that CML8 contributes to defense mechanisms against pathogenic bacteria and oomycetes. CML8 gene expression is finely regulated at the root level and manipulated during infection with Ralstonia, and CML8 overexpression confers better plant tolerance. To understand the processes controlled by CML8, genes differentially expressed at the root level in the first hours of infection have been identified. Overexpression of CML8 also confers better tolerance against Xanthomonas and Phytophtora, and most of the genes differentially expressed in response to Ralstonia are differentially expressed in these different pathosystems. Collectively, CML8 acts as a positive regulator against Ralstonia solanaceraum and against other vascular or root pathogens, suggesting that CML8 is a multifunctional protein that regulates common downstream processes involved in the defense response of plants to several pathogens.

Two ancestral genes shaped the Xanthomonas campestris TAL effector gene repertoire.

Xanthomonas transcription activator-like effectors (TALEs) are injected inside plant cells to promote host susceptibility by enhancing transcription of host susceptibility genes. TALE-encoding (tal) genes were thought to be absent from Brassicaceae-infecting Xanthomonas campestris (Xc) genomes based on four reference genomic sequences. We discovered tal genes in 26 of 49 Xc strains isolated worldwide and used a combination of single molecule real time (SMRT) and tal amplicon sequencing to yield a near-complete description of the TALEs found in Xc (Xc TALome). The 53 sequenced tal genes encode 21 distinct DNA binding domains that sort into seven major DNA binding specificities. In silico analysis of the Brassica rapa promoterome identified a repertoire of predicted TALE targets, five of which were experimentally validated using quantitative reverse transcription polymerase chain reaction. The Xc TALome shows multiple signs of DNA rearrangements that probably drove its evolution from two ancestral tal genes. We discovered that Tal12a and Tal15a of Xcc strain Xca5 contribute together in the development of disease symptoms on susceptible B. oleracea var. botrytis cv Clovis. This large and polymorphic repertoire of TALEs opens novel perspectives for elucidating TALE-mediated susceptibility of Brassicaceae to black rot disease and for understanding the molecular processes underlying TALE evolution.

Immunity at Cauliflower Hydathodes Controls Systemic Infection by Xanthomonas campestris pv campestris.

Hydathodes are water pores found on leaves of a wide range of vascular plants and are the sites of guttation. We report here on the detailed anatomy of cauliflower (Brassicaoleracea) and Arabidopsis (Arabidopsis thaliana) hydathodes. Hydathode surface presents pores resembling stomata giving access to large cavities. Beneath, the epithem is composed of a lacunar and highly vascularized parenchyma offering a direct connection between leaf surface and xylem vessels. Arabidopsis hydathode pores were responsive to ABA and light similar to stomata. The flg22 flagellin peptide, a well-characterized elicitor of plant basal immunity, did not induce closure of hydathode pores in contrast to stomata. Because hydathodes are natural infection routes for several pathogens, we investigated hydathode infection by the adapted vascular phytopathogenic bacterium Xanthomonas campestris pv campestris (Xcc), the causal agent of black rot disease of Brassicaceae. Microscopic observations of hydathodes six days postinoculation indicated a digestion of the epithem cells and a high bacterial multiplication. Postinvasive immunity was shown to limit pathogen growth in the epithem and is actively suppressed by the type III secretion system and its effector proteins. Altogether, these results give a detailed anatomic description of Brassicaceae hydathodes and highlight the efficient use of this tissue as an initial niche for subsequent vascular systemic dissemination of Xcc in distant plant tissues.

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 ß-xylosidase NixI (GH3), which is involved in the cleavage of the ß-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.

Genomics and transcriptomics of Xanthomonas campestris species challenge the concept of core type III effectome.

BACKGROUND: The bacterial species Xanthomonas campestris infects a wide range of Brassicaceae. Specific pathovars of this species cause black rot (pv. campestris), bacterial blight of stock (pv. incanae) or bacterial leaf spot (pv. raphani). RESULTS: In this study, we extended the genomic coverage of the species by sequencing and annotating the genomes of strains from pathovar incanae (CFBP 1606R and CFBP 2527R), pathovar raphani (CFBP 5828R) and a pathovar formerly named barbareae (CFBP 5825R). While comparative analyses identified a large core ORFeome at the species level, the core type III effectome was limited to only three putative type III effectors (XopP, XopF1 and XopAL1). In Xanthomonas, these effector proteins are injected inside the plant cells by the type III secretion system and contribute collectively to virulence. A deep and strand-specific RNA sequencing strategy was adopted in order to experimentally refine genome annotation for strain CFBP 5828R. This approach also allowed the experimental definition of novel ORFs and non-coding RNA transcripts. Using a constitutively active allele of hrpG, a master regulator of the type III secretion system, a HrpG-dependent regulon of 141 genes co-regulated with the type III secretion system was identified. Importantly, all these genes but seven are positively regulated by HrpG and 56 of those encode components of the Hrp type III secretion system and putative effector proteins. CONCLUSIONS: This dataset is an important resource to mine for novel type III effector proteins as well as for bacterial genes which could contribute to pathogenicity of X. campestris.

Bacterial host adaptation through sequence and structural variations of a single type III effector gene.

Molecular mechanisms underlying quantitative variations of pathogenicity remain elusive. Here, we identified the Xanthomonas campestris XopJ6 effector that triggers disease resistance in cauliflower and Arabidopsis thaliana. XopJ6 is a close homolog of the Ralstoniapseudosolanacearum PopP2 YopJ family acetyltransferase. XopJ6 is recognized by the RRS1-R/RPS4 NLR pair that integrates a WRKY decoy domain mimicking effector targets. We identified a XopJ6 natural variant carrying a single residue substitution in XopJ6 WRKY-binding site that disrupts interaction with WRKY proteins. This mutation allows XopJ6 to evade immune perception while retaining some XopJ6 virulence functions. Interestingly, xopJ6 resides in a Tn3-family transposon likely contributing to xopJ6 copy number variation (CNV). Using synthetic biology, we demonstrate that xopJ6 CNV tunes pathogen virulence on Arabidopsis through gene dosage-mediated modulation of xopJ6 expression. Together, our findings highlight how sequence and structural genetic variations restricted at a particular effector gene contribute to bacterial host adaptation.

Genome sequence of Xanthomonas fuscans subsp. fuscans strain 4834-R reveals that flagellar motility is not a general feature of xanthomonads.

BACKGROUND: Xanthomonads are plant-associated bacteria responsible for diseases on economically important crops. Xanthomonas fuscans subsp. fuscans (Xff) is one of the causal agents of common bacterial blight of bean. In this study, the complete genome sequence of strain Xff 4834-R was determined and compared to other Xanthomonas genome sequences. RESULTS: Comparative genomics analyses revealed core characteristics shared between Xff 4834-R and other xanthomonads including chemotaxis elements, two-component systems, TonB-dependent transporters, secretion systems (from T1SS to T6SS) and multiple effectors. For instance a repertoire of 29 Type 3 Effectors (T3Es) with two Transcription Activator-Like Effectors was predicted. Mobile elements were associated with major modifications in the genome structure and gene content in comparison to other Xanthomonas genomes. Notably, a deletion of 33 kbp affects flagellum biosynthesis in Xff 4834-R. The presence of a complete flagellar cluster was assessed in a collection of more than 300 strains representing different species and pathovars of Xanthomonas. Five percent of the tested strains presented a deletion in the flagellar cluster and were non-motile. Moreover, half of the Xff strains isolated from the same epidemic than 4834-R was non-motile and this ratio was conserved in the strains colonizing the next bean seed generations. CONCLUSIONS: This work describes the first genome of a Xanthomonas strain pathogenic on bean and reports the existence of non-motile xanthomonads belonging to different species and pathovars. Isolation of such Xff variants from a natural epidemic may suggest that flagellar motility is not a key function for in planta fitness.

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.

Genomic insights into strategies used by Xanthomonas albilineans with its reduced artillery to spread within sugarcane xylem vessels.

BACKGROUND: Xanthomonas albilineans causes leaf scald, a lethal disease of sugarcane. X. albilineans exhibits distinctive pathogenic mechanisms, ecology and taxonomy compared to other species of Xanthomonas. For example, this species produces a potent DNA gyrase inhibitor called albicidin that is largely responsible for inducing disease symptoms; its habitat is limited to xylem; and the species exhibits large variability. A first manuscript on the complete genome sequence of the highly pathogenic X. albilineans strain GPE PC73 focused exclusively on distinctive genomic features shared with Xylella fastidiosa-another xylem-limited Xanthomonadaceae. The present manuscript on the same genome sequence aims to describe all other pathogenicity-related genomic features of X. albilineans, and to compare, using suppression subtractive hybridization (SSH), genomic features of two strains differing in pathogenicity. RESULTS: Comparative genomic analyses showed that most of the known pathogenicity factors from other Xanthomonas species are conserved in X. albilineans, with the notable absence of two major determinants of the "artillery" of other plant pathogenic species of Xanthomonas: the xanthan gum biosynthesis gene cluster, and the type III secretion system Hrp (hypersensitive response and pathogenicity). Genomic features specific to X. albilineans that may contribute to specific adaptation of this pathogen to sugarcane xylem vessels were also revealed. SSH experiments led to the identification of 20 genes common to three highly pathogenic strains but missing in a less pathogenic strain. These 20 genes, which include four ABC transporter genes, a methyl-accepting chemotaxis protein gene and an oxidoreductase gene, could play a key role in pathogenicity. With the exception of hypothetical proteins revealed by our comparative genomic analyses and SSH experiments, no genes potentially involved in any offensive or counter-defensive mechanism specific to X. albilineans were identified, supposing that X. albilineans has a reduced artillery compared to other pathogenic Xanthomonas species. Particular attention has therefore been given to genomic features specific to X. albilineans making it more capable of evading sugarcane surveillance systems or resisting sugarcane defense systems. CONCLUSIONS: This study confirms that X. albilineans is a highly distinctive species within the genus Xanthomonas, and opens new perpectives towards a greater understanding of the pathogenicity of this destructive sugarcane pathogen.

A ß-glucuronidase (GUS) Based Bacterial Competition Assay to Assess Fine Differencesin Fitness during Plant Infection.

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.

Xanthomonas transcriptome inside cauliflower hydathodes reveals bacterial virulence strategies and physiological adaptations at early infection stages.

Xanthomonas campestris pv. campestris (Xcc) is a seed-transmitted vascular pathogen causing black rot disease on cultivated and wild Brassicaceae. Xcc enters the plant tissues preferentially via hydathodes, which are organs localized at leaf margins. To decipher both physiological and virulence strategies deployed by Xcc during early stages of infection, the transcriptomic profile of Xcc was analysed 3 days after entry into cauliflower hydathodes. Despite the absence of visible plant tissue alterations and despite a biotrophic lifestyle, 18% of Xcc genes were differentially expressed, including a striking repression of chemotaxis and motility functions. The Xcc full repertoire of virulence factors had not yet been activated but the expression of the HrpG regulon composed of 95 genes, including genes coding for the type III secretion machinery important for suppression of plant immunity, was induced. The expression of genes involved in metabolic adaptations such as catabolism of plant compounds, transport functions, sulphur and phosphate metabolism was upregulated while limited stress responses were observed 3 days postinfection. We confirmed experimentally that high-affinity phosphate transport is needed for bacterial fitness inside hydathodes. This analysis provides information about the nutritional and stress status of bacteria during the early biotrophic infection stages and helps to decipher the adaptive strategy of Xcc to the hydathode environment.

Insights into the extracytoplasmic stress response of Xanthomonas campestris pv. campestris: role and regulation of {sigma}E-dependent activity.

Xanthomonas campestris pv. campestris is an epiphytic bacterium that can become a vascular pathogen responsible for black rot disease of crucifers. To adapt gene expression in response to ever-changing habitats, phytopathogenic bacteria have evolved signal transduction regulatory pathways, such as extracytoplasmic function (ECF) σ factors. The alternative sigma factor σ(E), encoded by rpoE, is crucial for envelope stress response and plays a role in the pathogenicity of many bacterial species. Here, we combine different approaches to investigate the role and mechanism of σ(E)-dependent activation in X. campestris pv. campestris. We show that the rpoE gene is organized as a single transcription unit with the anti-σ gene rseA and the protease gene mucD and that rpoE transcription is autoregulated. rseA and mucD transcription is also controlled by a highly conserved σ(E)-dependent promoter within the σ(E) gene sequence. The σ(E)-mediated stress response is required for stationary-phase survival, resistance to cadmium, and adaptation to membrane-perturbing stresses (elevated temperature and ethanol). Using microarray technology, we started to define the σ(E) regulon of X. campestris pv. campestris. These genes encode proteins belonging to different classes, including periplasmic or membrane proteins, biosynthetic enzymes, classical heat shock proteins, and the heat stress σ factor σ(H). The consensus sequence for the predicted σ(E)-regulated promoter elements is GGAACTN(15-17)GTCNNA. Determination of the rpoH transcription start site revealed that rpoH was directly regulated by σ(E) under both normal and heat stress conditions. Finally, σ(E) activity is regulated by the putative regulated intramembrane proteolysis (RIP) proteases RseP and DegS, as previously described in many other bacteria. However, our data suggest that RseP and DegS are not only dedicated to RseA cleavage and that the proteolytic cascade of RseA could involve other proteases.

Analysis of the xylem sap proteome of Brassica oleracea reveals a high content in secreted proteins.

Xylem plays a major role in plant development and is considered part of the apoplast. Here, we studied the proteome of Brassica oleracea cv Bartolo and compared it to the plant cell wall proteome of another Brassicaceae, the model plant Arabidopsis thaliana. B. oleracea was chosen because it is technically difficult to harvest enough A. thaliana xylem sap for proteomic analysis. We studied the whole proteome and an N-glycoproteome obtained after Concanavalin A affinity chromatography. Altogether, 189 proteins were identified by LC-MS/MS using Brassica EST and cDNA sequences. A predicted signal peptide was found in 164 proteins suggesting that most proteins of the xylem sap are secreted. Eighty-one proteins were identified in the N-glycoproteome, with 25 of them specific of this fraction, suggesting that they were concentrated during the chromatography step. All the protein families identified in this study were found in the cell wall proteomes. However, proteases and oxido-reductases were more numerous in the xylem sap proteome, whereas enzyme inhibitors were rare. The origin of xylem sap proteins is discussed. All the experimental data including the MS/MS data were made available in the WallProtDB cell wall proteomic database.

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 complete genome sequence of Xanthomonas albilineans provides new insights into the reductive genome evolution of the xylem-limited Xanthomonadaceae.

BACKGROUND: The Xanthomonadaceae family contains two xylem-limited plant pathogenic bacterial species, Xanthomonas albilineans and Xylella fastidiosa. X. fastidiosa was the first completely sequenced plant pathogen. It is insect-vectored, has a reduced genome and does not possess hrp genes which encode a Type III secretion system found in most plant pathogenic bacteria. X. fastidiosa was excluded from the Xanthomonas group based on phylogenetic analyses with rRNA sequences. RESULTS: The complete genome of X. albilineans was sequenced and annotated. X. albilineans, which is not known to be insect-vectored, also has a reduced genome and does not possess hrp genes. Phylogenetic analysis using X. albilineans genomic sequences showed that X. fastidiosa belongs to the Xanthomonas group. Order of divergence of the Xanthomonadaceae revealed that X. albilineans and X. fastidiosa experienced a convergent reductive genome evolution during their descent from the progenitor of the Xanthomonas genus. Reductive genome evolutions of the two xylem-limited Xanthomonadaceae were compared in light of their genome characteristics and those of obligate animal symbionts and pathogens. CONCLUSION: The two xylem-limited Xanthomonadaceae, during their descent from a common ancestral parent, experienced a convergent reductive genome evolution. Adaptation to the nutrient-poor xylem elements and to the cloistered environmental niche of xylem vessels probably favoured this convergent evolution. However, genome characteristics of X. albilineans differ from those of X. fastidiosa and obligate animal symbionts and pathogens, indicating that a distinctive process was responsible for the reductive genome evolution in this pathogen. The possible role in genome reduction of the unique toxin albicidin, produced by X. albilineans, is discussed.

Use of an in-house approach to study the three-dimensional structures of various outer membrane proteins: structure of the alcaligin outer membrane transporter FauA from Bordetella pertussis.

Bordetella pertussis is the bacterial agent of whooping cough in humans. Under iron-limiting conditions, it produces the siderophore alcaligin. Released to the extracellular environment, alcaligin chelates iron, which is then taken up as a ferric alcaligin complex via the FauA outer membrane transporter. FauA belongs to a family of TonB-dependent outer membrane transporters that function using energy derived from the proton motive force. Using an in-house protocol for membrane-protein expression, purification and crystallization, FauA was crystallized in its apo form together with three other TonB-dependent transporters from different organisms. Here, the protocol used to study FauA is described and its three-dimensional structure determined at 2.3 A resolution is discussed.