Molecular and pathogenic characterization of new Xanthomonas oryzae pv. oryzae strains from the coastline region of Fangchenggang city in China

Virulence assays and DNA polymorphism analyses were used to characterize 33 Xanthomonas oryzae pv. oryzae (Xoo) strains collected from the coastline region of Fangchenggang city in China. Two new pathogenic races (FXP1 and FXP2), were determined by leaf-clipping inoculation of 12 near-isogenic International Rice-Bacterial Blight (IRBB) rice lines, each containing a single resistance gene. Race FXP1 consisted of twenty-eight strains that were incompatible on IRBB5 and IRBB7, while race FXP2 included five strains that were incompatible on IRBB5 and IRBB7 and moderately virulent on IRBB8 containing the xa8 gene. Restriction fragment length polymorphism (RFLP) analysis revealed that each probe of avrXa10 and IS1112 resolved two haplotypes. In a dendrogram generated from the combined RFLP data, the 33 Xoo strains were resolved into two clusters. There was a weak correlation (r = 0.53) between race and haplotype. All of the rice cultivars planted in the coastline region of Fangchenggang city were susceptible to the representative Xoo strains tested above. However, we found that four rice cultivars used as breeding materials in the laboratory could fully resist infection by the Xoo strains, suggesting that the isolated Xoo strains could be used to detect resistant rice cultivars suitable for planting in the local rice field.     

Requirement of a mip-like gene for virulence in the phytopathogenic bacterium Xanthomonas campestris pv. campestris

Macrophage infectivity potentiators (Mips) are FKBP domain-containing proteins reported as virulence factors in several human pathogens, such as members of genera Legionella, Salmonella and Chlamydia. The putative peptidylprolyl cis-trans isomerase (PPIase) encoded by XC2699 of the plant bacterial pathogen Xanthomonas campestris pv. campestris 8004 exhibits a 49% similarity at the amino-acid level to the Mip protein of Legionella pneumophila. This mip-like gene, XC2699, was overexpressed in Escherichia coli and the purified (His)6-tagged Mip-like protein encoded by XC2699 exhibited a PPIase activity specifically inhibited by FK-506. A mutation in the mip-like gene XC2699 led to significant reductions in virulence and replication capacity in the host plant Chinese radish (Raphanus sativus L. var. radiculus Pers.). Furthermore, the production of exopolysaccharide and the activity of extracellular proteases, virulence factors of X. campestris pv. campestris, were significantly decreased in the mip-like mutant. These results reveal that the mip-like gene is involved in the pathogenesis of X. campestris pv. campestris through an effect on the production of these virulence factors.     

Cyclic di-GMP signalling in the virulence and environmental adaptation of Xanthomonas campestris

Cyclic di-GMP is a second messenger with a role in regulation of a range of cellular functions in diverse bacteria including the virulence of pathogens. Cellular levels of cyclic di-GMP are controlled through synthesis, catalysed by the GGDEF protein domain, and degradation by EAL or HD-GYP domains. Here we report a comprehensive study of cyclic di-GMP signalling in bacterial disease in which we examine the contribution of all proteins with GGDEF, EAL or HD-GYP domains to virulence and virulence factor production in the phytopathogen Xanthomonas campestris pathovar campestris (Xcc). Genes with significant roles in virulence to plants included those encoding proteins whose probable function is in cyclic-di-GMP synthesis as well as others (including the HD-GYP domain regulator RpfG) implicated in cyclic di-GMP degradation. Furthermore, RpfG controlled expression of a subset of these genes. A partially overlapping set of elements controlled the production of virulence factors in vitro. Other GGDEF-EAL domain proteins had no effect on virulence factor synthesis but did influence motility. These findings indicate the existence of a regulatory network that may allow Xcc to integrate information from diverse environmental inputs to modulate virulence factor synthesis as well as of cyclic di-GMP signalling systems dedicated to other specific tasks.     

Comparative and functional genomics reveals genetic diversity and determinants of host specificity among reference strains and a large collection of Chinese isolates of the phytopathogen Xanthomonas campestris pv. campestris

Background

Xanthomonas campestris pathovar campestris (Xcc) is the causal agent of black rot disease of crucifers worldwide. The molecular genetic diversity and host specificity of Xcc are poorly understood.

Results

We constructed a microarray based on the complete genome sequence of Xcc strain 8004 and investigated the genetic diversity and host specificity of Xcc by array-based comparative genome hybridization analyses of 18 virulent strains. The results demonstrate that a genetic core comprising 3,405 of the 4,186 coding sequences (CDSs) spotted on the array are conserved and a flexible gene pool with 730 CDSs is absent/highly divergent (AHD). The results also revealed that 258 of the 304 proved/presumed pathogenicity genes are conserved and 46 are AHD. The conserved pathogenicity genes include mainly the genes involved in type I, II and III secretion systems, the quorum sensing system, extracellular enzymes and polysaccharide production, as well as many other proved pathogenicity genes, while the AHD CDSs contain the genes encoding type IV secretion system (T4SS) and type III-effectors. A Xcc T4SS-deletion mutant displayed the same virulence as wild type. Furthermore, three avirulence genes (avrXccC, avrXccE1 and avrBs1) were identified. avrXccC and avrXccE1 conferred avirulence on the hosts mustard cultivar Guangtou and Chinese cabbage cultivar Zhongbai-83, respectively, and avrBs1 conferred hypersensitive response on the nonhost pepper ECW10R.

Conclusion

About 80% of the Xcc CDSs, including 258 proved/presumed pathogenicity genes, is conserved in different strains. Xcc T4SS is not involved in pathogenicity. An efficient strategy to identify avr genes determining host specificity from the AHD genes was developed.     

IL-2与金黄色葡萄球菌肠毒素A和B融合基因的克隆及表达

目的：IL-2与金黄色葡萄球菌肠毒素A和B融合基因的克隆及表达。方法：分别在金黄色葡萄球菌肠毒素A227Ala、B基因的两端克隆上两个酶切点Hind Ⅲ,Kpn Ⅰ。将IL-2基因突变,设计一段linker使之分别与SEA227Ala和SEB相连并克隆到PET表达载体中,在大肠杆菌DH5a（DE3）-Pass中表达。结果：表达的蛋白占总蛋白15%。结论：IL-2与金黄色葡萄球菌肠毒素A和B融合蛋白能在大肠杆菌中有效表达。     

金黄色葡萄球菌肠毒素B基因的克隆和在大肠杆菌中高效表达

利用PCR方法从金黄色葡萄球菌TSTw基因组DNA中扩增出约700bp的DNA片段,将之克隆到pGEM7Zf(+)载体上并转化大肠杆菌 DH5α菌株。重组质粒的测序结果表明克隆到了seb基因,它含有717bp（不包括N端81bp的信号肽编码区）,其核苷酸序列与文献报道完全一致。将其连接于表达载体7ZTS上,转化到大肠杆菌JM109（DE3）内。表达的SEB占总蛋白33.5%。      

转拟南芥AtNPR1基因增强水稻对水稻白叶枯病和稻瘟病的抗性

AtNPR1基因是拟南芥系统获得抗性的一个重要调节基因,在拟南芥中过量表达AtNPR1基因能使拟南芥对细菌和真菌的抗性同时增强.为了研究在水稻中过量表达AtNPR1基因对水稻抗病性的影响,将该基因转入到广西主栽籼稻恢复系品种桂99中.经PCR验证得到了79株转基因植株,DNA斑点杂交表明ATNPR1基因已经整合到桂99染色体DNA中.Northern杂交和RT-PCR分析表明,AtNPR1基因在桂99中已经表达;同时还检测了转基因植株对水稻白叶枯病和稻瘟病的抗性,结果表明转基因植株对该两种病害的抗性均显著增强.     

Novel Carbohydrate-Binding Module Identified in a Ruminal Metagenomic Endoglucanase

Endoglucanase C5614-1 comprises a catalytic module (CM) and an X module (XM). The XM showed no significant homology with known carbohydrate-binding modules (CBMs). Recombinant full-length endoglucanase could bind Avicel, whereas the CM could not. The XM could bind various polysaccharides. The results demonstrated that the XM was a new CBM.Most cellulases are modular proteins that comprise two or more discrete modules, such as catalytic modules (CMs) and carbohydrate-binding modules (CBMs), each of which can function independently (9). CBMs are classified into 59 families based on their amino acid similarity in the CAZY database (http://www.cazy.org/fam/acc_CBM.html). The main functions of CBMs are to recognize and bind polysaccharides and to increase the hydrolytic activities of the enzymes against insoluble and soluble substrates (3). Endoglucanase C5614-1 (GenBank accession no. {"type":"entrez-protein","attrs":{"text":"ACA61140","term_id":"169640132","term_text":"ACA61140"}}ACA61140), which was identified from the metagenome of the contents of buffalo rumen (5), is a modular enzyme comprising an N-terminal signal peptide (amino acids [aa] 1 to 20), a CM belonging to the glycosyl hydrolase family 5 (aa 40 to 334), and a C-terminal X module (XM) of unknown function (aa 335 to 537) (Fig. ​(Fig.1A).1A). No linker region rich in Ser/Pro/Thr was found between the CM and the XM. In this study, we aimed to ascertain the function of the XM in the endoglucanase C5614-1.Open in a separate windowFIG. 1.Endoglucanase C5614-1 and its derivatives. (A) Modular organization of C5614-1 and its truncated derivatives. Abbreviations: SP, signal peptide; GHF5, GHF5 catalytic module; X, the X module (XM). (B) SDS-PAGE of purified recombinant proteins. Protein samples were analyzed on a 10% gel. Lane 1, protein molecular mass standard (molecular masses are shown on the left); lane 2, rC5614-1; lane 3, rGHF5; lane 4, rX.The XM showed no significant homology to known CBMs. The XM shared 25% to 33% identities and 41% to 45% similarities with about 200 amino acids at the C terminus of a xylanase (GenBank accession no. {"type":"entrez-protein","attrs":{"text":"AAC36862","term_id":"143974","term_text":"AAC36862"}}AAC36862) from the ruminal bacterium Prevotella ruminicola, uncultured ruminal microbial cellulases ({"type":"entrez-protein","attrs":{"text":"ABX76045","term_id":"161898163","term_text":"ABX76045"}}ABX76045, {"type":"entrez-protein","attrs":{"text":"ACA61132","term_id":"169640121","term_text":"ACA61132"}}ACA61132, {"type":"entrez-protein","attrs":{"text":"ACA61135","term_id":"169640125","term_text":"ACA61135"}}ACA61135, {"type":"entrez-protein","attrs":{"text":"ACA61137","term_id":"169640128","term_text":"ACA61137"}}ACA61137, and {"type":"entrez-protein","attrs":{"text":"ABB46200","term_id":"78522584","term_text":"ABB46200"}}ABB46200), and an uncultured bacterial bifunctional mannanase-xyloglucanase ({"type":"entrez-protein","attrs":{"text":"ADA62505","term_id":"281335881","term_text":"ADA62505"}}ADA62505). None of these homologous polypeptides was confirmed to show carbohydrate-binding activity. An alignment of XM with these homologous sequences using ClustalW (http://www.ebi.ac.uk/Tools/clustalw) is shown in Fig. ​Fig.2.2. Seven conserved aromatic amino acid residues were found in the respective sequences. Aromatic amino acid residues in CBMs play critical roles in recognizing and binding polysaccharides (4).Open in a separate windowFIG. 2.Multiple sequence alignment of the XM (203 aa) in endoglucanase C5614-1 (aa 335 to 537), with its homologous sequences. The identical and similar amino acid residues are indicated by asterisks and dots, respectively, below the alignment. The conserved aromatic amino acid residues are indicated by arrows. GenBank accession no. {"type":"entrez-protein","attrs":{"text":"AAC36862","term_id":"143974","term_text":"AAC36862"}}AAC36862, Prevotella ruminicola xylanase (aa 376 to 584); {"type":"entrez-protein","attrs":{"text":"ABB46200","term_id":"78522584","term_text":"ABB46200"}}ABB46200, ruminal uncultured bacterium endoglycosidase precursor protein (aa 719 to 917); {"type":"entrez-protein","attrs":{"text":"ABX76045","term_id":"161898163","term_text":"ABX76045"}}ABX76045, ruminal uncultured microorganism endo-1,4-beta-d-glucanase (aa 342 to 552); {"type":"entrez-protein","attrs":{"text":"ADA62505","term_id":"281335881","term_text":"ADA62505"}}ADA62505, ruminal uncultured bacterium bifunctional mannanase-xyloglucanase (aa 721 to 919); {"type":"entrez-protein","attrs":{"text":"ACA61132","term_id":"169640121","term_text":"ACA61132"}}ACA61132, ruminal uncultured microorganism cellulase C29-2 (aa 341 to 553); {"type":"entrez-protein","attrs":{"text":"ACA61135","term_id":"169640125","term_text":"ACA61135"}}ACA61135, ruminal uncultured microorganism cellulase C35-2 (aa 337 to 552); {"type":"entrez-protein","attrs":{"text":"ACA61137","term_id":"169640128","term_text":"ACA61137"}}ACA61137, ruminal uncultured microorganism cellulase C67-1 (aa 335 to 546). The amino acid numbers in each of the parentheses above define the range of the homologous region in each sequence.A PCR-based approach was used to produce constructs expressing C5614-1 derivatives (Fig. ​(Fig.1A).1A). Plasmid C5614 (5), carrying the endoglucanase gene C5614-1, was used as a PCR template. The primer pairs used to amplify the portions of C5614-1 encoding C5614-1 amino acids 20 to 537 (recombinant C5614-1 [rC5614-1], comprising the CM and the XM), 20 to 348 (rGHF5, containing the CM only), and 349 to 537 (rX, containing the XM only) are, respectively, as follows: C5614-1F (5′CAGCCATGGAGGCACAAGATTTTGAGACTGCTACCGAA-3′) and C5614-1R (5′-GACCTCGAGTTGTGCTATGTATTTTTTGCCGTTCTGG-3′), C5614-1F and C5614-1CM (5′-GGGCTCGAGGGTTAATGTCTCAGCCAGGTCAGGCTG-3′), and C5614-1X (5′-GGGCCATGGCCAAAGCCTATCATGGCAGCGCGTTC-3′) and C5614-1R. The underlined sequences in the primers are NcoI and XhoI restriction sites.The digested PCR products were ligated into the same digested expression vector, pET-30a(+) (Novagen, Madison, WI). The recombinant plasmids were transformed into Escherichia coli Rosetta(DE3)pLysS (Novagen), and the cloned fragments were expressed as proteins with 6×His tags at both the N and C termini. The recombinant C5614-1 derivatives were purified by affinity chromatography with Cobalt immobilized metal chromatography resin (Clontech, Palo Alto, CA), according to the user manual. Each of the purified proteins produced a single band on an SDS-PAGE gel (8), and their molecular sizes were in agreement with those of the deduced polypeptides (Fig. ​(Fig.1B1B).The hydrolytic activities of rC5614-1 and rGHF5 toward carboxymethyl cellulose (CMC) were determined essentially as described by Duan et al. (5). The pH profiles of the enzymatic reactions of rC5614-1 and rGHF5 were similar, and both showed maximum activities at pH 5.0 (data not shown). However, rC5614-1 and rGHF5 showed different temperature profiles (Fig. ​(Fig.3A).3A). The rGHF5 showed narrower pH stability and lower temperature stability profiles than those of rC5614-1 (Fig. 3B and C). These results indicated that the XM is required for the stability of rC5614-1.Open in a separate windowFIG. 3.Effects of pH and temperature on the activities and stability of rC5614-1 and rGHF5. (A) Influence of temperature on the activities of rC5614-1 and rGHF5. Cellulase activity was measured at pH 5.0 in citrate-phosphate buffer at the indicated temperatures for 10 min. Values are expressed as percentages of maximal activity at 50°C and 35°C for rC5614-1 and rGHF5, respectively. (B) Influence of pH on rC5614-1 and rGHF5 stability. Purified enzyme was first incubated in citrate-phosphate buffer (pH 2.5 to 7.0), 0.1 M Tris-HCl buffer (pH 7.5 to 8.5), or 0.1 M glycine-NaOH buffer (pH 9.0 to 12) at 4°C for 24 h, and activity was measured under optimal conditions for 10 min. Values are expressed as percentages of maximal activity when the sample was incubated under pH 5.5 and pH 6.0 for rC5614-1 and rGHF5, respectively. (C) Thermal stability of recombinant rC5614-1 and rGHF5. Purified enzymes were first incubated at the indicated temperatures for 1 h; activity was then measured under optimal conditions for 10 min. Values are expressed as percentages of untreated-sample activity.The hydrolytic activities of rC5614-1 toward particle substrates, including birch wood xylan, lichenan, and acid-swollen cellulose (ASC), were about three times greater than those of rGHF5; however, rC5614-1 and rGHF5 showed similar hydrolytic activities toward soluble substrates (Table ​(Table1).1). This result indicated that the presence of the XM enhanced the hydrolytic activity of the enzyme toward insoluble substrates but not toward soluble substrates. Similar phenomena were reported for Irpex lacteus exocellulase I (7), Clostridium thermocellum Xyn10C (1), and Clostridium stercorarium Xyn10B (2).

TABLE 1.

Specific activities of rC5614-1 and rGHF5 toward various substrates