Cellular Fatty Acid Composition of Nine Pathovars of Xanthomonas campestris

Cellular fatty acids of 80 strains of Xanthomonas campestris, representing 9 different pathovars, were analyzed by gas-liquid chromatography and mass spectrometry. A total of 48 fatty acids were identified, the most important being the 16:0 (averaging at least 4.5 % of the total), the cis- and trans- 9 16 : 1 (over 14.4 %), and the iso and anteiso 15 : 0 (over 30 %). Other major fatty acids (averaging over 1 % of total) were the saturated 14 : 0 and 15 : 0, the hydroxy-substituted iso 3-OH 11 : 0, 3-OH 12 : 0 and iso 3-OH 13 : 0, and the branch-chained iso 11 : 0, iso 16 : 0, iso 17 : 1, iso 17 : 0 and anteiso 17 : 0. Of 33 minor fatty acids detected and identified, only 7 have been previously reported in the xanthomonads. Significant differences in mean percentages of 5 major fatty acids and 4 (chemical) class totals were detected among pathovars, which statistically segregated into three groups by rank analysis. X. campestris pv. dieffenbachiae was in a group by itself; pvs. campestris, citri (pathotypes A and B), manihotis, phaseoli, pruni and vesicatoria were in a seond group, and pvs. glycines, begonia and citri (pathotype E) were in a third.     

Outer Membrane Proteins and Lipopolysaccharides in Pathovars of Xanthomonas campestris

Variations in the outer membrane proteins (OMPs) and lipopolysaccharides (LPSs) of 54 isolates belonging to 16 different pathovars of Xanthomonas campestris were characterized. OMP samples prepared by sarcosyl extraction of cell walls and LPS samples prepared by proteinase K treatment of sonicated cells were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of 4 M urea. In general, the OMP and LPS profiles within each pathovar were very similar but different from the profiles of other pathovars. Heterogeneity in OMP and LPS profiles was observed within X. campestris pv. campestris, X. campestris pv. translucens, and X. campestris pv. vesicatoria. LPSs were isolated from six X. campestris pathovars, which fell into two major groups on the basis of O antigenicity. The O antigens of X. campestris pv. begoniae, X. campestris pv. graminis, and X. campestris pv. translucens cross-reacted with each other; the other group consisted of X. campestris pv. campestris, X. campestris pv. pelargonii, and X. campestris pv. vesicatoria. A chemical analysis revealed a significant difference between the compositions of the neutral sugars of the LPSs of those two groups; the LPSs of the first group contained xylose and a 6-deoxy-3-O-methyl hexose, whereas the LPSs of the other group lacked both sugars.     

Induction of Hydrolytic Enzymes in Brassica campestris in Response to Pathovars of Xanthomonas campestris

Inoculation of mature leaves of turnip (Brassica campestris) with the incompatible Xanthomonas campestris pv vitians resulted in the induction of β-1,3-glucanase and chitinase/lysozyme (CHL) activity. No increase in the basal activity of β-1,3-glucanase was observed after inoculation of leaves with heat- or rifampicin-killed X. c. vitians, Escherichia coli, or sterile water. Inoculation with the compatible X. campestris pv campestris resulted in a slower induction of glucanase than that seen with X. c. vitians. In contrast, all bacteria caused an induction of CHL activity. One major β-1,3-glucanase (molecular mass 36.5 kilodaltons, isoelectric point [pl] ~8.5) was purified from both inoculated and untreated leaves by ion-exchange chromatography. The enzyme degraded laminarin by an endo-glycolytic mechanism. Two major CHL isozymes (CHL 1 and CHL 2, molecular mass 30 kilodaltons and pl 9.4 and 10.2, respectively) were purified from X. c. vitians inoculated leaves by affinity chromatography on a chitin column followed by ion-exchange chromatography. Both enzymes degraded chitin by an endo-glycolytic mechanism although the ratio of lysozyme to chitinase specific activities for CHL 1 and CHL2 were different. The induction of CHL 1 was associated with the hypersensitive reaction caused by X. c. vitians whereas all other treatments induced largely CHL 2.     

Fingerprinting of Xanthomonas campestris pv. pelargonii and Related Pathovars Using Random-Primed PCR

Polymerase chain reaction (PCR) amplification of total DNA was evaluated as a method to distinguish Xanthomonas campestris pv. pelargonii from other pathovars within this species. Two sets of highly conserved enterobacterial consensus sequences were used as targets for PCR amplification: (a) enterobacterial repetitive intergenic consensus [ERIC] and (b) repetitive extragenic palindromic [REP] sequences. Nucleic acid was extracted from a total of 37 isolates of bacteria: 19 isolates ofX campestris pv. pelargonii and 18 isolates representing 10 other pathovars of X. campestris. After PCR amplification using the ERIC primer pair the DNA fingerprints of X. campestris pv, pelargonii contained two major DNA products (estimated size 500 and 740 pp) that were conserved among all 19 isolates. With the REP primer pair, the fingerprints were more complex and major DNA products ranging from -690 to 1650 bp were detected. Using information from both ERIC- and REP-primed Imgerprints, the X. campestris pv. pelargonii fingerprints were distinguishable from the fingerprints of the other pathovars examined: pvs. citrumelo. citri, beganiae, vittans B and C. phaseoli. campestris, manihotis, juglandis, carotae and pruni.     

TAIL-PCR方法快速分离Xcc致病相关基因序列

以mini-Tn5 gfp-km转座子中nptⅡ片段作为探针,对已获得的五株野油菜黄单胞菌野油菜黑腐病致病型（Xcc)非致病突变体进行了Southern blot分析,结果表明,这五株突变体确由mini-Tn5 gfp-km转座子插入致病相关基因所致,且为单拷贝不同位点的插入。提取这五株突变体总DNA作为模板,采用改进的热不对称交错PCR（TAIL－PCR）方法从其中克隆到了各自转座子插入区侧翼序列,对这些侧翼序列进行了序列测定并将分析结果与GenBank database及Xcc全基因组序列做了比较,结果表明,五个侧翼序列所在的基因确与Xcc致病性有关。这种改进后的TAIL－PCR方法为突变体特别是转座子插入突变体中目的基因的克隆提供了一种简要高效的新方法。     

十字花科黑腐病菌hrpG基因原核表达

在十字花科黑腐病菌(Xcc)中,hrp基因对寄主的致病性和非寄主的超敏反应中起核心作用,而hrpG对整个hrp基因簇起调控作用.HrpG为OmpR家族的双组分系统感受调控蛋白,含有两个结构域,分别是N端Response_reg和C端Trans reg_C.本研究利用表达载体pQE-30 Xa,成功构建了HrpG的表达重组子,在E.coli M15 [pREP4]中进行诱导表达.通过调节诱导温度、IPTG浓度和诱导时间最终确定在温度为20℃,IPTG浓度为0.8 mmol/L,诱导表达4 h.hrpG基因在宿主细胞E.coli M15获得高效可溶性表达.目前尚未有可溶性HrpG蛋白获得成功表达的报导,本研究中获得HrpG蛋白在大肠杆菌获得大量可溶性的表达,将为in vitro研究HrpG的生理活性,特异的结合位点和调控功能研究打下良好基础.     

Characterization of a Unique Chromosomal Copper Resistance Gene Cluster from Xanthomonas campestris pv. vesicatoria

We characterized the copper resistance genes in strain XvP26 of Xanthomonas campestris pv. vesicatoria, which was originally isolated from a pepper plant in Taiwan. The copper resistance genes were localized to a 7,652-bp region which, based on pulsed-field gel electrophoresis and Southern hybridization, was determined to be located on the chromosome. These genes hybridized only weakly, as determined by Southern analysis, to other copper resistance genes in Xanthomonas and Pseudomonas strains. We identified five open reading frames (ORFs) whose products exhibited high levels of amino acid sequence identity to the products of previously reported copper genes. Mutations in ORF1, ORF3, and ORF4 removed copper resistance, whereas mutations in ORF5 resulted in an intermediate copper resistance phenotype and insertions in ORF2 had no effect on resistance conferred to a copper-sensitive recipient in transconjugant tests. Based on sequence analysis, ORF1 was determined to have high levels of identity with the CopR (66%) and PcoR (63%) genes in Pseudomonas syringae pv. tomato and Escherichia coli, respectively. ORF2 and ORF5 had high levels of identity with the PcoS gene in E. coli and the gene encoding a putative copper-containing oxidoreductase signal peptide protein in Sinorhizobium meliloti, respectively. ORF3 and ORF4 exhibited 23% identity to the gene encoding a cation efflux system membrane protein, CzcC, and 62% identity to the gene encoding a putative copper-containing oxidoreductase protein, respectively. The latter two ORFs were determined to be induced following exposure to low concentrations of copper, while addition of Co, Cd, or Zn resulted in no significant induction. PCR analysis of 51 pepper and 34 tomato copper-resistant X. campestris pv. vesicatoria strains collected from several regions in Taiwan between 1987 and 2000 and nine copper-resistant strains from the United States and South America showed that successful amplification of DNA was obtained only for strain XvP26. The organization of this set of copper resistance genes appears to be uncommon, and the set appears to occur rarely in X. campestris pv. vesicatoria.     

野油菜黄单胞菌6-磷酸果糖激酶基因的初步分析

6-磷酸果糖激酶是糖酵解途径中的关键酶,它催化糖酵解途径中第一个不可逆反应。本研究利用pK18mobsacB自杀质粒采用同源双交换的方法对野油菜黄单胞菌Xcc8004中的6-磷酸果糖激酶基因(XC_0872)进行缺失突变,获得无标记的缺失突变体DM0872。表型检测结果显示DM0872突变体不影响野油菜黄单胞菌对葡萄糖和果糖的利用,不影响胞外多糖的合成,也不影响其致病性。该结果显示糖酵解途径在野油菜黄单胞菌的地位并不重要。另外,我们利用RT-PCR方法检测了XC_0872的转录情况,结果显示XC_0872在Xcc8004中是转录的。而之前曾有报道称黄单胞菌中无法检测出6-磷酸果糖激酶活性,这表明XC_0872进行了转录后调控从而使6-磷酸果糖激酶活性受到限制。本研究为野油菜黄单胞菌中糖酵解途径的调控提供了理论依据,对揭示野油菜黄单胞菌中该途径的调控机制具有一定的意义。     

Difference Between Xanthomonas campestris pv. phlei and X.c pv. graminis Revealed by Genomic Fingerprinting and Restriction Fragment Length Polymorphism Patterns

Genomic DNA was prepared from 16 strains of Xanthomonas campestris pv. graminis and Xanthomonas campestris pv. phlei isolated from six species of forage grasses in four countries. The two pathovars could be distinguished clearly by genomic fingerprints generated by EcoRI, BamHI or HindIII digestion. DNA profiles produced by HindIII digestion could differentiate not only between the two pathoars but also among strains within the same pahtovar from different countries. A 1.6 kb EcoRI fragment was cloned from genomic DNA of strain LMG726 and used to detect restriction fragment-length polymorphism among the same strains. EcoRI and BamHI polymorphisms were seen between the two pathovars probed with this 1.6 kb EcoRI fragment (p726EI probe). These polymorphisms appeared to be highly conserved and unique for each pathovar, consistent with previous grouping of the strains based on other criteria.     

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。     

Characterization of stress-responsive genes, hrcA-grpE-dnaK-dnaJ, from phytopathogenic Xanthomonas campestris

Sequencing of a 6.4-kb DNA fragment, cloned from the plant pathogenic bacterium Xanthomonas campestris pv. campestris 17 revealed five ORFs whose deduced amino acid sequences show strong similarities to the bacterial HrcA, GrpE, DnaK, DnaJ, and PdxK. The four heat shock genes are organized in the order hrcA-grpE-dnaK-dnaJ, a genome organization found in many gram-positive bacteria, but only in one gram-negative species (Xylella fastidiosa). These observations suggest that the HrcA-CIRCE system, comprising at least four genes arranged in this order, already existed for the regulation of stress responses before bacteria diverged into gram-negative and gram-positive groups. Primer-extension results suggested the presence of promoters at the regions upstream of grpE and dnaK. In the presence of stress, heat or ethanol (4%), the X. campestris pv. campestris 17 grpE and dnaK promoters were induced two- to three-fold over controls. Since the grpE and dnaK promoters possess E. coli sigma(32) promoter-like sequences, they are functional in E. coli, although at levels much lower than in X. campestris pv. campestris 17. Furthermore, expression of the X. campestris pv. campestris 17 dnaK promoter in E. coli was elevated by the cloned X. campestris sigma(32) gene, indicating that the cognate sigma(32) works more efficiently for the X. campestris promoters.     

Flocculation properties of xanthan produced by Xanthomonas campestris

Summary Xanthan had a flocculating activity in a kaolin suspension and high flocculating activity was obtained in the suspension (pH 7.0) adding Al³⁺, Fe³⁺ or Fe²⁺. Xanthan had high flocculating activity not only in other inorganic suspensions such as active carbon and acid clay but also in organic suspensions of cellulose and yeast. From these flocculation properties, xanthan is anticipated to be utilized in wide areas as a new biodegradable, harmless biopolymer flocculant.     

采用两种突变方法对甘蓝黑腐病菌XC1348基因的功能解析

为研究甘蓝黑腐病菌Xcc8004基因组中一个功能未知的假定蛋白XC₁348的基因功能,本研究用自杀质粒pK18mob和pK18mobSacB分别通过同源单交换和同源双交换构建XC₁348的非极性整合突变体1348nk和有标记的缺失突变体DM1348Gm,并对两种突变体的表型进行检测,结果显示该基因突变后不影响细菌的胞外多糖（EPS）产量、胞外酶活性以及细菌游动性。有趣的是该基因的两种突变体的致病力存在较大差异,原因可能是Xcc经两种方法定点突变后分别具有的不同基因组结构而影响到某些基因的表达所致。通过研究初步了解了XC₁348基因的功能并为今后改进定点突变方法提供现实依据。