RESUMO
BACKGROUND: Molecular typing of pathogen populations is an important tool for the development of effective strategies for disease control. Diverse molecular markers have been used to characterize populations of Xanthomonas axonopodis pv. manihotis (Xam), the main bacterial pathogen of cassava. Recently, diversity and population dynamics of Xam in the Colombian Caribbean coast were estimated using AFLPs, where populations were found to be dynamic, diverse and with haplotypes unstable across time. Aiming to examine the current state of pathogen populations located in the Colombian Eastern Plains, we also used AFLP markers and we evaluated the usefulness of Variable Number Tandem Repeats (VNTRs) as new molecular markers for the study of Xam populations. RESULTS: The population analyses showed that AFLP and VNTR provide a detailed and congruent description of Xam populations from the Colombian Eastern Plains. These two typing strategies clearly separated strains from the Colombian Eastern Plains into distinct populations probably because of geographical distance. Although the majority of analyses were congruent between typing markers, fewer VNTRs were needed to detect a higher number of genetic populations of the pathogen as well as a higher genetic flow among sampled locations than those detected by AFLPs. CONCLUSIONS: This study shows the advantages of VNTRs over AFLPs in the surveillance of pathogen populations and suggests the implementation of VNTRs in studies that involve large numbers of Xam isolates in order to obtain a more detailed overview of the pathogen to improve the strategies for disease control.
Assuntos
Variação Genética , Manihot/microbiologia , Tipagem Molecular/métodos , Doenças das Plantas/microbiologia , Xanthomonas axonopodis/classificação , Xanthomonas axonopodis/genética , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Análise por Conglomerados , Colômbia , DNA Bacteriano/química , DNA Bacteriano/genética , Genótipo , Repetições Minissatélites , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNA , Xanthomonas axonopodis/isolamento & purificaçãoRESUMO
Cassava bacterial blight, caused by Xanthomonas axonopodis pv. manihotis (Xam), is the most important bacterial disease affecting this crop. A continuous surveillance of the pathogen population dynamics is required to develop an efficient disease management program. During the 1990s, Xam populations showed high levels of genetic variation and relevant migratory processes that were important determinants of the distribution of the pathogen diversity in Colombia. Aiming to characterize the current population structure of the pathogen and the evolutionary forces that shape these populations, sampling collections were carried out from September 2008 until November 2010 in the Colombian Caribbean Region. One hundred and sixty bacterial isolates were characterized using amplified fragment length polymorphism (AFLP) markers. Additionally, a subset of effector genes were sequenced in some isolates to determine their usefulness in Xam population studies and to provide additional information to that obtained with AFLPs. Virulence patterns of ten isolates were determined in nine cassava accessions. Our results show a complex architecture of population and confirm migratory process previously reported in the Caribbean Region. Chinú, one of the locations sampled, presented remarkable features in population dynamics such as longer genetic distances, higher diversity indices, and a genetically differentiated population when it was compared with other locations. Virulence tests showed that MCOL2215, one of the most cultivated cassava varieties in the Caribbean coast, was susceptible to the majority of Xam isolates tested. This study shows the current condition of populations of Xam in the Caribbean Region of Colombia, and it contributes to improve the existing bacterial blight control practices.
Assuntos
Genética Populacional , Manihot/microbiologia , Doenças das Plantas/microbiologia , Xanthomonas axonopodis/genética , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Colômbia , DNA Bacteriano/genética , Evolução Molecular , Genes Bacterianos , Marcadores Genéticos , Haplótipos , Polimorfismo Genético , Seleção Genética , Virulência/genética , Xanthomonas axonopodis/patogenicidadeRESUMO
Xanthomonas axonopodis pv. manihotis (Xam) is the causal agent of bacterial blight of cassava, which is among the main components of human diet in Africa and South America. Current information about the molecular pathogenicity factors involved in the infection process of this organism is limited. Previous studies in other bacteria in this genus suggest that advanced draft genome sequences are valuable resources for molecular studies on their interaction with plants and could provide valuable tools for diagnostics and detection. Here we have generated the first manually annotated high-quality draft genome sequence of Xam strain CIO151. Its genomic structure is similar to that of other xanthomonads, especially Xanthomonas euvesicatoria and Xanthomonas citri pv. citri species. Several putative pathogenicity factors were identified, including type III effectors, cell wall-degrading enzymes and clusters encoding protein secretion systems. Specific characteristics in this genome include changes in the xanthomonadin cluster that could explain the lack of typical yellow color in all strains of this pathovar and the presence of 50 regions in the genome with atypical nucleotide composition. The genome sequence was used to predict and evaluate 22 variable number of tandem repeat (VNTR) loci that were subsequently demonstrated as polymorphic in representative Xam strains. Our results demonstrate that Xanthomonas axonopodis pv. manihotis strain CIO151 possesses ten clusters of pathogenicity factors conserved within the genus Xanthomonas. We report 126 genes that are potentially unique to Xam, as well as potential horizontal transfer events in the history of the genome. The relation of these regions with virulence and pathogenicity could explain several aspects of the biology of this pathogen, including its ability to colonize both vascular and non-vascular tissues of cassava plants. A set of 16 robust, polymorphic VNTR loci will be useful to develop a multi-locus VNTR analysis scheme for epidemiological surveillance of this disease.
Assuntos
Xanthomonas axonopodis/genética , Genoma Bacteriano/genética , Repetições Minissatélites/genética , Virulência/genética , Xanthomonas axonopodis/patogenicidadeRESUMO
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.
Assuntos
Proteínas de Bactérias/química , Biofilmes , Citrus/microbiologia , Doenças das Plantas/microbiologia , Proteômica , Xanthomonas axonopodis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Xanthomonas axonopodis/química , Xanthomonas axonopodis/fisiologiaRESUMO
Xanthomonas axonopodis pv. citri (Xac) is the phytopathogen responsible for citrus canker, one of the most devastating citrus diseases in the world. A broad range of pathogens is recognized by plants through so-called pathogen-associated molecular patterns (PAMPs), which are highly conserved fragments of pathogenic molecules. In plant pathogenic bacteria, lipopolisaccharyde (LPS) is considered a virulence factor and it is being recognized as a PAMP. The study of the participation of Xac LPS in citrus canker establishment could help to understand the molecular bases of this disease. In the present work we investigated the role of Xac LPS in bacterial virulence and in basal defense during the interaction with host and non host plants. We analyzed physiological features of Xac mutants in LPS biosynthesis genes (wzt and rfb303) and the effect of these mutations on the interaction with orange and tobacco plants. Xac mutants showed an increased sensitivity to external stresses and differences in bacterial motilities, in vivo and in vitro adhesion and biofilm formation. Changes in the expression levels of the LPS biosynthesis genes were observed in a medium that mimics the plant environment. Xacwzt exhibited reduced virulence in host plants compared to Xac wild-type and Xacrfb303. However, both mutant strains produced a lower increase in the expression levels of host plant defense-related genes respect to the parental strain. In addition, Xac LPS mutants were not able to generate HR during the incompatible interaction with tobacco plants. Our findings indicate that the structural modifications of Xac LPS impinge on other physiological attributes and lead to a reduction in bacterial virulence. On the other hand, Xac LPS has a role in the activation of basal defense in host and non host plants.
Assuntos
Citrus/microbiologia , Lipopolissacarídeos/metabolismo , Doenças das Plantas/microbiologia , Xanthomonas axonopodis/metabolismo , Xanthomonas axonopodis/patogenicidade , Aderência Bacteriana/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes , Regulação Bacteriana da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Interações Hospedeiro-Patógeno , Mutação , Fenótipo , Doenças das Plantas/imunologia , Folhas de Planta/genética , Folhas de Planta/microbiologia , Estresse Fisiológico , Virulência , Xanthomonas axonopodis/genéticaRESUMO
Recent studies have demonstrated that an appropriate light environment is required for the establishment of efficient vegetal resistance responses in several plant-pathogen interactions. The photoreceptors implicated in such responses are mainly those belonging to the phytochrome family. Data obtained from bacterial genome sequences revealed the presence of photosensory proteins of the BLUF (Blue Light sensing Using FAD), LOV (Light, Oxygen, Voltage) and phytochrome families with no known functions. Xanthomonas axonopodis pv. citri is a Gram-negative bacterium responsible for citrus canker. The in silico analysis of the X. axonopodis pv. citri genome sequence revealed the presence of a gene encoding a putative LOV photoreceptor, in addition to two genes encoding BLUF proteins. This suggests that blue light sensing could play a role in X. axonopodis pv. citri physiology. We obtained the recombinant Xac-LOV protein by expression in Escherichia coli and performed a spectroscopic analysis of the purified protein, which demonstrated that it has a canonical LOV photochemistry. We also constructed a mutant strain of X. axonopodis pv. citri lacking the LOV protein and found that the loss of this protein altered bacterial motility, exopolysaccharide production and biofilm formation. Moreover, we observed that the adhesion of the mutant strain to abiotic and biotic surfaces was significantly diminished compared to the wild-type. Finally, inoculation of orange (Citrus sinensis) leaves with the mutant strain of X. axonopodis pv. citri resulted in marked differences in the development of symptoms in plant tissues relative to the wild-type, suggesting a role for the Xac-LOV protein in the pathogenic process. Altogether, these results suggest the novel involvement of a photosensory system in the regulation of physiological attributes of a phytopathogenic bacterium. A functional blue light receptor in Xanthomonas spp. has been described for the first time, showing an important role in virulence during citrus canker disease.
Assuntos
Proteínas de Bactérias/metabolismo , Citrus sinensis/microbiologia , Interações Hospedeiro-Patógeno/fisiologia , Xanthomonas axonopodis/crescimento & desenvolvimento , Xanthomonas axonopodis/fisiologia , Sequência de Aminoácidos , Aderência Bacteriana , Proteínas de Bactérias/química , Biofilmes , Contagem de Colônia Microbiana , Biologia Computacional , Deleção de Genes , Genes Bacterianos/genética , Histidina Quinase , Dados de Sequência Molecular , Movimento/fisiologia , Processos Fotoquímicos , Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia , Polissacarídeos Bacterianos/biossíntese , Proteínas Quinases/metabolismo , Proteínas Recombinantes/metabolismo , Xanthomonas axonopodis/enzimologia , Xanthomonas axonopodis/genéticaRESUMO
Xanthomonas axonopodis pv. passiflorae causes bacterial spot in passion fruit. It attacks the purple and yellow passion fruit as well as the sweet passion fruit. The diversity of 87 isolates of pv. passiflorae collected from across 22 fruit orchards in Brazil was evaluated using molecular profiles and statistical procedures, including an unweighted pair-group method with arithmetical averages-based dendrogram, analysis of molecular variance (AMOVA), and an assigning test that provides information on genetic structure at the population level. Isolates from another eight pathovars were included in the molecular analyses and all were shown to have a distinct repetitive sequence-based polymerase chain reaction profile. Amplified fragment length polymorphism technique revealed considerable diversity among isolates of pv. passiflorae, and AMOVA showed that most of the variance (49.4%) was due to differences between localities. Cluster analysis revealed that most genotypic clusters were homogeneous and that variance was associated primarily with geographic origin. The disease adversely affects fruit production and may kill infected plants. A method for rapid diagnosis of the pathogen, even before the disease symptoms become evident, has value for producers. Here, a set of primers (Xapas) was designed by exploiting a single-nucleotide polymorphism between the sequences of the intergenic 16S-23S rRNA spacer region of the pathovars. Xapas was shown to effectively detect all pv. passiflorae isolates and is recommended for disease diagnosis in passion fruit orchards.
Assuntos
DNA Bacteriano/genética , Variação Genética , Passiflora/microbiologia , Doenças das Plantas/microbiologia , Reação em Cadeia da Polimerase/métodos , Xanthomonas axonopodis/classificação , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Brasil , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , DNA Espaçador Ribossômico , Geografia , Passiflora/genética , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Virulência , Xanthomonas axonopodis/genética , Xanthomonas axonopodis/isolamento & purificação , Xanthomonas axonopodis/patogenicidadeRESUMO
Amplified fragment length polymorphism (AFLP) was used to analyze the genetic diversity of 14 strains of Xanthomonas arboricola pv. pruni and seven strains of X. axonopodis pv. phaseoli, which are used in xanthan production studies. Relationships identified by the AFLP profiles were assessed for xanthan production capacity, geographical location and host plant. Strains were isolated from 10 different geographic regions in South and Southeast States in Brazil. Data were analyzed for genetic similarity using the Dice coefficient and subjected to UPGMA cluster analysis. A total of 128 AFLP fragments were generated from four primer combinations: EcoRI+C/MseI+0, EcoRI+A/MseI+0, EcoRI+G/MseI+T and EcoRI+G/MseI+A. Of these, 96.1 percent were polymorphic. X. axonopodis pv. phaseoli (S D = 0.27) was shown to be more polymorphic than X. arboricola pv. pruni (S D = 0.58). All 14 pathovar pruni strains were included in a single main group (S D = 0.58), while the pathovar phaseoli strains were divided into three separate groups, with one group containing five strains (S D = 0.38) and two isolated groups (S D = 0.31 and 0.27) composed of only one strain each. Species were distinguished by three and eight specific AFLP markers present in the pathovar phaseoli and the pathovar pruni, respectively. For the unique strain without xanthan production capacity (X. axonopodis pv. phaseoli str. 48), nine specific AFLP bands were found. There was no evidence that geographic area or host plant influenced genetic heterogeneity. Correlations between AFLP patterns and xanthan production capacity were found in some strains, but were not consistent enough to establish a relationship.
Assuntos
Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Impressões Digitais de DNA , Variação Genética , Xanthomonas axonopodis/genética , Xanthomonas axonopodis/isolamento & purificação , Xanthomonas/genética , Xanthomonas/isolamento & purificação , Métodos , Métodos , VirulênciaRESUMO
Lateral gene transfer (LGT) is considered as one of the drivers in bacterial genome evolution, usually associated with increased fitness and/or changes in behavior, especially if one considers pathogenic vs. non-pathogenic bacterial groups. The genomes of two phytopathogens, Xanthomonas campestris pv. campestris and Xanthomonas axonopodis pv. citri, were previously inspected for genome islands originating from LGT events, and, in this work, potentially early and late LGT events were identified according to their altered nucleotide composition. The biological role of the islands was also assessed, and pathogenicity, virulence and secondary metabolism pathways were functions highly represented, especially in islands that were found to be recently transferred. However, old islands are composed of a high proportion of genes related to cell primary metabolic functions. These old islands, normally undetected by traditional atypical composition analysis, but confirmed as product of LGT by atypical phylogenetic reconstruction, reveal the role of LGT events by replacing core metabolic genes normally inherited by vertical processes.
Assuntos
Transferência Genética Horizontal , Ilhas Genômicas , Xanthomonas axonopodis/genética , Xanthomonas campestris/genética , DNA Bacteriano/genética , Evolução Molecular , Redes e Vias Metabólicas/genética , Filogenia , Homologia de Sequência de Aminoácidos , Fatores de Virulência/genética , Xanthomonas axonopodis/metabolismo , Xanthomonas axonopodis/patogenicidade , Xanthomonas campestris/metabolismo , Xanthomonas campestris/patogenicidadeRESUMO
The oligopeptide-binding protein, OppA, binds and ushers oligopeptide substrates to the membrane-associated oligopeptide permease (Opp), a multi-component ABC-type transporter involved in the uptake of oligopeptides expressed by several bacterial species. In the present study, we report the cloning, purification, refolding and conformational analysis of a recombinant OppA protein derived from Xanthomonas axonopodis pv. citri (X. citri), the etiological agent of citrus canker. The oppA gene was expressed in Escherichia coli BL21 (DE3) strain under optimized inducing conditions and the recombinant protein remained largely insoluble. Solubilization was achieved following refolding of the denatured protein. Circular dichroism analysis indicated that the recombinant OppA protein preserved conformational features of orthologs expressed by other bacterial species. The refolded recombinant OppA represents a useful tool for structural and functional analyses of the X. citri protein.
Assuntos
Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Membrana Transportadoras/isolamento & purificação , Proteínas de Membrana Transportadoras/metabolismo , Dobramento de Proteína , Xanthomonas axonopodis/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Dicroísmo Circular , Clonagem Molecular , Biologia Computacional/métodos , Escherichia coli/genética , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Dados de Sequência Molecular , Óperon , Plasmídeos , Conformação Proteica , Desnaturação Proteica , Renaturação Proteica , Sinais Direcionadores de Proteínas , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Solubilidade , Especificidade por Substrato , Xanthomonas axonopodis/metabolismoRESUMO
Xanthomonas axonopodis pv. citri (Xac) causes citrus canker and the completion of the Xac genome sequence has opened up the possibility of investigating basic cellular mechanisms at the genomic level. Copper compounds have been extensively used in agriculture to control plant diseases. The copA and copB genes, identified by annotation of the Xac genome, encode homologues of proteins involved in copper resistance. A gene expression assay by Northern blotting revealed that copA and copB are expressed as a unique transcript specifically induced by copper. Synthesis of the gene products was also induced by copper, reaching a maximum level at 4 h after addition of copper to the culture medium. CopA was a cytosolic protein and CopB was detected in the cytoplasmic membrane. The gene encoding CopA was disrupted by the insertion of a transposon, leading to mutant strains that were unable to grow in culture medium containing copper, even at the lowest CuSO(4) concentration tested (0.25 mM), whereas the wild-type strain was able to grow in the presence of 1 mM copper. Cell suspensions of the wild-type and mutant strains in different copper concentrations were inoculated in lemon leaves to analyse their ability to induce citrus canker symptoms. Cells of mutant strains showed higher sensitivity than the wild-type strain in the presence of copper, i.e. they were not able to induce citrus canker symptoms at high copper concentrations and exhibited a more retarded growth in planta.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Citrus/microbiologia , Cobre/metabolismo , Inativação Gênica , Doenças das Plantas/microbiologia , Xanthomonas axonopodis/genética , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/análise , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , Sequência de Bases , Proteínas de Transporte de Cátions/análise , Proteínas de Transporte de Cátions/genética , Fracionamento Celular , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Dados de Sequência Molecular , Mutagênese Insercional , Óperon , Folhas de Planta/microbiologia , Transposases/genética , Xanthomonas axonopodis/crescimento & desenvolvimento , Xanthomonas axonopodis/metabolismoRESUMO
The oligopeptide-binding protein, OppA, binds and ushers oligopeptide substrates to the membrane-associated oligopeptide permease (Opp), a multi-component ABC-type transporter involved in the uptake of oligopeptides expressed by several bacterial species. In the present study, we report the cloning, purification, refolding and conformational analysis of a recombinant OppA protein derived from Xanthomonas axonopodis pv. citri (X. citri), the etiological agent of citrus canker. The oppA gene was expressed in Escherichia coli BL21 (DE3) strain under optimized inducing conditions and the recombinant protein remained largely insoluble. Solubilization was achieved following refolding of the denatured protein. Circular dichroism analysis indicated that the recombinant OppA protein preserved conformational features of orthologs expressed by other bacterial species. The refolded recombinant OppA represents a useful tool for structural and functional analyses of the X. citri protein.
Assuntos
Dobramento de Proteína , Proteínas de Bactérias/isolamento & purificação , Proteínas de Membrana Transportadoras/isolamento & purificação , Proteínas de Transporte/metabolismo , Xanthomonas axonopodis/genética , Sequência de Aminoácidos , Sequência de Bases , Biologia Computacional/métodos , Dicroísmo Circular , Clonagem Molecular , Escherichia coli/genética , Dados de Sequência Molecular , Óperon , Plasmídeos , Conformação Proteica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Xanthomonas axonopodis/metabolismoRESUMO
In Xanthomonas axonopodis pv. citri (Xac or X. citri), the modA gene codes for a periplasmic protein (ModA) that is capable of binding molybdate and tungstate as part of the ABC-type transporter required for the uptake of micronutrients. In this study, we report the crystallographic structure of the Xac ModA protein with bound molybdate. The Xac ModA structure is similar to orthologs with known three-dimensional structures and consists of two nearly symmetrical domains separated by a hinge region where the oxyanion-binding site lies. Phylogenetic analysis of different ModA orthologs based on sequence alignments revealed three groups of molybdate-binding proteins: bacterial phytopathogens, enterobacteria and soil bacteria. Even though the ModA orthologs are segregated into different groups, the ligand-binding hydrogen bonds are mostly conserved, except for Archaeglobus fulgidus ModA. A detailed discussion of hydrophobic interactions in the active site is presented and two new residues, Ala38 and Ser151, are shown to be part of the ligand-binding pocket.
Assuntos
Molibdênio/química , Molibdênio/metabolismo , Proteínas Periplásmicas de Ligação/química , Proteínas Periplásmicas de Ligação/metabolismo , Xanthomonas axonopodis/química , Xanthomonas axonopodis/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Sequência Conservada , Cristalografia por Raios X , Ligantes , Dados de Sequência Molecular , Proteínas Periplásmicas de Ligação/genética , Filogenia , Doenças das Plantas/microbiologia , Ligação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia Estrutural de Proteína , Xanthomonas axonopodis/genética , Xanthomonas axonopodis/patogenicidadeRESUMO
The phytopathogenic bacterium Xanthomonas axonopodis pv. citri is responsible for the canker disease affecting citrus plants throughout the world. Here, we have evaluated the role of bacterial attachment and biofilm formation in leaf colonization during canker development on lemon leaves. Crystal violet staining and confocal laser scanning microscopy analysis of X. axonopodis pv. citri strains expressing the green fluorescent protein were used to evaluate attachment and biofilm formation on abiotic and biotic (leaf) surfaces. Wild-type X. axonopodis pv. citri attached to and formed a complex, structured biofilm on glass in minimal medium containing glucose. Similar attachment and structured biofilm formation also were seen on lemon leaves. An X. axonopodis pv. citri gumB mutant strain, defective in production of the extracellular polysaccharide xanthan, did not form a structured biofilm on either abiotic or biotic surfaces. In addition, the X. axonopodis pv. citri gumB showed reduced growth and survival on leaf surfaces and reduced disease symptoms. These findings suggest an important role for formation of biofilms in the epiphytic survival of X. axonopodis pv. citri prior to development of canker disease.
Assuntos
Biofilmes , Citrus/microbiologia , Doenças das Plantas/microbiologia , Xanthomonas axonopodis/fisiologia , Xanthomonas axonopodis/patogenicidade , Aderência Bacteriana , Citrus/metabolismo , Folhas de Planta/microbiologia , Polissacarídeos Bacterianos/biossíntese , Virulência , Xanthomonas axonopodis/genéticaRESUMO
Genome annotation of the plant pathogen Xanthomonas axonopodis pv. citri (Xac), identified flagellar genes in a 15.7 kb gene cluster. However, FlgN, a secretion chaperone for hook-associated proteins FlgK and FlgL, was not identified. We performed extensive screening of the X. axonopodis pv. citri genome with the yeast two-hybrid system to identify a protein with the characteristics of the flagellar chaperone FlgN. We found a candidate (XAC1990) encoded by an operon for components of the flagellum apparatus that interacted with FlgK. In order to further support this finding, Xac FlgK and XAC1990 were cloned, expressed, and purified. The recombinant proteins were characterized by spectroscopic methods and their interaction in vitro confirmed by pull-down assays. We, therefore, conclude that XAC1990 and its homologs in other Xanthomonas species are, in fact, FlgN proteins. These observations extend the sequence diversity covered by this family of proteins.
Assuntos
Proteínas de Bactérias/metabolismo , Flagelos/fisiologia , Xanthomonas axonopodis/fisiologia , Sequência de Aminoácidos , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Técnicas do Sistema de Duplo-Híbrido , Xanthomonas axonopodis/genética , Xanthomonas axonopodis/metabolismoRESUMO
Xanthan-deficient mutants of Xanthomonas axonopodis pv. citri, the bacterium responsible for citrus canker, were generated by deletion and marker exchange of the region encoding the carboxy-terminal end of the first glycosyltransferase, GumD. Mutants of gumD did not produce xanthan and remained pathogenic in citrus plants to the same extent as wild-type bacteria. The kinetics of appearance of initial symptoms, areas of plant material affected, and growth of bacteria inside plant tissue throughout the disease process were similar for both wild-type and mutant inoculations. Moreover, exopolysaccharide deficiency did not impair the ability of the bacteria to induce hypersensitive response on non-host plants. Apart from variations in phenotypic aspects, no differences in growth or survival under different stress conditions were observed between the xanthan-deficient mutant and wild-type bacteria. However, gumD mutants displayed impaired survival under oxidative stress during stationary phase as well as impaired epiphytic survival on citrus leaves. Our results suggest that xanthan does not play an essential role in citrus canker at the initial stages of infection or in the incompatible interactions between X. axonopodis pv. citri and non-host plants, but facilitates the maintenance of bacteria on the host plant, possibly improving the efficiency of colonization of distant tissue.
Assuntos
Citrus/microbiologia , Doenças das Plantas/microbiologia , Polissacarídeos Bacterianos/fisiologia , Xanthomonas axonopodis/patogenicidade , Genes Bacterianos , Mutagênese , Folhas de Planta/microbiologia , Virulência , Xanthomonas axonopodis/química , Xanthomonas axonopodis/genética , Xanthomonas axonopodis/fisiologiaRESUMO
Xanthomonas axonopodis pv. citri (Xac) causes citrus canker in plantations around the world and is of particular significance in Brazil where its incidence has risen exponentially over the past decade. Approximately one third of the predicted Xac open reading frames show no homology, or homology with very low score with that of known sequences. It is believed that Xac utilizes secretion systems to transfer virulence proteins into susceptible eukaryotic cells. This process is assisted by secretion chaperones that maintain virulence proteins partly or completely unfolded during translocation. We have cloned three of these hypothetical secretion chaperones: XAC0419 and XAC1346 from type III secretion system (TTSS) and XACb0033 from type IV secretion system (TFSS). All proteins were cloned in a pET23a vector (Novagen), expressed at 37 degrees C using a BL21(DE3)pLysS Escherichia coli strain and purified by ion exchange and gel-filtration chromatographic methods. Pure proteins were characterized using spectroscopic measurements: circular dichroism, and both static and lifetime emission fluorescence in the case of XACb0033. The analyzed proteins are stable at elevated temperatures (up to 65 degrees C) and exhibit alpha-helix content from approximately 30% (XACb003) to approximately 87% (XAC1346). XACb0033 exhibits lifetimes in the fluorescence experiments that indicate different neighborhoods for its tryptophan residues. These chaperones have the characteristics of TTSS and TFSS: all are small, with a high alpha-helix content, and without ATP-binding or ATP-hydrolyzing activity.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Chaperonas Moleculares/genética , Chaperonas Moleculares/isolamento & purificação , Xanthomonas axonopodis/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Dicroísmo Circular , Citrus/microbiologia , Clonagem Molecular , Escherichia coli/genética , Expressão Gênica , Genes Bacterianos , Chaperonas Moleculares/química , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Doenças das Plantas/microbiologia , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Espectrometria de Fluorescência , Virulência , Xanthomonas axonopodis/patogenicidadeRESUMO
We report here, the cloning, expression, and purification of a broad specificity aminopeptidase from Xanthomonas campestris pv. citri in fusion with a hexa-histidine tag at the N-terminal portion of the protein to facilitate purification. The protein was expressed in the soluble fraction and could be purified in one step by IMAC, yielding approximately 50mg pure protein per liter of cells. We show that the protein is folded and presents aminopeptidase activity against synthetic substrates. Also, we present the characterization of its specificity, showing that the protein was, indeed, able to catalyze the removal of N-terminal residues from synthetic substrates.
Assuntos
Aminopeptidases/genética , Aminopeptidases/metabolismo , Xanthomonas axonopodis/enzimologia , Sequência de Aminoácidos , Aminopeptidases/química , Aminopeptidases/isolamento & purificação , Sequência de Bases , Sequência Conservada , Primers do DNA , Cinética , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência , Espectrofotometria Ultravioleta , Especificidade por Substrato , Xanthomonas axonopodis/genéticaRESUMO
The oligopeptide-binding protein, OppA, ushers oligopeptide substrates to the membrane-associated oligopeptide permease (Opp), a multi-component ABC-type transporter involved in the uptake of oligopeptides by several bacterial species. In the present study, we report a structural model and an oligopeptide docking analysis of the OppA protein expressed by Xanthomonas axonopodis pv. citri (X. citri), the etiological agent of citrus canker. The X. citri OppA structural model showed a conserved three-dimensional structure, irrespective of the low amino acid identities with previously defined structures of Bacillus subtilis and Salmonella typhimurium orthologs. Oligopeptide docking analysis carried out with the proposed model indicated that the X. citri OppA preferentially binds tri- and tetrapeptides. The present study represents the first structural analysis of an OppA ortholog expressed by a phytopathogen and contributes to the understanding of the physiology and nutritional strategies of X. citri.