Characterisation of IS1126 from Porphyromonas gingivalis W83: a new member of the IS4 family of insertion sequence elements

Abstract The nucleotide sequence of IS 1126 , the only insertion sequence so far isolated from the oral pathogen Porphyromonas gingivalis , has been determined. It had a nucleotide sequence of 1338 base pair (bp) flanked by 12 bp perfect inverted repeats and generated a 5 bp target site duplication. The single major open reading frame encoded a predicted protein of 361 amino acids and molecular mass of 41 kDa. The gene encoding the transpsosase was subcloned into pUC18 and the transposase expressed in Escherichia coli minicells. The predicted amino acid sequence of the transposashad homology to putative transposases of IS 1106 and IS 1186 both of which belong to the IS 5 group within the IS₄ super-family of insertion elements. On the basis of this homology we propose that IS 1126 should also be included in the IS 5 group. Southern-blot analysis of a number of P. gingivalis strains using IS 1126 as a probe revealed a unique pattern of hybridisation for each strain and the absence of IS 1126 from other closely related Porphyromonas species. This should allow IS 1126 to be used as a rapid epidemiological tool in studying oral infections by P. gingivalis .     

High-quality mutant libraries of Xanthomonas oryzae pv. oryzae and X. campestris pv. campestris generated by an efficient transposon mutagenesis system

A novel transposon mutagenesis system for the phytopathogenic bacteria Xanthomonas oryzae pv. oryzae (Xoo) and X. campestris pv. campestris (Xcc) was developed using a Tn5-based transposome. A highly efficient transformation up to 10(6) transformants per microg transposon DNA was obtained. Southern blot and thermal asymmetric interlaced polymerase chain reaction analyses of Tn5 insertion sites suggested a random mode of transposition. The transposition was stable in the transformants for 20 subcultures. Eighteen thousand and 17000 transformants for Xoo and Xcc, respectively, were generated, corresponding to 4X ORF coverage of the genomes. The libraries will facilitate the identification of pathogenicity-related genes as well as functional genomic analysis in Xoo and Xcc.     

野油菜黄单胞菌野油菜致病变种8004菌株wxcA基因与EPS的产量有关

在以前的工作中,采用转座子Tn5 gusA5对野油菜黄单胞菌野油菜致病变种(Xcc)8004菌株进行诱变,获得一批胞外多糖(EPS)合成减少的突变体,对这些突变体的Tn5 gusA5的插入位点进行分析后,发现有两株突变体是wxcA基因不同插入位点的突变体。以前认为wxcA基因与脂多糖(LPS)的O-抗原合成有关而与EPS的合成无关。为明确wxc4基因的功能,对8004菌株的wxcA基因进行缺失,获得的△wxcA突变体的EPS产量与野生型菌株相比,减少了50％,并且一段PCR合成的包含wxcA基因的DNA片段能反式互补△wxcA突变体,恢复突变体的EPS产量。这证实了8004菌株的wxcA基因与EPS的合成产量有关。     

Sensitive and specific detection of Xanthomonas campestris pv campestris by PCR using species-specific primers based on hrpF gene sequences

A sensitive and specific assay was developed to detect bacterial black rot of crucifers caused by Xanthomonas campestris pv. campestris (X. c. pv. campestris), in cabbage seed and plant. Primers XCF and XCR from hrpF homologous to nolX, host recognition protein, were used to amplify a 525 bp DNA fragment. PCR technique was applied to detect the pathogen in naturally infected seed and plant of cabbage. The PCR product was only produced from X. c. pv. campestris among 40 isolates of Xanthomonas strains, Escherichia coli (O157:H7), Pectobacterium carotovorum subsp. carotovorum, and other reference bacteria.     

Genetic and Proteomic Analyses of a Xanthomonas campestris pv. campestris purC Mutant Deficient in Purine Biosynthesis and Virulence

Bacterial proliferation in hosts requires activation of a number of housekeeping pathways, including purine de novo biosynthesis. Although inactivation of purine biosynthesis genes can attenuate virulence, it is unclear which biochemical or virulence factors are associated with the purine biosynthesis pathway in vivo. We report that inactivation of purC, a gene encoding phosphoribosylaminoimidazole-succinocarboxamide synthase, caused complete loss of virulence in Xanthomonas campestris pv. cam- pestris, the causal agent of black rot disease of cruciferous plants. The purC mutant was a purine auxotroph; it could not grow on minimal medium, whereas addition of purine derivatives, such as hypoxanthine or adenine plus guanine, restored growth of the mutant. The purC mutation also significantly enhanced the production of an unknown purine synthesis associated pigment and extracellular polysaccharides by the bacterium. In addition, comparative proteomic analyses of bacteria grown on rich and minimal media revealed that the purC mutation affected the expression levels of diverse proteins involved in purine and pyrimidine synthesis, carbon and energy metabolisms, iron uptake, proteolysis, protein secretion, and signal transduction. These results provided clues to understanding the contributions of purine synthesis to bacterial virulence and interactions with host immune systems.     

Virulence analysis and genetic diversity of Xanthomonas campestris pv. campestris causing black rot of crucifers

Xanthomonas campestris pv. campestris (Xcc) is a devastating bacterium to cause black rot disease in crucifers. To study the genetic diversity and virulence analysis, 24 isolates of Xcc were collected from cole crops including cauliflower, cabbage, broccoli and knol khol from different agro-climatic regions of India ranging from temperate to subtropical climates. For virulence analysis, 24 isolates of Xcc were tested on 27 cultivars of crucifers including seven species of Brassica spp. (B. campestris, B. carinata, B. juncea, B. napus, B. nigra, B. oleracea and B. rapa), Sinapsis alba, Eruca sativa and Raphanus sativus under field conditions at IARI, New Delhi, during November 2010–March 2011. Maximum disease incidence 85.15% was found in the cultivars of crucifers caused by strains Xcc-C124, Xcc-C6, Xcc-C125, Xcc-C111 and Xcc-C131 after 15 days of inoculation and significantly increased after 30 days. Black rot severity in cultivars of crucifers varied from 0 to 6.9 and 0 to 7.9 out of 9 scale after 15 and 30 days of inoculation, respectively. But, no disease incidence was recorded on all the tested cultivars of B. juncea (Pusa Bold, Varuna, Pusa Mustard-21 and Pusa Vijay) against all the strains of Xcc after 15 days. Genetic diversity of 24 strains of Xcc was studied using REP- and BOX-PCR, indicating the existence of wide range of genetic diversity among the strains. The strains were clustered into two groups at 50% similarity coefficient and designated as Group 1 and Group 2. The majority of the strains (23 strains) were clustered under Group 1 except Xcc-C120, which formed separate group (Group 2). In the present study, genetic diversity and virulence pattern in Indian strains of Xcc were established, which will be helpful in the development of resistant genotypes against this bacterial pathogen.     

Optimierung der verfahren zum nachweis des pelargonienwelkeerregers (Xanthomonas campestris pv. pelargonii)

Die weit verbreitete Pelargonienwelke wird von Xanthomonas campestris pv. pelargonii hervorgerufen. In Betrieben mit groß angelegten Pelargonienkulturen kann eine Verbreitung des Erregers große wirtschaftliche Einbußen verursachen. Daher ist schon beim Aufbau der gesunden Mutterpflanzenbestände erforderlich, die Pflanzen auf ihren Gesundheitszustands hin zu kontrollieren, um gegebenenfalls eine rechtzeitige Eliminierung erkrankter Pflanzen einzuleiten. Eine exakte und frühzeitige Diagnose der Bakterien ist notwendig. Mit den bisher konventionell angewendeten Methoden über Differenzialmedien ist der Nachweis der Pathogene nicht immer eindeutig. Es wurden Versuche durchgeführt, X. campestris pv. pelargonii auf serologischem Wege mit Hilfe des ELISA (enzyme‐linked immunosorbent assay) und Immunfluoreszenztest frühzeitig zu erkennen. Das Testsverfahren wurde soweit verbessert, daß ein Nachweis unterhalb von 10⁴ Bakterien/ml möglich ist.     

Loss of chloroplast‐localized protein phosphatase 2Cs in Arabidopsis thaliana leads to enhancement of plant immunity and resistance to Xanthomonas campestris pv. campestris infection

Protein phosphatases (PPs) counteract kinases in reversible phosphorylation events during numerous signal transduction pathways in eukaryotes. PP2Cs, one of the four major classes of the serine/threonine‐specific PP family, are greatly expanded in plants. Thus, PP2Cs are thought to play a specific role in signal transduction pathways. Some rice PP2Cs classified in subgroup K are responsive to infection by the compatible Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight. In Arabidopsis thaliana, orthologous PP2C genes (AtPP2C62 and AtPP2C26) classified to subgroup K are also responsive to Xanthomonas campestris pv. campestris (Xcc, causal agent of black rot) infection. To elucidate the function of these subgroup K PP2Cs, atpp2c62‐ and atpp2c26‐deficient A. thaliana mutants were characterized. A double mutant plant which was inoculated with a compatible Xcc showed reduced lesion development, as well as the suppression of bacterial multiplication. AtPP2C62 and AtPP2C26 localized to the chloroplast. Furthermore, the photosynthesis‐related protein, chaperonin‐60, was indicated as the potential candidate for the dephosphorylated substrate catalysed by AtPP2C62 and AtPP2C26 using two‐dimensional isoelectric focusing sodium dodecylsulfate‐polyacrylamide gel electrophoresis (2D‐IDF‐SDS‐PAGE). Taken together, AtPP2C62 and AtPP2C26 are suggested to be involved in both photosynthesis and suppression of the plant immune system. These results imply the occurrence of crosstalk between photosynthesis and the plant defence system to control productivity under pathogen infection.     

Avirulence gene and insertion element-based RFLP as well as RAPD markers reveal high levels of genomic polymorphism in the rice pathogen Xanthomonas oryzae pv. oryzae

Genetic polymorphism within the genomes of bacterial pathogens determines their evolutionary potential during long-term interaction with their hosts. To investigate the level of genetic variation in Xanthomonas oryzae pv. oryzae (Xoo), the causative agent of rice bacterial blight disease, three DNA marker systems, including (i) restriction fragment length polymorphism (RFLP) of the avrBs3/PthA family genes (avrXa27), (ii) RFLP of insertion (IS) elements and (iii) random amplified polymorphic DNA (RAPD) markers, were used to detect polymorphism among 32 Xoo strains that differed in their virulence patterns. All these strains contained multiple avrXa27 homologs that were variable in copy number and genomic location. RFLP of six IS elements revealed that these mobile sequences were abundant in Xoo genomes, with 150 of the total of 165 discernable markers being variable. Thirty-eight decamer primers of RAPD amplified a total of 691 bands, with 100% of them being variable. In addition, analysis of molecular variance (AMOVA) of data from RFLP analysis of IS elements and from RAPD analysis showed that most of the genetic variation residues were within Xoo populations, rather than between populations. Although all three DNA marker systems supported that substantial variation was maintained in Xoo genomes, Mantel tests did not identify significant correlation between the similarity coefficients calculated from them. The results of the present study indicated that Xoo genomes contain a high level of genetic polymorphism, which greatly facilitates the evolution of this important pathogen during interaction with its host rice plant.     

Induction of defence-related enzymes in tomato (Solanum lycopersicum) plants treated with Bacillus subtilis CBR05 against Xanthomonas campestris pv. vesicatoria

Bacterial spot disease caused by Xanthomonas campestris pv. vesicatoria is one of the most important destructive diseases of tomato in many parts of the agricultural world. Therefore, the present study aims to determine the effects of Bacillus subtilis CBR05 inoculation on bacterial spot disease severity and the induction of defence-related enzymes response in tomato. Tomato leaves were evaluated to determine the activities of antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and polyphenol oxidase (PPO)) and the content of malondialdehyde (MDA). A reduction in bacterial spot severity was observed in plants inoculated with B. subtilis, compared with those of uninoculated controls. A significant increase in SOD, CAT, POD, and PPO activities was observed in plants treated with B. subtilis after 24?h inoculation compared with non-inoculated pathogen control and mock-inoculated controls. Moreover, the MDA content was induced by pathogen infection, and its amount in B. subtilis inoculated plants was significantly lower than that in pathogen control. Our results suggest that early increases in antioxidant enzymes and the reduction in MDA content with B. subtilis inoculation may play a pivotal role in mitigating oxidative stress, thereby induced systemic resistance against bacterial spot disease in tomato.     

Comparative microscopic and enzymatic characterization of the leaf necrosis induced in Arabidopsis thaliana by lead nitrate and by Xanthomonas campestris pv. campestris after foliar spray

The heavy metal lead was administered to Arabidopsis thaliana plants by foliar spray. At a concentration of l4mol m^?3, the lead nitrate suspension induced densely distributed necrotic lesions on A. thaliana leaves. A number of Arabidopsis ecotypes were tested and a differential response to heavy-metal toxicity was noted. The necrosis provoked as a result of the phytotoxic effect of lead had a similar appearance to the necrotic lesions observed in a hypersensitive response of A. thaliana to inoculation with Xanthomonas campestris pv. campestris (Lummerzheim et al. 1993, Molecular Plant-Microbe Interactions 6, 532–544). In addition to this phenotypic resemblance, accumulation of polyphenols and callose depositions observed by microscopic analysis, as well as increases in the activities of the stress-related proteins β1,3-glucanases, chitinases and peroxidases, revealed significant similarities in the plant response to the two treatments examined, lead toxicity and bacterial infection. The results allow the establishment of markers for both types of stress.     

Biochemical and molecular identification of Xanthomonas translucens pv. undulosa causing bacterial leaf streak of wheat in Punjab,Pakistan

Xanthomonas translucens pv. undulosa, (Xtu.), causal agent of Bacterial leaf streak (BLS) of wheat, was characterised through pathogencity, hypersensitivity, biochemical and molecular assays. Fifty symptomatic leaves of wheat were collected from eight agro-ecological zones of Punjab out of which 25 were isolated and purified. Maximum incidence and severity in Faisalabad were followed by Multan and Rahim Yar Khan. The pathogen isolated from diseased leaves was identified on the basis of colonies pattern, colour, biochemical and pathogencity test as X. translucens pv. undulosa and confirmed its pathogencity through pathogencity test. For molecular characterization, the bacterial 16S–23S rDNA spacer fragments were amplified by PCR with conserved primers (C1 and C2) and then in combination with specific primers (T1 & T2). 300?bp product amplified by C1 and C2 primer pair confirmed the presence of Xanthomonas, while specific primers T1 and T2 amplified a product of 200?bp, confirmed the presence of X. translucens pv. undulosa. This work will be quite helpful for wheat pathologist and breeders for future management strategy for this disease.     

Structural analyses of novel glycerophosphorylated alpha-cyclosophorohexadecaoses isolated from X. campestris pv. campestris

Novel periplasmic anionic cyclic glucans produced by Xanthomonas campestris pv. campestris were isolated by trichloroacetic acid treatment and various chromatographic techniques. No report has been made on the presence of substituted cyclic glucans of the Xanthomonas species. We show, for the first time, that X. campestris pv. campestris produces the anionic cyclic glucans with phosphoglycerol residues, the presence of which can be predicted by analyzing the sequence database with the aid of the NCBI RefSeq database. To analyze the structure of isolated anionic cyclic glucans analyses, we used NMR spectroscopy, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOFMS) and electrospray-ionization mass spectrometry (ESIMS). The results suggest that the novel anionic forms of the cyclic glucans of X. campestris pv. campestris are glycerophosphorylated alpha-cyclosophorohexadecaose with one or two phosphoglycerol substituents at the C-6 positions of the glucose residues.     

Genetic and structural characterization of the avirulence gene avrBs3 from Xanthomonas campestris pv. vesicatoria

Summary The avirulence gene avrBs3 from Xanthomonas campestris pv. vesicatoria was cloned and found to be localized on a self-transmissable plasmid. Genetic analysis of an avrBs3 insertion mutation revealed that avrBs3 constitutes a single locus, specifying the resistant phenotype on pepper plants. Southern blot experiments showed that no DNA sequences homologous to avrBs3 were present in other races of X. c. pv. vesicatoria, which are unable to induce a hypersensitive reaction on ECW-30R. However, the DNA of several different pathovars of X. campestris hybridized to the avrBs3 probe. A deletion analysis defined a region of 3.6–3.7 kb essential for avrBs3 activity. The nucleotide sequence of this region was determined. A 3561 nucleotide open reading frame (ORF1), encoding a 125000 dalton protein, was found in the 3.7 kb region that was sufficient for avrBs3 activity. A second long ORF (2351 nucleotides) was identified on the other strand. A remarkable feature of both ORFs is the presence of 17 direct repeats of 102 bp which share 91%–100% homology with each other.     

水稻条斑病菌gacA基因的克隆及功能初析

根据黄单胞菌gacA基因的同源性设计简并引物,采用PCR方法从水稻条斑病菌(Xanthomonas oryzae pv.oryzicola,Xooc)中克隆了gacA同源基因,命名为gacAXooc。序列比较显示,该基因在黄单胞菌中是相对保守的。通过同源重组的方法,构建了gacAXooc的插入突变株。对0.1% Tryptone的趋化应答能力检测发现,gacA突变株的趋化能力明显降低,证明gacA与Xooc的趋化性相关。     

Gene expression in Brassica campestris showing a hypersensitive response to the incompatible pathogen Xanthomonas campestris pv. vitians

Xanthomonas campestris pv. vitians, a pathogen of lettuce, elicits a hypersensitive response within 12 hours of inoculation into Brassica leaves, characterized by tissue collapse, loss of membrane integrity, vein blockage and melanin production. In contrast, the compatible pathogen, X. c. pv. campestris, has no visible effects on leaves for 48 hours, after which inoculated areas show chlorosis which eventually spreads, followed by rotting.mRNA was prepared from leaves inoculated with suspensions of both pathovars or with sterile medium up to 24 hours following inoculation. In vitro translation of total and poly A⁺ RNA in rabbit reticulocyte lysate in the presence of ³⁵S methionine followed by separation of the polypeptide products by 2D-PAGE, allowed comparison of the effects of these treatments on plant gene expression. Major changes in gene expression were observed as a consequence of the inoculation technique. In addition, after inoculation with X. c. vitians, up to fifteen additional major polypeptides appeared or greatly increased by four hours. Some of these had disappeared by nine hours and several more had appeared. No major polypeptides disappeared or decreased greatly in intensity following inoculation with X. c. vitians.