The histone-like protein HupB influences biofilm formation and virulence in Xanthomonas citri ssp. citri through the regulation of flagellar biosynthesis

Citrus canker is an important disease of citrus, whose causal agent is the bacterium Xanthomonas citri ssp. citri (Xcc). In previous studies, we found a group of Xcc mutants, generated by the insertion of the Tn5 transposon, which showed impaired ability to attach to an abiotic substrate. One of these mutants carries the Tn5 insertion in hupB, a gene encoding a bacterial histone-like protein, homologue to the β-subunit of the Heat-Unstable (HU) nucleoid protein of Escherichia coli. These types of protein are necessary to maintain the bacterial nucleoid organization and the global regulation of gene expression. Here, we characterized the influence of the mutation in hupB regarding Xcc biofilm formation and virulence. The mutant strain hupB was incapable of swimming in soft agar, whereas its complemented strain partially recovered this phenotype. Electron microscope imaging revealed that impaired motility of hupB was a consequence of the absence of the flagellum. Comparison of the expression of flagellar genes between the wild-type strain and hupB showed that the mutant exhibited decreased expression of fliC (encoding flagellin). The hupB mutant also displayed reduced virulence compared with the wild-type strain when they were used to infect Citrus lemon plants using different infection methods. Our results therefore show that the histone-like protein HupB plays an essential role in the pathogenesis of Xcc through the regulation of biofilm formation and biosynthesis of the flagellum.     

Xanthomonas citri ssp. citri requires the outer membrane porin OprB for maximal virulence and biofilm formation

Xanthomonas citri ssp. citri (Xcc) causes canker disease in citrus, and biofilm formation is critical for the disease cycle. OprB (Outer membrane protein B) has been shown previously to be more abundant in Xcc biofilms compared with the planktonic state. In this work, we showed that the loss of OprB in an oprB mutant abolishes bacterial biofilm formation and adherence to the host, and also compromises virulence and efficient epiphytic survival of the bacteria. Moreover, the oprB mutant is impaired in bacterial stress resistance. OprB belongs to a family of carbohydrate transport proteins, and the uptake of glucose is decreased in the mutant strain, indicating that OprB transports glucose. Loss of OprB leads to increased production of xanthan exopolysaccharide, and the carbohydrate intermediates of xanthan biosynthesis are also elevated in the mutant. The xanthan produced by the mutant has a higher viscosity and, unlike wild‐type xanthan, completely lacks pyruvylation. Overall, these results suggest that Xcc reprogrammes its carbon metabolism when it senses a shortage of glucose input. The participation of OprB in the process of biofilm formation and virulence, as well as in metabolic changes to redirect the carbon flux, is discussed. Our results demonstrate the importance of environmental nutrient supply and glucose uptake via OprB for Xcc virulence.     

The wxacO gene of Xanthomonas citri ssp. citri encodes a protein with a role in lipopolysaccharide biosynthesis, biofilm formation, stress tolerance and virulence

Xanthomonas citri ssp. citri (Xcc) causes citrus canker, one of the most economically damaging diseases affecting citrus worldwide. Biofilm formation is important for the pathogen to survive epiphytically in planta prior to the induction of canker symptoms. In this study, two EZ-Tn5 transposon mutants of Xcc strain 306, affected in biofilm formation, were isolated; subsequent analyses led to the identification of a novel gene locus XAC3596 (designated as wxacO), encoding a putative transmembrane protein, and the rfbC gene, encoding a truncated O-antigen biosynthesis protein. Sodium dodecylsulphate-polyacrylamide gel electrophoresis revealed that lipopolysaccharide (LPS) biosynthesis was affected in both wxacO and rfbC mutants. The wxacO mutant was impaired in the formation of a structured biofilm on glass or host plant leaves, as shown in confocal laser scanning microscopy analysis of strains containing a plasmid expressing the green fluorescent protein. Both wxacO and rfbC mutants were more sensitive than the wild-type strain to different environmental stresses, and more susceptible to the antimicrobial peptide polymyxin B. The two mutants were attenuated in swimming motility, but not in flagellar formation. The mutants also showed reduced virulence and decreased growth on host leaves when spray inoculated. The affected phenotypes of the wxacO and rfbC mutants were complemented to wild-type levels by the intact wxacO and rfbC genes, respectively. This report identifies a new gene influencing LPS production by Xcc. In addition, our results suggest that a structurally intact LPS is critical for survival in the phyllosphere and for the virulence of Xcc.     

The novel virulence-related gene nlxA in the lipopolysaccharide cluster of Xanthomonas citri ssp. citri is involved in the production of lipopolysaccharide and extracellular polysaccharide, motility, biofilm formation and stress resistance

Lipopolysaccharide (LPS) is an important virulence factor of Xanthomonas citri ssp. citri, the causative agent of citrus canker disease. In this research, a novel gene, designated as nlxA (novel LPS cluster gene of X. citri ssp. citri), in the LPS cluster of X. citri ssp. citri 306, was characterized. Our results indicate that nlxA is required for O‐polysaccharide biosynthesis by encoding a putative rhamnosyltransferase. This is supported by several lines of evidence: (i) NlxA shares 40.14% identity with WsaF, which acts as a rhamnosyltransferase; (ii) sodium dodecylsulphate‐polyacrylamide gel electrophoresis analysis showed that four bands of the O‐antigen part of LPS were missing in the LPS production of the nlxA mutant; this is also consistent with a previous report that the O‐antigen moiety of LPS of X. citri ssp. citri is composed of a rhamnose homo‐oligosaccharide; (iii) mutation of nlxA resulted in a significant reduction in the resistance of X. citri ssp. citri to different stresses, including sodium dodecylsulphate, polymyxin B, H₂O₂, phenol, CuSO₄ and ZnSO₄. In addition, our results indicate that nlxA plays an important role in extracellular polysaccharide production, biofilm formation, stress resistance, motility on semi‐solid plates, virulence and in planta growth in the host plant grapefruit.     

Comparative proteomic analysis of Xanthomonas citri ssp. citri periplasmic proteins reveals changes in cellular envelope metabolism during in vitro pathogenicity induction

Citrus canker is a plant disease caused by Gram‐negative bacteria from the genus Xanthomonas. The most virulent species is Xanthomonas citri ssp. citri (XAC), which attacks a wide range of citrus hosts. Differential proteomic analysis of the periplasm‐enriched fraction was performed for XAC cells grown in pathogenicity‐inducing (XAM‐M) and pathogenicity‐non‐inducing (nutrient broth) media using two‐dimensional electrophoresis combined with liquid chromatography‐tandem mass spectrometry. Amongst the 40 proteins identified, transglycosylase was detected in a highly abundant spot in XAC cells grown under inducing condition. Additional up‐regulated proteins related to cellular envelope metabolism included glucose‐1‐phosphate thymidylyltransferase, dTDP‐4‐dehydrorhamnose‐3,5‐epimerase and peptidyl‐prolyl cis–trans‐isomerase. Phosphoglucomutase and superoxide dismutase proteins, known to be involved in pathogenicity in other Xanthomonas species or organisms, were also detected. Western blot and quantitative real‐time polymerase chain reaction analyses for transglycosylase and superoxide dismutase confirmed that these proteins were up‐regulated under inducing condition, consistent with the proteomic results. Multiple spots for the 60‐kDa chaperonin and glyceraldehyde‐3‐phosphate dehydrogenase were identified, suggesting the presence of post‐translational modifications. We propose that substantial alterations in cellular envelope metabolism occur during the XAC infectious process, which are related to several aspects, from defence against reactive oxygen species to exopolysaccharide synthesis. Our results provide new candidates for virulence‐related proteins, whose abundance correlates with the induction of pathogenicity and virulence genes, such as hrpD6, hrpG, hrpB7, hpa1 and hrpX. The results present new potential targets against XAC to be investigated in further functional studies.     

Identification of Xanthomonas citri ssp. citri host specificity genes in a heterologous expression host

We provide the first conclusive evidence that Xanthomonas axonopodis pv. citri Asiatic strain (Xac-A) and, in particular, Xac-A^w, a unique citrus canker A strain isolated from Key lime in Wellington, Florida, induces a hypersensitive reaction (HR) in grapefruit leaves. Using the heterologous tomato pathogen X. perforans , as a recipient of the Xac-A^w genomic library, we identified a 1599-bp open reading frame responsible for HR in grapefruit, but not Key lime, and designated it avrGf 1. Xac-A^wΔ avrGf 1 produced typical, although visibly reduced, citrus canker symptoms (i.e. raised pustules) in grapefruit and typical canker symptoms in Key lime. We also determined that the X. perforans transconjugant carrying an Xac-A^w hrpG elicited HR in grapefruit and Key lime leaves, and that xopA in X. perforans was partly responsible for HR. Xac-A transconjugants carrying the X. perforans xopA were reduced in ability to grow in grapefruit leaves relative to wild-type Xac-A. The X. perforans xopA appears to be a host-limiting factor. An avrBs3 homologue, which contained 18.5 repeats and induced HR in tomato, was designated avrTaw . This gene, when expressed in a pustule-minus Xac-A^w, did not complement pustule formation; however, pthA^w , a functional pthA homologue, complemented the mutant strain to produce typical pustules in Key lime, but markedly reduced pustules in grapefruit. Both avrBs3 homologues, when expressed in a typical Xac-A strain, resulted in typical citrus canker pustules in grapefruit, indicating that neither homologue suppressed pustule size in grapefruit. Xac-A^w contains other unidentified factors that suppress development in grapefruit.     

XacFhaB adhesin,an important Xanthomonas citri ssp. citri virulence factor,is recognized as a pathogen‐associated molecular pattern

Adhesion to host tissue is one of the key steps of the bacterial pathogenic process. Xanthomonas citri ssp. citri possesses a non‐fimbrial adhesin protein, XacFhaB, required for bacterial attachment, which we have previously demonstrated to be an important virulence factor for the development of citrus canker. XacFhaB is a 4753‐residue‐long protein with a predicted β‐helical fold structure, involved in bacterial aggregation, biofilm formation and adhesion to the host. In this work, to further characterize this protein and considering its large size, XacFhaB was dissected into three regions based on bioinformatic and structural analyses for functional studies. First, the capacity of these protein regions to aggregate bacterial cells was analysed. Two of these regions were able to form bacterial aggregates, with the most amino‐terminal region being dispensable for this activity. Moreover, XacFhaB shows features resembling pathogen‐associated molecular patterns (PAMPs), which are recognized by plants. As PAMPs activate plant basal immune responses, the role of the three XacFhaB regions as elicitors of these responses was investigated. All adhesin regions were able to induce basal immune responses in host and non‐host plants, with a stronger activation by the carboxyl‐terminal region. Furthermore, pre‐infiltration of citrus leaves with XacFhaB regions impaired X. citri ssp. citri growth, confirming the induction of defence responses and restraint of citrus canker. This work reveals that adhesins from plant pathogens trigger plant defence responses, opening up new pathways for the development of protective strategies for disease control.     

Recent advances in the understanding of Xanthomonas citri ssp. citri pathogenesis and citrus canker disease management

Taxonomic status : Bacteria; Phylum Proteobacteria; Class Gammaproteobacteria; Order Xanthomonadales; Family Xanthomonadaceae; Genus Xanthomonas; Species Xanthomonas citri ssp. citri (Xcc). Host range : Compatible hosts vary in their susceptibility to citrus canker (CC), with grapefruit, lime and lemon being the most susceptible, sweet orange being moderately susceptible, and kumquat and calamondin being amongst the least susceptible. Microbiological properties : Xcc is a rod‐shaped (1.5–2.0 × 0.5–0.75 µm), Gram‐negative, aerobic bacterium with a single polar flagellum. The bacterium forms yellow colonies on culture media as a result of the production of xanthomonadin. Distribution : Present in South America, the British Virgin Islands, Africa, the Middle East, India, Asia and the South Pacific islands. Localized incidence in the USA, Argentina, Brazil, Bolivia, Uruguay, Senegal, Mali, Burkina Faso, Tanzania, Iran, Saudi Arabia, Yemen and Bangladesh. Widespread throughout Paraguay, Comoros, China, Japan, Malaysia and Vietnam. Eradicated from South Africa, Australia and New Zealand. Absent from Europe.     

An Xanthomonas citri pathogenicity gene, pthA, pleiotropically encodes gratuitous avirulence on nonhosts.

The pathogenicity gene, pthA, of Xanthomonas citri is required to elicit symptoms of Asiatic citrus canker disease; introduction of pthA into Xanthomonas strains that are mildly pathogenic or opportunistic on citrus confers the ability to induce cankers on citrus (S. Swarup, R. De Feyter, R. H. Brlansky, and D. W. Gabriel, Phytopathology 81:802-809, 1991). The structure and the function of pthA in other xanthomonads and in X. citri were further investigated. When pthA was introduced into strains of X. phaseoli and X. campestris pv. malvacearum (neither pathogenic to citrus), the transconjugants remained nonpathogenic to citrus and elicited a hypersensitive response (HR) on their respective hosts, bean and cotton. In X. c. pv. malvacearum, pthA conferred cultivar-specific avirulence. Structurally, pthA is highly similar to avrBs3 and avrBsP from X. c. pv. vesicatoria and to avrB4, avrb6, avrb7, avrBIn, avrB101, and avrB102 from X. c. pv. malvacearum. Surprisingly, marker-exchanged pthA::Tn5-gusA mutant B21.2 of X. citri specifically lost the ability to induce the nonhost HR on bean, but retained the ability to induce the nonhost HR on cotton. The loss of the ability of B21.2 to elicit an HR on bean was restored by introduction of cloned pthA, indicating that the genetics of the nonhost HR may be the same as that found in homologous interactions involving specific avr genes. In contrast with expectations of homologous HR reactions, however, elimination of pthA function (resulting in loss of HR) did not result in water-soaking or even moderate levels of growth in planta of X. citri on bean; the nonhost HR, therefore, may not be responsible for the "resistance" of bean to X. citri and may not limit the host range of X. citri on bean. The pleiotropic avirulence function of pthA and the heterologous HR of bean to X. citri are both evidently gratuitous.     

Genome-wide mutagenesis of Xanthomonas axonopodis pv. citri reveals novel genetic determinants and regulation mechanisms of biofilm formation

Xanthomonas axonopodis pv. citri (Xac) causes citrus canker disease, a major threat to citrus production worldwide. Accumulating evidence suggests that the formation of biofilms on citrus leaves plays an important role in the epiphytic survival of this pathogen prior to the development of canker disease. However, the process of Xac biofilm formation is poorly understood. Here, we report a genome-scale study of Xac biofilm formation in which we identified 92 genes, including 33 novel genes involved in biofilm formation and 7 previously characterized genes, colR, fhaB, fliC, galU, gumD, wxacO, and rbfC, known to be important for Xac biofilm formation. In addition, 52 other genes with defined or putative functions in biofilm formation were identified, even though they had not previously reported been to be associated with biofilm formation. The 92 genes were isolated from 292 biofilm-defective mutants following a screen of a transposon insertion library containing 22,000 Xac strain 306 mutants. Further analyses indicated that 16 of the novel genes are involved in the production of extracellular polysaccharide (EPS) and/or lipopolysaccharide (LPS), 7 genes are involved in signaling and regulatory pathways, and 5 genes have unknown roles in biofilm formation. Furthermore, two novel genes, XAC0482, encoding a haloacid dehalogenase-like phosphatase, and XAC0494 (designated as rbfS), encoding a two-component sensor protein, were confirmed to be biofilm-related genes through complementation assays. Our data demonstrate that the formation of mature biofilm requires EPS, LPS, both flagellum-dependent and flagellum-independent cell motility, secreted proteins and extracellular DNA. Additionally, multiple signaling pathways are involved in Xac biofilm formation. This work is the first report on a genome-wide scale of the genetic processes of biofilm formation in plant pathogenic bacteria. The report provides significant new information about the genetic determinants and regulatory mechanism of biofilm formation.     

A novel Xanthomonas citri subsp. citri NADPH quinone reductase involved in salt stress response and virulence

BackgroundXanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker is maintained as an epiphyte on citrus leaves until entering the plant tissue. During epiphytic survival, bacteria may encounter low water availability that challenges the infection process. Proteomics analyses of Xcc under saline stress, mimicking the conditions found during epiphytic survival, showed increased abundance of a putative NAD(P)H dehydrogenase encoded by XAC2229.MethodsExpression levels of XAC2229 and a Xcc mutant in XAC2229 were analyzed in salt and oxidative stress and during plant-pathogen interaction. An Escherichia coli expressing XAC2229 was obtained, and the role of this protein in oxidative stress resistance and in reactive oxygen species production was studied. Finally, Xac2229 protein was purified, spectrophotometric and cofactor analyses were done and enzymatic activities determined.ResultsXAC2229 was expressed under salt stress and during plant-pathogen interaction. ΔXAC2229 mutant showed less number of cankers and impaired epiphytic survival than the wild type strain. ΔXAC2229 survived less in the presence of H₂O₂ and produced more reactive oxygen species and thiobarbituric acid-reactive substances than the wild type strain. Similar results were observed for E. coli expressing XAC2229. Xac2229 is a FAD containing flavoprotein, displays diaphorase activity with an optimum at pH 6.0 and has quinone reductase activity using NADPH as an electron donor.ConclusionsA FAD containing flavoprotein from Xcc is a new NADPH quinone reductase required for bacterial virulence, particularly in Xcc epiphytic survival on citrus leaves.General significanceA novel protein involved in the worldwide disease citrus canker was characterized.     

Biofilm formation, epiphytic fitness, and canker development in Xanthomonas axonopodis pv. citri

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.     

The relationship between PthA expression and the pathogenicity of Xanthomonas axonopodis pv. citri

Citrus canker disease, caused by Xanthomonas axonopodis pv. citri, affects almost all citrus species and cultivars and hascaused severe damage to the citrus industry worldwide. PthA is considered the main pathogenesis effector of the pathogen. This research aimed to temporally and spatially analyze the expression of the PthA protein of the bactrium during its culture, and then try to understand the relationship between the PthA expression levels and the pathogenicity. The relationship between the expression of PthA and the pathogenicity of X. axonopodis pv. citri was fully investigated by using SDS-PAGE, Western blot, ELISA and field inoculation, It was found that bacteria cultured for 36 h had the highest expression of PthA and showed the most virulent pathogenicity. The conservation duration of the pathogen isolates influenced their PthA expression and the pathogenicity, and negative relationship between the duration and the expression of PthA and pathogenicity. When the stored pathogen bacteria were cultured in liquid LB medium, they were able to regain activated, showing higher PthA expression level and enhanced pathogenicity, even though the activity was inferior, in terms of both PthA expression and pathogenicity, than the freshly isolated ones. Seven isolates from different citrus orchards displayed almost identical protein expression profiles. It could conclude that the expressions of PthA was positively related to pathogenicity.     

High-throughput screening and analysis of genes of Xanthomonas citri subsp. citri involved in citrus canker symptom development

Citrus canker is caused by Xanthomonas citri subsp. citri and is one of the most devastating diseases on citrus plants. To investigate the virulence mechanism of this pathogen, a mutant library of strain 306 containing approximately 22,000 mutants was screened for virulence-deficient mutants in grapefruit (Citrus paradise). Eighty-two genes were identified that contribute to citrus canker symptom development caused by X. citri subsp. citri. Among the 82 identified genes, 23 genes were classified as essential genes, as mutation of these genes caused severe reduction of bacterial growth in M9 medium. The remaining 59 genes were classified as putative virulence-related genes that include 32 previously reported virulence-related genes and 27 novel genes. The 32 known virulence-related genes include genes that are involved in the type III secretion system (T3SS) and T3SS effectors, the quorum-sensing system, extracellular polysaccharide and lipopolysaccharide synthesis, and general metabolic pathways. The contribution to pathogenesis by nine genes (pthA4, trpG, trpC, purD, hrpM, peh-1, XAC1230, XAC1548, and XAC3049) was confirmed by complementation assays. We further validated the mutated genes and their phenotypes by analyzing the EZ-Tn5 insertion copy number using Southern blot analysis. In conclusion, we have significantly advanced our understanding of the putative genetic determinants of the virulence mechanism of X. citri subsp. citri by identifying 59 putative virulence-related genes, including 27 novel genes.     

New genes of Xanthomonas citri subsp. citri involved in pathogenesis and adaptation revealed by a transposon-based mutant library

Background  

Citrus canker is a disease caused by the phytopathogens Xanthomonas citri subsp. citri, Xanthomonas fuscans subsp. aurantifolli and Xanthomonas alfalfae subsp. citrumelonis. The first of the three species, which causes citrus bacterial canker type A, is the most widely spread and severe, attacking all citrus species. In Brazil, this species is the most important, being found in practically all areas where citrus canker has been detected. Like most phytobacterioses, there is no efficient way to control citrus canker. Considering the importance of the disease worldwide, investigation is needed to accurately detect which genes are related to the pathogen-host adaptation process and which are associated with pathogenesis.     

Stenotrophomonas maltophilia flagellin is involved in bacterial adhesion and biofilm formation

Stenotrophomonas maltophilia is an emerging drug-resistant pathogen and an important opportunistic pathogen. S. maltophilia flagellin was purified using serial ultracentrifugation. The purity of flagellin was checked by SDS-PAGE. The antibodies were raised in rabbits. The presence of anti-flagellin and the titer of flagellin were detected by immunoblotting and bacterial agglutination techniques. Two methods (viable bacterial count and spectrophotometric methods) were applied to evaluate bacterial adhesion and biofilm formation. Pretreatment of S. maltophilia with dilutions of anti-flagellin (from 1/40 to 1/640) reduced the ability of S. maltophilia to adhere and form biofilms on polystyrene (P < 0.05). In the present study, the inhibition of bacterial adhesion to polystyrene was dose-dependent. The positive correlation was observed between the antibody dilutions and bacterial adhesion (CFU/mL) (r > +0.5, P < 0.05), while, the negative correlation (r < ?0.5, P < 0.05) was observed between the percentage of adhesion inhibition and anti-flagellin dilutions. The current study proved the direct role of S. maltophilia flagellin in bacterial adhesion to and biofilm formation on polystyrene.     

The enterococcal surface protein, Esp, is involved in Enterococcus faecalis biofilm formation

The enterococcal surface protein, Esp, is a high-molecular-weight surface protein of unknown function whose frequency is significantly increased among infection-derived Enterococcus faecalis isolates. In this work, a global structural similarity was found between Bap, a biofilm-associated protein of Staphylococcus aureus, and Esp. Analysis of the relationship between the presence of the Esp-encoding gene (esp) and the biofilm formation capacity in E. faecalis demonstrated that the presence of the esp gene is highly associated (P < 0.0001) with the capacity of E. faecalis to form a biofilm on a polystyrene surface, since 93.5% of the E. faecalis esp-positive isolates were capable of forming a biofilm. Moreover, none of the E. faecalis esp-deficient isolates were biofilm producers. Depending on the E. faecalis isolate, insertional mutagenesis of esp caused either a complete loss of the biofilm formation phenotype or no apparent phenotypic defect. Complementation studies revealed that Esp expression in an E. faecalis esp-deficient strain promoted primary attachment and biofilm formation on polystyrene and polyvinyl chloride plastic from urine collection bags. Together, these results demonstrate that (i) biofilm formation capacity is widespread among clinical E. faecalis isolates, (ii) the biofilm formation capacity is restricted to the E. faecalis strains harboring esp, and (iii) Esp promotes primary attachment and biofilm formation of E. faecalis on abiotic surfaces.