Non-gamma-proteobacteria gene islands contribute to the Xanthomonas genome

Horizontal gene transfer, a process through which genomes acquire sequences from distantly related organisms, is believed to be a major source of genetic diversity in bacteria. A central question concerning the impact of gene transfer on bacterial genome evolution is the proportion of horizontally transferred sequences within genomes. Through BLAST search, we found that the genomes of two phytopathogens, Xanthomonas campestris pv. campestris and Xanthomonas axonopodis pv. citri, have close to 40% of the genes with the highest similarity to genes from phylogenetically distant organisms (non-gamma-proteobacteria). Most of these genes are found to be contiguous in the genome, forming genome islands, which may have been transferred from other organisms. Overall, the total number of genes within genome islands corresponds to almost one quarter of the entire xanthomonad genomes. Interestingly, many of the genes in these islands are functionally related to plant pathogenesis and virulence. Thus, these results suggest that horizontally transferred genes are clustered in the genome, and may facilitate fitness in new environments, as in the case of plant-bacteria interaction.     

Replacement of the Arginine Biosynthesis Operon in Xanthomonadales by Lateral Gene Transfer

The role of lateral gene transfer (LGT) in prokaryotes has been shown to rapidly change the genome content, providing new gene tools for environmental adaptation. Features related to pathogenesis and resistance to strong selective conditions have been widely shown to be products of gene transfer between bacteria. The genomes of the γ-proteobacteria from the genus Xanthomonas, composed mainly of phytopathogens, have potential genomic islands that may represent imprints of such evolutionary processes. In this work, the evolution of genes involved in the pathway responsible for arginine biosynthesis in Xanthomonadales was investigated, and several lines of evidence point to the foreign origin of the arg genes clustered within a potential operon. Their presence inside a potential genomic island, bordered by a tRNA gene, the unusual ranking of sequence similarity, and the atypical phylogenies indicate that the metabolic pathway for arginine biosynthesis was acquired through LGT in the Xanthomonadales group. Moreover, although homologues were also found in Bacteroidetes (Flavobacteria group), for many of the genes analyzed close homologues are detected in different life domains (Eukarya and Archaea), indicating that the source of these arg genes may have been outside the Bacteria clade. The possibility of replacement of a complete primary metabolic pathway by LGT events supports the selfish operon hypothesis and may occur only under very special environmental conditions. Such rare events reveal part of the history of these interesting mosaic Xanthomonadales genomes, disclosing the importance of gene transfer modifying primary metabolism pathways and extending the scenario for bacterial genome evolution.     

Insights into genome plasticity and pathogenicity of the plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria revealed by the complete genome sequence

The gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria is the causative agent of bacterial spot disease in pepper and tomato plants, which leads to economically important yield losses. This pathosystem has become a well-established model for studying bacterial infection strategies. Here, we present the whole-genome sequence of the pepper-pathogenic Xanthomonas campestris pv. vesicatoria strain 85-10, which comprises a 5.17-Mb circular chromosome and four plasmids. The genome has a high G+C content (64.75%) and signatures of extensive genome plasticity. Whole-genome comparisons revealed a gene order similar to both Xanthomonas axonopodis pv. citri and Xanthomonas campestris pv. campestris and a structure completely different from Xanthomonas oryzae pv. oryzae. A total of 548 coding sequences (12.2%) are unique to X. campestris pv. vesicatoria. In addition to a type III secretion system, which is essential for pathogenicity, the genome of strain 85-10 encodes all other types of protein secretion systems described so far in gram-negative bacteria. Remarkably, one of the putative type IV secretion systems encoded on the largest plasmid is similar to the Icm/Dot systems of the human pathogens Legionella pneumophila and Coxiella burnetii. Comparisons with other completely sequenced plant pathogens predicted six novel type III effector proteins and several other virulence factors, including adhesins, cell wall-degrading enzymes, and extracellular polysaccharides.     

Identification of Xanthomonas campestris pv. campestris galactose utilization genes from transcriptome data

A 70 mer oligonucleotide microarray was constructed to analyze genome-wide expression profiles of Xanthomonas campestris pv. campestris B100, a plant-pathogenic bacterium that is industrially employed to produce the exopolysaccharide xanthan gum which has many applications as a stabilizing, thickening, gelling, and emulsifying agent in food, pharmaceutical, and cosmetic industries. As an application example, global changes of gene expression were monitored during growth of X. campestris pv. campestris B100 on two different carbon sources. Exponential growing bacterial cultures were incubated either for 1h or permanently in minimal medium supplemented with 1% galactose in comparison to growth in minimal medium supplemented with 1% glucose. Six genes were identified that were significantly increased in gene expression under both growth conditions. These genes were located in three distinguished chromosomal regions in operon-like gene clusters. Genes from these clusters encode secreted glycosidases, which were predicted to be specific for galactose-containing carbohydrates, as well as transport proteins probably located in the outer and inner cell membrane. Finally genes from one cluster code for cytoplasmic enzymes of a metabolic pathway specific for the breakdown of galactose to intermediates of glycolysis.     

Two Xanthomonas extracellular polygalacturonases, PghAxc and PghBxc, are regulated by type III secretion regulators HrpX and HrpG and are required for virulence

Xanthomonas campestris pv. campestris, the causal agent of black rot disease, produces a suite of extracellular cell-wall degrading enzymes (CWDE) that are involved in bacterial virulence. Polygalacturonase (PG) is an important CWDE and functions to degrade the pectic layers of plant cell walls. Although previous studies have documented the virulence functions of PG in Erwinia and Ralstonia species, the regulation of PG genes still needs to be elucidated. In this study, we identified two novel PG genes (pghAxc and pghBxc) encoding functional PG from X. campestris pv. campestris 8004. The expressions of these two PG genes are regulated by the type III secretion regulators HrpX and HrpG and the global regulator Clp. These PG genes could be efficiently induced in planta and were required for the full virulence of X. campestris pv. campestris to Arabidopsis. In addition, these PG were confirmed to be secreted via the type II secretion system in an Xps-dependent manner.     

Genome-scale mutagenesis and phenotypic characterization of two-component signal transduction systems in Xanthomonas campestris pv. campestris ATCC 33913

The gram-negative bacterium Xanthomonas campestris pv. campestris is the causal agent of black rot disease of cruciferous plants. Its genome encodes a large repertoire of two-component signal transduction systems (TCSTSs), which consist of histidine kinases and response regulators (RR) to monitor and respond to environmental stimuli. To investigate the biological functions of these TCSTS genes, we aimed to inactivate all 54 RR genes in X. campestris pv. campestris ATCC 33913, and successfully generated 51 viable mutants using the insertion inactivation method. Plant inoculation identified two novel response regulator genes (XCC1958 and XCC3107) that are involved in virulence of this strain. Genetic complementation demonstrated that XCC3107, designated as vgrR (virulence and growth regulator), also affects bacterial growth and activity of extracellular proteases. In addition, we assessed the survival of these mutants under various stresses, including osmotic stress, high sodium concentration, heat shock, and sodium dodecyl sulfate exposure, and identified a number of genes that may be involved in the general stress response of X. campestris pv. campestris. Mutagenesis and phenotypic characterization of RR genes in this study will facilitate future studies on signaling networks in this important phytopathogenic bacterium.     

Genome-enabled determination of amino acid biosynthesis in Xanthomonas campestris pv. campestris and identification of biosynthetic pathways for alanine, glycine, and isoleucine by 13C-isotopologue profiling

To elucidate the biosynthetic pathways for all proteinogenic amino acids in Xanthomonas campestris pv. campestris, this study combines results obtained by in silico genome analysis and by (13)C-NMR-based isotopologue profiling to provide a panoramic view on a substantial section of bacterial metabolism. Initially, biosynthesis pathways were reconstructed from an improved annotation of the complete genome of X. campestris pv. campestris B100. This metabolic reconstruction resulted in the unequivocal identification of biosynthesis routes for 17 amino acids in total: arginine, asparagine, aspartate, cysteine, glutamate, glutamine, histidine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Ambiguous pathways were reconstructed from the genome data for alanine, glycine, and isoleucine biosynthesis. (13)C-NMR analyses supported the identification of the metabolically active pathways. The biosynthetic routes for these amino acids were derived from the precursor molecules pyruvate, serine, and pyruvate, respectively. By combining genome analysis and isotopologue profiling, a comprehensive set of biosynthetic pathways covering all proteinogenic amino acids was unraveled for this plant pathogenic bacterium, which plays an important role in biotechnology as a producer of the exopolysaccharide xanthan. The data obtained lay ground for subsequent functional analyses in post-genomics and biotechnology, while the innovative combination of in silico and wet lab technology described here is promising as a general approach to elucidate metabolic pathways.     

Mini-Tn7 vectors as genetic tools for gene cloning at a single copy number in an industrially important and phytopathogenic bacteria, Xanthomonas spp.

Transposon mini-Tn 7 vectors insert into the chromosome of several Gram-negative bacteria in a site-specific manner. Here, we showed the application of mini-Tn 7 as single copy site-specific integration vector system for Xanthomonas campestris pv. campestris . The transposition of the mini-Tn 7 into the bacterial genome was detected at a Tn 7 attachment ( att Tn 7 ) site located downstream of glmS1 . Furthermore, using a newly constructed vector pBBR1FLP2 containing the flipase (FLP) recombinase for site-specific excision of the sequence between the FLP recombinase target (FRT) sites, and a sacB counter selection marker, an unmarked mini-Tn 7 insertion mutant was created. Mini-Tn 7 insertion did not affect bacterial virulence on the tested plant. The mini-Tn 7 and FLP–FRT systems also work well in Xanthomonas oryzae pv. oryzae .     

Transaldolase exhibits a protective role against menadione toxicity in Xanthomonas campestris pv. phaseoli

A talA gene encoded transaldolase, a rate-limiting enzyme in the non-oxidative branch of the pentose-phosphate pathway, was cloned from Xanthomonas campestris pv. phaseoli. talA located in a region of the bacterial genome rich in genes involved in oxidative stress protection and regulation. TalA from X. campestris pv. phaseoli showed a high degree of homology to many previously reported transaldolases from both prokaryotic and eukaryotic sources. The expression of X. campestris pv. phaseoli talA was high at log-phase of growth, then declined at stationary phase, and could not be induced by oxidants. A talA mutant constructed by insertional inactivation did not possess any detectable transaldolase activity. Lack of a functional talA gene did not affect bacterial growth in a rich medium containing glucose or sucrose as a carbon source. However, the talA knockout mutant showed increased sensitivity to the superoxide generator menadione, but not to other oxidants. This increased menadione sensitivity phenotype could be complemented by expression of talA in a plasmid vector. The data demonstrated a novel and essential role of transaldolase in protection against menadione toxicity in X. campestris.     

Characterization of stress-responsive genes, hrcA-grpE-dnaK-dnaJ, from phytopathogenic Xanthomonas campestris

Sequencing of a 6.4-kb DNA fragment, cloned from the plant pathogenic bacterium Xanthomonas campestris pv. campestris 17 revealed five ORFs whose deduced amino acid sequences show strong similarities to the bacterial HrcA, GrpE, DnaK, DnaJ, and PdxK. The four heat shock genes are organized in the order hrcA-grpE-dnaK-dnaJ, a genome organization found in many gram-positive bacteria, but only in one gram-negative species (Xylella fastidiosa). These observations suggest that the HrcA-CIRCE system, comprising at least four genes arranged in this order, already existed for the regulation of stress responses before bacteria diverged into gram-negative and gram-positive groups. Primer-extension results suggested the presence of promoters at the regions upstream of grpE and dnaK. In the presence of stress, heat or ethanol (4%), the X. campestris pv. campestris 17 grpE and dnaK promoters were induced two- to three-fold over controls. Since the grpE and dnaK promoters possess E. coli sigma(32) promoter-like sequences, they are functional in E. coli, although at levels much lower than in X. campestris pv. campestris 17. Furthermore, expression of the X. campestris pv. campestris 17 dnaK promoter in E. coli was elevated by the cloned X. campestris sigma(32) gene, indicating that the cognate sigma(32) works more efficiently for the X. campestris promoters.     

Comparative genomic analysis of Xanthomonas axonopodis pv. citrumelo F1, which causes citrus bacterial spot disease, and related strains provides insights into virulence and host specificity

Xanthomonas axonopodis pv. citrumelo is a citrus pathogen causing citrus bacterial spot disease that is geographically restricted within the state of Florida. Illumina, 454 sequencing, and optical mapping were used to obtain a complete genome sequence of X. axonopodis pv. citrumelo strain F1, 4.9 Mb in size. The strain lacks plasmids, in contrast to other citrus Xanthomonas pathogens. Phylogenetic analysis revealed that this pathogen is very close to the tomato bacterial spot pathogen X. campestris pv. vesicatoria 85-10, with a completely different host range. We also compared X. axonopodis pv. citrumelo to the genome of citrus canker pathogen X. axonopodis pv. citri 306. Comparative genomic analysis showed differences in several gene clusters, like those for type III effectors, the type IV secretion system, lipopolysaccharide synthesis, and others. In addition to pthA, effectors such as xopE3, xopAI, and hrpW were absent from X. axonopodis pv. citrumelo while present in X. axonopodis pv. citri. These effectors might be responsible for survival and the low virulence of this pathogen on citrus compared to that of X. axonopodis pv. citri. We also identified unique effectors in X. axonopodis pv. citrumelo that may be related to the different host range as compared to that of X. axonopodis pv. citri. X. axonopodis pv. citrumelo also lacks various genes, such as syrE1, syrE2, and RTX toxin family genes, which were present in X. axonopodis pv. citri. These may be associated with the distinct virulences of X. axonopodis pv. citrumelo and X. axonopodis pv. citri. Comparison of the complete genome sequence of X. axonopodis pv. citrumelo to those of X. axonopodis pv. citri and X. campestris pv. vesicatoria provides valuable insights into the mechanism of bacterial virulence and host specificity.     

Genome sequencing and comparative analysis of the carrot bacterial blight pathogen, Xanthomonas hortorum pv. carotae M081, for insights into pathogenicity and applications in molecular diagnostics

Xanthomonas hortorum pv. carotae (Xhc) is an economically important pathogen of carrots. Its ability to epiphytically colonize foliar surfaces and infect seeds can result in bacterial blight of carrots when grown in warm and humid regions. We used high-throughput sequencing to determine the genome sequence of isolate M081 of Xhc. The short reads were de novo assembled and the resulting contigs were ordered using a syntenic reference genome sequence from X. campestris pv. campestris ATCC 33913. The improved, high-quality draft genome sequence of Xhc M081 is the first for its species. Despite its distance from other sequenced xanthomonads, Xhc M081 still shared a large inventory of orthologous genes, including many clusters of virulence genes common to other foliar pathogenic species of Xanthomonas. We also mined the genome sequence and identified at least 21 candidate type III effector genes. Two were members of the avrBs2 and xopQ families that demonstrably elicit effector-triggered immunity. We showed that expression in planta of these two type III effectors from Xhc M081 was sufficient to elicit resistance gene-mediated hypersensitive responses in heterologous plants, indicating a possibility for resistance gene-mediated control of Xhc. Finally, we identified regions unique to the Xhc M081 genome sequence, and demonstrated their potential in the design of molecular diagnostics for this pathogen.     

The genome of Xanthomonas campestris pv. campestris B100 and its use for the reconstruction of metabolic pathways involved in xanthan biosynthesis

The complete genome sequence of the Xanthomonas campestris pv. campestris strain B100 was established. It consisted of a chromosome of 5,079,003bp, with 4471 protein-coding genes and 62 RNA genes. Comparative genomics showed that the genes required for the synthesis of xanthan and xanthan precursors were highly conserved among three sequenced X. campestris pv. campestris genomes, but differed noticeably when compared to the remaining four Xanthomonas genomes available. For the xanthan biosynthesis genes gumB and gumK earlier translational starts were proposed, while gumI and gumL turned out to be unique with no homologues beyond the Xanthomonas genomes sequenced. From the genomic data the biosynthesis pathways for the production of the exopolysaccharide xanthan could be elucidated. The first step of this process is the uptake of sugars serving as carbon and energy sources wherefore genes for 15 carbohydrate import systems could be identified. Metabolic pathways playing a role for xanthan biosynthesis could be deduced from the annotated genome. These reconstructed pathways concerned the storage and metabolization of the imported sugars. The recognized sugar utilization pathways included the Entner-Doudoroff and the pentose phosphate pathway as well as the Embden-Meyerhof pathway (glycolysis). The reconstruction indicated that the nucleotide sugar precursors for xanthan can be converted from intermediates of the pentose phosphate pathway, some of which are also intermediates of glycolysis or the Entner-Doudoroff pathway. Xanthan biosynthesis requires in particular the nucleotide sugars UDP-glucose, UDP-glucuronate, and GDP-mannose, from which xanthan repeat units are built under the control of the gum genes. The updated genome annotation data allowed reconsidering and refining the mechanistic model for xanthan biosynthesis.     

Comparative analyses of Xanthomonas and Xylella complete genomes

Computational analyses of four bacterial genomes of the Xanthomonadaceae family reveal new unique genes that may be involved in adaptation, pathogenicity, and host specificity. The Xanthomonas genus presents 3636 unique genes distributed in 1470 families, while Xylella genus presents 1026 unique genes distributed in 375 families. Among Xanthomonas-specific genes, we highlight a large number of cell wall degrading enzymes, proteases, and iron receptors, a set of energy metabolism genes, second copy of the type II secretion system, type III secretion system, flagella and chemotactic machinery, and the xanthomonadin synthesis gene cluster. Important genes unique to the Xylella genus are an additional copy of a type IV pili gene cluster and the complete machinery of colicin V synthesis and secretion. Intersections of gene sets from both genera reveal a cluster of genes homologous to Salmonella's SPI-7 island in Xanthomonas axonopodis pv citri and Xylella fastidiosa 9a5c, which might be involved in host specificity. Each genome also presents important unique genes, such as an HMS cluster, the kdgT gene, and O-antigen in Xanthomonas axonopodis pv citri; a number of avrBS genes and a distinct O-antigen in Xanthomonas campestris pv campestris, a type I restriction-modification system and a nickase gene in Xylella fastidiosa 9a5c, and a type II restriction-modification system and two genes related to peptidoglycan biosynthesis in Xylella fastidiosa temecula 1. All these differences imply a considerable number of gene gains and losses during the divergence of the four lineages, and are associated with structural genome modifications that may have a direct relation with the mode of transmission, adaptation to specific environments and pathogenicity of each organism.     

Development of an engineered bioluminescent reporter phage for detection of bacterial blight of crucifers

Bacterial blight, caused by the phytopathogen Pseudomonas cannabina pv. alisalensis, is an emerging disease afflicting important members of the Brassicaceae family. The disease is often misdiagnosed as pepper spot, a much less severe disease caused by the related pathogen Pseudomonas syringae pv. maculicola. We have developed a phage-based diagnostic that can both identify and detect the causative agent of bacterial blight and differentiate the two pathogens. A recombinant "light"-tagged reporter phage was generated by integrating bacterial luxAB genes encoding luciferase into the genome of P. cannabina pv. alisalensis phage PBSPCA1. The PBSPCA1::luxAB reporter phage is viable and stable and retains properties similar to those of the wild-type phage. PBSPCA1::luxAB rapidly and sensitively detects P. cannabina pv. alisalensis by conferring a bioluminescent signal response to cultured cells. Detection is dependent on cell viability. Other bacterial pathogens of Brassica species such as P. syringae pv. maculicola, Pseudomonas marginalis, Pectobacterium carotovorum, Xanthomonas campestris pv. campestris, and X. campestris pv. raphani either do not produce a response or produce significantly attenuated signals with the reporter phage. Importantly, the reporter phage detects P. cannabina pv. alisalensis on diseased plant specimens, indicating its potential for disease diagnosis.     

Characteristics of a PCR-Based Assay for In Planta Detection of Xanthomonas campestris pv. pelargonii

Polymerase chain reaction (PCR) amplification was carried out with a primer pair targeting a sequence in the genome of Xanthomonas campestris pv. pelargonii , the causative agent of bacterial blight in geraniums. PCR amplification with the primer pair XcpMl/XcpM2 using total nucleic acid preparations from 22 geographicallydiverse isolates of X. campestris pv. pelargonii generated a major 197 bp DNA product. In contrast, no major amplification products were consistently generated from 12 other pathovars of X. campestris or from 19 isolates representing 10 different plant pathogenic bacteria, including two other bacterial pathogens of geraniums, Corynebacterium fascians and Pseudomonas cichorii . After PCR using this primer pair, between 1380 and 13800 copies of the X, campestris pv. pelargonii bacterial DNA target as template were detected by ethidium bromide staining of agarose gels, and between 13.8 and 138 copies by blot hybridization to a pathovar-specific biotinylated probe. Similarly, between 630 and 6300 colonyforming units (CFU) of X. campestris pv. pelargonii could be detected after ethidium bromide staining of agarose gels, and between 63 and 630 CFU after blot hybridization. The PCR-based assay was used to identify X. campestris pv. pelargonii in diseased geraniums; whereas discrete amplification products were not obtained with healthy plants.     

Identification of new early markers of the hypersensitive response in Arabidopsis thaliana(1).

New molecular markers of the hypersensitive response (HR) of Arabidopsis thaliana to the bacterial pathogen Xanthomonas campestris pv. campestris (X.c.c.) have been identified by differential screening of a cDNA library constructed from suspension cells inoculated by an HR-inducing strain in the presence of cycloheximide. Seven families of genes (called Athsr) have been isolated, show similarities to voltage-dependent anion channels (VDAC) and alternative oxidases, or are novel proteins. Athsr genes have shown to be specifically or preferentially expressed during the HR. These data suggest that Athsr genes might be involved in early events conditioning the establishment of the HR.