Crystal structures and mutagenesis of sucrose hydrolase from Xanthomonas axonopodis pv. glycines: insight into the exclusively hydrolytic amylosucrase fold

Neisseria polysaccharea amylosucrase (NpAS), a transglucosidase of glycoside hydrolase family 13, is a hydrolase and glucosyltransferase that catalyzes the synthesis of amylose-like polymer from a sucrose substrate. Recently, an NpAS homolog from Xanthomonas axonopodis pv. glycines was identified as a member of the newly defined carbohydrate utilization locus that regulates the utilization of plant sucrose in phytopathogenic bacteria. Interestingly, this enzyme is exclusively a hydrolase and not a glucosyltransferase; it is thus known as sucrose hydrolase (SUH). Here, we elucidated the novel functional features of SUH using X-ray crystallography and site-directed mutagenesis. Four different crystal structures of SUH, including the SUH-Tris and the SUH-sucrose and SUH-glucose complexes, represent structural snapshots along the catalytic reaction coordinate. These structures show that SUH is distinctly different from NpAS in that ligand-induced conformational changes in SUH cause the formation of a pocket-shaped active site and in that SUH lacks the three arginine residues found in the NpAS active site that appear to be crucial for NpAS glucosyltransferase activity. Mutation of SUH to insert these arginines failed to confer glucosyltransferase activity, providing evidence that its enzymatic activity is limited to sucrose hydrolysis by its pocket-shaped active site and the identity of residues in the vicinity of the active site.     

A LOV protein modulates the physiological attributes of Xanthomonas axonopodis pv. citri relevant for host plant colonization

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.     

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.     

Characterization of the Xanthomonas axonopodis pv. glycines Hrp pathogenicity island

We sequenced an approximately 29-kb region from Xanthomonas axonopodis pv. glycines that contained the Hrp type III secretion system, and we characterized the genes in this region by Tn3-gus mutagenesis and gene expression analyses. From the region, hrp (hypersensitive response and pathogenicity) and hrc (hrp and conserved) genes, which encode type III secretion systems, and hpa (hrp-associated) genes were identified. The characteristics of the region, such as the presence of many virulence genes, low G+C content, and bordering tRNA genes, satisfied the criteria for a pathogenicity island (PAI) in a bacterium. The PAI was composed of nine hrp, nine hrc, and eight hpa genes with seven plant-inducible promoter boxes. The hrp and hrc mutants failed to elicit hypersensitive responses in pepper plants but induced hypersensitive responses in all tomato plants tested. The Hrp PAI of X. axonopodis pv. glycines resembled the Hrp PAIs of other Xanthomonas species, and the Hrp PAI core region was highly conserved. However, in contrast to the PAI of Pseudomonas syringae, the regions upstream and downstream from the Hrp PAI core region showed variability in the xanthomonads. In addition, we demonstrate that HpaG, which is located in the Hrp PAI region of X. axonopodis pv. glycines, is a response elicitor. Purified HpaG elicited hypersensitive responses at a concentration of 1.0 micro M in pepper, tobacco, and Arabidopsis thaliana ecotype Cvi-0 by acting as a type III secreted effector protein. However, HpaG failed to elicit hypersensitive responses in tomato, Chinese cabbage, and A. thaliana ecotypes Col-0 and Ler. This is the first report to show that the harpin-like effector protein of Xanthomonas species exhibits elicitor activity.     

An hrcU-homologous gene mutant of Xanthomonas campestris pv. glycines 8ra that lost pathogenicity on the host plant but was able to elicit the hypersensitive response on nonhosts.

Transposon mutagenesis was used to isolate nonpathogenic mutants of Xanthomonas campestris pv. glycines 8ra, which causes bacterial pustule disease in soybean. A 6.1-kb DNA region in which a mutation gave loss of pathogenicity was isolated and found to carry six open reading frames (ORFs). Four ORFs had homology with hrcU, hrcV, hrcR, and hrcS genes of Ralstonia solanacearum and X. campestris pv. vesicatoria. One nonpathogenic mutant, X. campestris pv. glycines H80, lost pathogenicity on soybean but was able to elicit the hypersensitive response (HR) on nonhost pepper and tomato plants. This mutant still multiplied as well as the wild type in the leaves or cotyledons of soybean. Although the DNA and amino acid sequences showed high homology with known hrp genes, the hrcU-homolog ORF is not required for HR induction on nonhost plants, pepper and tomato, or for the multiplication of bacteria in the host plant. This gene was only required for the pathogenic symptoms of X. campestris pv. glycines 8ra on soybean.     

A pthA homolog from Xanthomonas axonopodis pv. citri responsible for host-specific suppression of virulence

Strains of the plant-pathogenic bacterium Xanthomonas axonopodis pv. citri are differentiated into two groups with respect to aggressiveness (normal and weak) on Citrus grandis cultivars but not on other Citrus species such as Citrus sinensis. Random mutagenesis using the transposon Tn5 in X. axonopodis pv. citri strain KC21, which showed weak aggressiveness on a C. grandis cultivar, was used to isolate mutant KC21T46, which regained a normal level of aggressiveness on the cultivar. The gene inactivated by the transposon, hssB3.0, was shown to be responsible for the suppression of virulence on C. grandis. Sequence analysis revealed it to be a new member of the pthA homologs, which was almost identical in sequence to the other homologs except for the number of tandem repeats in the central region of the gene. hssB3.0 appears to be a chimera of other pthA homologs, pB3.1 and pB3.7, and could have been generated by recombination between these two genes. Importantly, in X. axonopodis pv. citri, hssB3.0 was found in all of the tested isolates belonging to the weakly aggressive group but not in the isolates of the normally aggressive group. Isolation of the virulence-deficient mutant KC21T14 from KC21, in which the pathogenicity gene pthA-KC21 was disrupted, showed that hssB3.0 induces a defense response on the host but partially interrupts canker development elicited by the pathogenicity gene in this bacterium.     

HrpG and HrpX play global roles in coordinating different virulence traits of Xanthomonas axonopodis pv. citri

Xanthomonas axonopodis pv. citri is the causal agent of citrus canker, which is one of the most serious diseases of citrus. To understand the virulence mechanisms of X. axonopodis pv. citri, we designed and conducted genome-wide microarray analyses to characterize the HrpG and HrpX regulons, which are critical for the pathogenicity of X. axonopodis pv. citri. Our analyses revealed that 232 and 181 genes belonged to the HrpG and HrpX regulons, respectively. In total, 123 genes were overlapped in the two regulons at any of the three selected timepoints representing three growth stages of X. axonopodis pv. citri in XVM2 medium. Our results showed that HrpG and HrpX regulated all 24 type III secretion system genes, 23 type III secretion system effector genes, and 29 type II secretion system substrate genes. Our data revealed that X. axonopodis pv. citri regulates multiple cellular activities responding to the host environment, such as amino acid biosynthesis; oxidative phosphorylation; pentose-phosphate pathway; transport of sugar, iron, and potassium; and phenolic catabolism, through HrpX and HrpG. We found that 124 and 90 unknown genes were controlled by HrpG and HrpX, respectively. Our results suggest that HrpG and HrpX interplay with a global signaling network and co-ordinate the expression of multiple virulence factors for modification and adaption of host environment during X. axonopodis pv. citri infection.     

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.     

Identification of new protein-protein interactions involving the products of the chromosome- and plasmid-encoded type IV secretion loci of the phytopathogen Xanthomonas axonopodis pv. citri

The recently sequenced genome of the bacterial plant pathogen Xanthomonas axonopodis pv. citri contains two virB gene clusters, one on the chromosome and one on a 64-kb plasmid, each of which codes for a previously uncharacterized type IV secretion system (T4SS). Here we used a yeast two-hybrid assay to identify protein-protein interactions in these two systems. Our results revealed interactions between known T4SS components as well as previously uncharacterized interactions involving hypothetical proteins coded by open reading frames in the two X. axonopodis pv. citri virB loci. Our results indicate that both loci may code for previously unidentified VirB7 proteins, which we show interact with either VirB6 or VirB9 or with a hypothetical protein coded by the same locus. Furthermore, a set of previously uncharacterized Xanthomonas proteins have been found to interact with VirD4, whose gene is adjacent to the chromosomal virB locus. The gene for one member of this family is found within the chromosomal virB locus. All these uncharacterized proteins possess a conserved 120-amino-acid domain in their C termini and may represent a family of cofactors or substrates of the Xanthomonas T4SS.     

Mutation in the xpsD gene of Xanthomonas axonopodis pv. citri affects cellulose degradation and virulence

The Gram-negative bacterium Xanthomonas axonopodis pv. citri, the causal agent of citrus canker, is a major threat to the citrus industry worldwide. Although this is a leaf spot pathogen, it bears genes highly related to degradation of plant cell walls, which are typically found in plant pathogens that cause symptoms of tissue maceration. Little is known on Xac capacity to cause disease and hydrolyze cellulose. We investigated the contribution of various open reading frames on degradation of a cellulose compound by means of a global mutational assay to selectively screen for a defect in carboxymethyl cellulase (CMCase) secretion in X. axonopodis pv. citri. Screening on CMC agar revealed one mutant clone defective in extracellular glycanase activity, out of nearly 3,000 clones. The insertion was located in the xpsD gene, a component of the type II secretion system (T2SS) showing an influence in the ability of Xac to colonize tissues and hydrolyze cellulose. In summary, these data show for the first time, that X. axonopodis pv. citri is capable of hydrolyzing cellulose in a T2SS-dependent process. Furthermore, it was demonstrated that the ability to degrade cellulose contributes to the infection process as a whole.     

The molybdate-binding protein (ModA) of the plant pathogen Xanthomonas axonopodis pv. citri

The modABC operon of phytopathogen Xanthomonas axonopodis pv. citri (X. citri) encodes a putative ABC transporter involved in the uptake of the molybdate and tungstate anions. Sequence analyses showed high similarity values of ModA orthologs found in X. campestris pv. campestris (X. campestris) and Escherichia coli. The X. citri modA gene was cloned in pET28a and the recombinant protein, expressed in the E. coli BL21 (DE3) strain, purified by immobilized metal affinity chromatography. The purified protein remained soluble and specifically bound molybdate and tungstate with K(d) 0.29+/-0.12 microM and 0.58+/-0.14 microM, respectively. Additionally binding of molybdate drastically enhanced the thermal stability of the recombinant ModA as compared to the apoprotein. This is the first characterization of a ModA ortholog expressed by a phytopathogen and represents an important tool for functional, biochemical and structural analyses of molybdate transport in Xanthomonas species.     

Avirulence gene diversity of Xanthomonas axonopodis pv. glycines isolated in Korea

The hybridization patterns with the avrBs3 gene that is known to determine the recognition of host specificity were used to study the diversity of Xanthomonas axonopodis pv. glycines causing bacterial leaf pustule in soybean. A total of 155 strains were isolated from diverse tissues of soybean cultivars collected in Korea and were classified into six different type strains of OcsF, SL1017, SL1018, SL1045, SL1157, and SL2098 according to the patterns of avrBs3-homologous bands. When these type strains were inoculated on various cultivars, most of the Korean strains mildly induced disease symptoms on the resistant CNS1 cultivars. Unlike other type strains, strain SL2098, which appeared not to contain any avrBs3 homolog, induced only a few pustules on even highly susceptible cultivars. When a plasmid carrying the 3.7-kb avrBs3-homologous gene from strain SL1045 was introduced into SL2098, the transformant could not recover the pathogenicity in susceptible host plants. However, when avrBs3-homologous genes of strain SL1018 were mutated by transposon mutagenesis, one of the mutants in which a 5.2-kb chromosomal band homologous to avrBs3 was disrupted could not induce the hypersensitive response on resistant cultivars such as William82 or CNS2. Our results suggest that the avrBs3 homologs may play important roles in the pathogenicity of Xanthomonas axonopodis pv. glycines and the recognition of soybean cultivars.     

A novel sucrose hydrolase from the bombycoid silkworms Bombyx mori,Trilocha varians,and Samia cynthia ricini with a substrate specificity for sucrose

Although membrane-associated sucrase activity has been detected in the midgut of various lepidopteran species, it has not yet been identified and characterized at the molecular level. In the present study, we identified a novel sucrose hydrolase (SUH) gene from the following three bombycoid silkworms: Bombyx mori, Trilocha varians, and Samia cynthia ricini and named them BmSuh, TvSuh, and ScSuh, respectively. The EST dataset showed that BmSuh is one of the major glycoside hydrolase genes in the larval midgut of B. mori. These genes were almost exclusively expressed in the larval midgut in all three species, mainly at the feeding stage. SUHs are classified into the glycoside hydrolase family 13 and show significant homology to insect maltases. Enzymatic assays revealed that recombinant SUHs were distinct from conventional maltases and exhibited substrate specificity for sucrose. The recombinant BmSUH was less sensitive to sugar-mimic alkaloids than TvSUH and ScSUH, which may explain the reason why the sucrase activity in the B. mori midgut was less affected by the sugar-mimic alkaloids derived from mulberry.     

Molecular detection of plant pathogenic bacteria using polymerase chain reaction single-strand conformation polymorphism

The application of polymerase chain reaction (PCR) technology to molecular diagnostics holds great promise for the early identification of agriculturally important plant pathogens. Ralstonia solanacearum, Xanthomoans axonopodis pv. vesicatoria, and Xanthomonas oryzae pv. oryzae are phytopathogenic bacteria, which can infect vegetables, cause severe yield loss. PCR-single-strand conformation polymorphism (PCR-SSCP) is a simple and powerful technique for identifying sequence changes in amplified DNA. The technique of PCR-SSCP is being exploited so far, only to detect and diagnose human bacterial pathogens in addition to plant pathogenic fungi. Selective media and serology are the commonly used methods for the detection of plant pathogens in infected plant materials. In this study, we developed PCR-SSCP technique to identify phytopathogenic bacteria. The PCR product was denatured and separated on a non-denaturing polyacrylamide gel. SSCP banding patterns were detected by silver staining of nucleic acids. We tested over 56 isolates of R. solanacearum, 44 isolates of X. axonopodis pv. vesicatoria, and 20 isolates of X. oryzae pv. oryzae. With the use of universal primer 16S rRNA, we could discriminate such species at the genus and species levels. Species-specific patterns were obtained for bacteria R. solanacearum, X. axonopodis pv. vesicatoria, and X. oryzae pv. oryzae. The potential use of PCR-SSCP technique for the detection and diagnosis of phytobacterial pathogens is discussed in the present paper.     

四种黄单胞菌的基因芯片检测方法的建立

结合双重PCR和基因芯片技术同时检测和鉴定我国检疫性细菌,包括水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,Xoo)、水稻细菌性条斑病菌(X.oryzae pv.oryzicola,Xooc)、柑桔溃疡病菌(X.axonopodis pv.citri,Xac)以及严重危害十字花科作物的甘蓝黑腐病菌(Xanthomonas campestris pv.campestris,Xcc)。以铁载体受体(Putative siderophore receptor)基因序列和RNA多聚酶西格玛因子(RNA polymerase sigma factor,rpoD)基因序列为靶标,设计引物和特异性探针能够同时检测这4种重要的病原菌。对17个细菌菌株进行芯片检测,仅4种靶标菌得到阳性结果,证明此方法具有很高的特异性。4种致病菌基因组DNA的检测灵敏度约为3 pg。检测结果表明,建立的基因芯片检测方法特异性强,能实现上述4种黄单胞菌的准确检测和鉴定,具有良好的应用前景。     

Characterization of ISXax1, a novel insertion sequence restricted to Xanthomonas axonopodis pv. phaseoli (variants fuscans and non-fuscans) and Xanthomonas axonopodis pv. vesicatoria

ISXax1 is a novel insertion sequence belonging to the IS256 and Mutator families. Dot blot, Southern blot, and PCR analyses revealed that ISXax1 is restricted to Xanthomonas axonopodis pv. phaseoli (variants fuscans and non-fuscans) and X. axonopodis pv. vesicatoria strains. Directed AFLP also showed that a high degree of polymorphism is associated with ISXax1 insertion in these strains.     

Reduced genetic variation occurs among genes of the highly clonal plant pathogen Xanthomonas axonopodis pv. vesicatoria, including the effector gene avrBs2

The bacterial plant pathogen Xanthomonas axonopodis pv. vesicatoria, also known as Xanthomonas campestris pv. vesicatoria group A, is the causal agent of bacterial spot in pepper and tomato. In order to test different models that may explain the coevolution of avrBs2 with its host plants, we sequenced avrBs2 and six chromosomal loci (total of 5.5 kb per strain) from a global sample of 55 X. axonopodis pv. vesicatoria strains collected from diseased peppers. We found an extreme lack of genetic variation among all X. axonopodis pv. vesicatoria genomic loci (average nucleotide diversity, pi = 9.1 x 10(-5)), including avrBs2. This lack of diversity is consistent with X. axonopodis pv. vesicatoria having undergone a recent population bottleneck and/or selective sweep followed by population expansion. Coalescent analysis determined that approximately 1.4 x 10(4) to 7.16 x 10(4) bacterial generations have passed since the most recent common ancestor (MRCA) of the current X. axonopodis pv. vesicatoria population. Assuming a range of 50 to 500 bacterial generations per year, only 28 to 1,432 years have passed since the MRCA. This time frame coincides with human intervention with the pathogen's host plants, from domestication to modern agricultural practices. Examination of 19 mutated (loss-of-function) avrBs2 alleles detected nine classes of mutations. All mutations affected protein coding, while no synonymous changes were found. The nature of at least one of the avrBs2 mutations suggests that it may be possible to observe one stage of an evolutionary arms race as X. axonopodis pv. vesicatoria responds to selection pressure to alter avrBs2 to escape host plant resistance.     

Specific detection of Xanthomonas axonopodis pv. dieffenbachiae in anthurium (Anthurium andreanum) tissues by nested PCR

Efficient control of Xanthomonas axonopodis pv. dieffenbachiae, the causal agent of anthurium bacterial blight, requires a sensitive and reliable diagnostic tool. A nested PCR test was developed from a sequence-characterized amplified region marker identified by randomly amplified polymorphic DNA PCR for the detection of X. axonopodis pv. dieffenbachiae. Serological and pathogenicity tests were performed concurrently with the nested PCR test with a large collection of X. axonopodis pv. dieffenbachiae strains that were isolated worldwide and are pathogenic to anthurium and/or other aroids. The internal primer pair directed amplification of the expected product (785 bp) for all 70 X. axonopodis pv. dieffenbachiae strains pathogenic to anthurium tested and for isolates originating from syngonium and not pathogenic to anthurium. This finding is consistent with previous studies which indicated that there is a high level of relatedness between strains from anthurium and strains from syngonium. Strains originating from the two host genera can be distinguished by restriction analysis of the amplification product. No amplification product was obtained with 98 strains of unrelated phytopathogenic bacteria or saprophytic bacteria from the anthurium phyllosphere, except for a weak signal obtained for one X. axonopodis pv. allii strain. Nevertheless, restriction enzyme analysis permitted the two pathovars to be distinguished. The detection threshold obtained with pure cultures or plant extracts (10(3) CFU ml(-1)) allowed detection of the pathogen from symptomless contaminated plants. This test could be a useful diagnostic tool for screening propagation stock plant material and for monitoring international movement of X. axonopodis pv. dieffenbachiae.