Project description:MultiLocus Variable number of tandem repeat Analysis (MLVA) has been extensively used to examine epidemiological and evolutionary issues on monomorphic human pathogenic bacteria, but not on bacterial plant pathogens of agricultural importance albeit such tools would improve our understanding of their epidemiology, as well as of the history of epidemics on a global scale. Xanthomonas citri pv. citri is a quarantine organism in several countries and a major threat for the citrus industry worldwide. We screened the genomes of Xanthomonas citri pv. citri strain IAPAR 306 and of phylogenetically related xanthomonads for tandem repeats. From these in silico data, an optimized MLVA scheme was developed to assess the global diversity of this monomorphic bacterium. Thirty-one minisatellite loci (MLVA-31) were selected to assess the genetic structure of 129 strains representative of the worldwide pathological and genetic diversity of X. citri pv. citri. Based on Discriminant Analysis of Principal Components (DAPC), four pathotype-specific clusters were defined. DAPC cluster 1 comprised strains that were implicated in the major geographical expansion of X. citri pv. citri during the 20th century. A subset of 12 loci (MLVA-12) resolved 89% of the total diversity and matched the genetic structure revealed by MLVA-31. MLVA-12 is proposed for routine epidemiological identification of X. citri pv. citri, whereas MLVA-31 is proposed for phylogenetic and population genetics studies. MLVA-31 represents an opportunity for international X. citri pv. citri genotyping and data sharing. The MLVA-31 data generated in this study was deposited in the Xanthomonas citri genotyping database (http://www.biopred.net/MLVA/).
Project description:The Xanthomonas citri pv. citri (X. citri) is a phytopathogenic bacterium that infects different species of citrus plants where it causes canker disease. The adaptation to different habitats is related to the ability of the cells to metabolize and to assimilate diverse compounds, including sulfur, an essential element for all organisms. In Escherichia coli, the necessary sulfur can be obtained by a set of proteins whose genes belong to the cys regulon. Although the cys regulon proteins and their importance have been described in many other bacteria, there are no data related to these proteins in X. citri or in the Xanthomonas genus. The study of the relevance of these systems in these phytopathogenic bacteria that have distinct mechanisms of infection is one essential step toward understanding their physiology. In this work, we used bioinformatics, molecular modeling and transcription analysis (RT-PCR) to identify and characterize the putative cys regulon genes in X. citri.We showed that the ATP Binding Cassette Transporter (ABC transporter) SbpCysUWA for sulfate uptake is conserved in X. citri and translated in presence of sulfate. On the other hand, differently from what is predicted in databases, according molecular modeling and phylogenetic analysis, X. citri does not show a proper taurine transporter, but two different ABC systems related to the alkanesulfonate/sulfonate transport that were recently acquired during evolution. RT-PCR analysis evidenced that these genes and their putative transcriptional regulator CysB are rather transcripted in XAM1, a medium with defined concentration of sulfate, than LB.The presence of at least three distinct systems for sulfate and sulfonates assimilation in X. citri evidenced the importance of these compounds for the bacterium. The transcription of genes involved with alkanesulfonate/sulfur compounds in XAM1 along to CysB suggests that despite the differences in the transporters, the regulation of these systems might be similar to the described for E. coli. Altogether, these results will serve as a foundation for further studies aimed to understanding the relevance of sulfur in growth, virulence and pathogenesis of X. citri and related bacteria.
Project description:We report the 5.1-Mb genome sequence of Xanthomonas citri pv. mangiferaeindicae strain LMG 941, the causal agent of bacterial black spot in mango. Apart from evolutionary studies, the draft genome will be a valuable resource for the epidemiological studies and quarantine of this phytopathogen.
Project description:Citrus bacterial canker caused by Xanthomonas axonopodis pv. citri is a serious disease that impacts citrus production worldwide, and X. axonopodis pv. citri is listed as a quarantine pest in certain countries. Biofilm formation is important for the successful development of a pathogenic relationship between various bacteria and their host(s). To understand the mechanisms of biofilm formation by X. axonopodis pv. citri strain XW19, the strain was subjected to transposon mutagenesis. One mutant with a mutation in a two-component response regulator gene that was deficient in biofilm formation on a polystyrene microplate was selected for further study. The protein was designated as BfdR for biofilm formation defective regulator. BfdR from strain XW19 shares 100% amino acid sequence identity with XAC1284 of X. axonopodis pv. citri strain 306 and 30-100% identity with two-component response regulators in various pathogens and environmental microorganisms. The bfdR mutant strain exhibited significantly decreased biofilm formation on the leaf surfaces of Mexican lime compared with the wild type strain. The bfdR mutant was also compromised in its ability to cause canker lesions. The wild-type phenotype was restored by providing pbfdR in trans in the bfdR mutant. Our data indicated that BfdR did not regulate the production of virulence-related extracellular enzymes including amylase, lipase, protease, and lecithinase or the expression of hrpG, rfbC, and katE; however, BfdR controlled the expression of rpfF in XVM2 medium, which mimics cytoplasmic fluids in planta. In conclusion, biofilm formation on leaf surfaces of citrus is important for canker development in X. axonopodis pv. citri XW19. The process is controlled by the two-component response regulator BfdR via regulation of rpfF, which is required for the biosynthesis of a diffusible signal factor.
Project description:BACKGROUND: Xanthomonas axonopodis pv. citri (X. a. pv. citri) causes citrus canker that can result in defoliation and premature fruit drop with significant production losses worldwide. Biofilm formation is an important process in bacterial pathogens and several lines of evidence suggest that in X. a. pv. citri this process is a requirement to achieve maximal virulence since it has a major role in host interactions. In this study, proteomics was used to gain further insights into the functions of biofilms. RESULTS: In order to identify differentially expressed proteins, a comparative proteomic study using 2D difference gel electrophoresis was carried out on X. a. pv. citri mature biofilm and planktonic cells. The biofilm proteome showed major variations in the composition of outer membrane proteins and receptor or transport proteins. Among them, several porins and TonB-dependent receptor were differentially regulated in the biofilm compared to the planktonic cells, indicating that these proteins may serve in maintaining specific membrane-associated functions including signaling and cellular homeostasis. In biofilms, UDP-glucose dehydrogenase with a major role in exopolysaccharide production and the non-fimbrial adhesin YapH involved in adherence were over-expressed, while a polynucleotide phosphorylase that was demonstrated to negatively control biofilm formation in E. coli was down-regulated. In addition, several proteins involved in protein synthesis, folding and stabilization were up-regulated in biofilms. Interestingly, some proteins related to energy production, such as ATP-synthase were down-regulated in biofilms. Moreover, a number of enzymes of the tricarboxylic acid cycle were differentially expressed. In addition, X. a. pv. citri biofilms also showed down-regulation of several antioxidant enzymes. The respective gene expression patterns of several identified proteins in both X. a. pv. citri mature biofilm and planktonic cells were evaluated by quantitative real-time PCR and shown to consistently correlate with those deduced from the proteomic study. CONCLUSIONS: Differentially expressed proteins are enriched in functional categories. Firstly, proteins that are down-regulated in X. a. pv. citri biofilms are enriched for the gene ontology (GO) terms 'generation of precursor metabolites and energy' and secondly, the biofilm proteome mainly changes in 'outer membrane and receptor or transport'. We argue that the differentially expressed proteins have a critical role in maintaining a functional external structure as well as enabling appropriate flow of nutrients and signals specific to the biofilm lifestyle.
Project description:pFL1 is a pUC9 derivative that contains a 572-bp EcoRI insert cloned from plasmid DNA of Xanthomonas campestris pv. citri XC62. The nucleotide sequence of pFL1 was determined, and the sequence information was used to design primers for application of the polymerase chain reaction (PCR) to the detection of X. campestris pv. citri, the causal agent of citrus bacterial canker disease. Seven 18-bp oligonucleotide primers were designed and tested with DNA from X. campestris pv. citri strains and other strains of X. campestris associated with Citrus spp. as templates in the PCR. Four primer pairs directed the amplification of target DNA from X. campestris pv. citri strains but not from strains of X. campestris associated with a different disease, citrus bacterial spot. Primer pair 2-3 directed the specific amplification of target DNA from pathotype A but not other pathotypes of X. campestris pv. citri. A pH 9.0 buffer that contained 1% Triton X-100 and 0.1% gelatin was absolutely required for the successful amplification of the target DNA, which was 61% G+C. Limits of detection after amplification and gel electrophoresis were 25 pg of purified target DNA and about 10 cells when Southern blots were made after gel electrophoresis and probed with biotinylated pFL1. This level of detection represents an increase in sensitivity of about 100-fold over that of dot blotting with the same hybridization probe. PCR products of the expected sizes were amplified from DNA extracted from 7-month-old lesions from which viable bacteria could not be isolated. These products were confirmed to be specific for X. campestris pv. citri by Southern blotting. This PCR-based detection protocol will be a useful addition to current methods of detection of this pathogen, which is currently the target of international quarantine measures.
Project description:BACKGROUND:The identification of factors involved in the host range definition and evolution is a pivotal challenge in the goal to predict and prevent the emergence of plant bacterial disease. To trace the evolution and find molecular differences between three pathotypes of Xanthomonas citri pv. citri that may explain their distinctive host ranges, 42 strains of X. citri pv. citri and one outgroup strain, Xanthomonas citri pv. bilvae were sequenced and compared. RESULTS:The strains from each pathotype form monophyletic clades, with a short branch shared by the A(w) and A pathotypes. Pathotype-specific recombination was detected in seven regions of the alignment. Using Ancestral Character Estimation, 426 SNPs were mapped to the four branches at the base of the A, A*, A(w) and A/A(w) clades. Several genes containing pathotype-specific nonsynonymous mutations have functions related to pathogenicity. The A pathotype is enriched for SNP-containing genes involved in defense mechanisms, while A* is significantly depleted for genes that are involved in transcription. The pathotypes differ by four gene islands that largely coincide with regions of recombination and include genes with a role in virulence. Both A* and A(w) are missing genes involved in defense mechanisms. In contrast to a recent study, we find that there are an extremely small number of pathotype-specific gene presences and absences. CONCLUSIONS:The three pathotypes of X. citri pv. citri that differ in their host ranges largely show genomic differences related to recombination, horizontal gene transfer and single nucleotide polymorphism. We detail the phylogenetic relationship of the pathotypes and provide a set of candidate genes involved in pathotype-specific evolutionary events that could explain to the differences in host range and pathogenicity between them.
Project description:Xanthomonas citri pv. vignicola strains cause bacterial blight of the legume crop cowpea. We report whole-genome sequences of three X. citri pv. vignicola strains obtained using PacBio single-molecule real-time sequencing. Such genomic data provide new information on pathogenicity factors, such as transcription activator-like effectors.
Project description:Xanthomonas axonopodis pv. citri is a phytopathogen bacterium that causes severe citrus canker disease. Similar to other phytopathogens, after infection by this bacterium, plants trigger a defense mechanism that produces reactive oxygen species. Ferredoxin-NADP(+) reductases (FNRs) are redox flavoenzymes that participate in several metabolic functions, including the response to reactive oxygen species. Xanthomonas axonopodis pv. citri has a gene (fpr) that encodes for a FNR (Xac-FNR) that belongs to the subclass I bacterial FNRs. The aim of this work was to search for the physiological role of this enzyme and to characterize its structural and functional properties. The functionality of Xac-FNR was tested by cross-complementation of a FNR knockout Escherichia coli strain, which exhibit high susceptibility to agents that produce an abnormal accumulation of (•)O(2)(-). Xac-FNR was able to substitute for the FNR in E. coli in its antioxidant role. The expression of fpr in X. axonopodis pv. citri was assessed using semiquantitative RT-PCR and Western blot analysis. A 2.2-fold induction was observed in the presence of the superoxide-generating agents methyl viologen and 2,3-dimethoxy-1,4-naphthoquinone. Structural and functional studies showed that Xac-FNR displayed different functional features from other subclass I bacterial FNRs. Our analyses suggest that these differences may be due to the unusual carboxy-terminal region. We propose a further classification of subclass I bacterial FNRs, which is useful to determine the nature of their ferredoxin redox partners. Using sequence analysis, we identified a ferredoxin (XAC1762) as a potential substrate of Xac-FNR. The purified ferredoxin protein displayed the typical broad UV-visible spectrum of [4Fe-4S] clusters and was able to function as substrate of Xac-FNR in the cytochrome c reductase activity. Our results suggest that Xac-FNR is involved in the oxidative stress response of Xanthomonas axonopodis pv. citri and performs its biological function most likely through the interaction with ferredoxin XAC1762.
Project description:BACKGROUND:Xanthomonas citri pv. citri (Xcc) is a citrus canker causing Gram-negative bacteria. Currently, little is known about the biological and molecular responses of Xcc to low temperatures. RESULTS:Results depicted that low temperature significantly reduced growth and increased biofilm formation and unsaturated fatty acid (UFA) ratio in Xcc. At low temperature Xcc formed branching structured motility. Global transcriptome analysis revealed that low temperature modulates multiple signaling networks and essential cellular processes such as carbon, nitrogen and fatty acid metabolism in Xcc. Differential expression of genes associated with type IV pilus system and pathogenesis are important cellular adaptive responses of Xcc to cold stress. CONCLUSIONS:Study provides clear insights into biological characteristics and genome-wide transcriptional analysis based molecular mechanism of Xcc in response to low temperature.