Glycosylation of flavonoids with a glycosyltransferase from Bacillus cereus

Microbial glycosyltransferases can convert many small lipophilic compounds such as phenolics, terpenoids, cyanohydrins and alkaloids into glycons using uridine-diphosphate-activated sugars. The main chemical functions of glycosylation processes are stabilization, detoxification and solubilization of the substrates. The gene encoding the UDP-glycosyltransferase from Bacillus cereus, BcGT-1, was cloned by PCR and sequenced. BcGT-1 was expressed in Escherichia coli BL21 (DE3) with a his-tag and purified using a His-tag affinity column. BcGT-1 could use apigenin, genistein, kaempferol, luteolin, naringenin and quercetin as substrates and gave two reaction products. The enzyme preferentially glycosylated at the 3-hydroxyl group, but it could transfer a glucose group onto the 7-hydroxyl group when the 3-hydroxyl group was not available. The reaction products made by biotransformation of flavonoids with E. coli expressing BcGT-1 are similar to those produced with the purified recombinant enzyme. Thus, this work provides a method that might be useful for the biosynthesis of flavonoid glucosides and for the glycosylation of related compounds.     

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.     

Identification of Xanthomonas campestris pv. campestris galactose utilization genes from transcriptome data

A 70 mer oligonucleotide microarray was constructed to analyze genome-wide expression profiles of Xanthomonas campestris pv. campestris B100, a plant-pathogenic bacterium that is industrially employed to produce the exopolysaccharide xanthan gum which has many applications as a stabilizing, thickening, gelling, and emulsifying agent in food, pharmaceutical, and cosmetic industries. As an application example, global changes of gene expression were monitored during growth of X. campestris pv. campestris B100 on two different carbon sources. Exponential growing bacterial cultures were incubated either for 1h or permanently in minimal medium supplemented with 1% galactose in comparison to growth in minimal medium supplemented with 1% glucose. Six genes were identified that were significantly increased in gene expression under both growth conditions. These genes were located in three distinguished chromosomal regions in operon-like gene clusters. Genes from these clusters encode secreted glycosidases, which were predicted to be specific for galactose-containing carbohydrates, as well as transport proteins probably located in the outer and inner cell membrane. Finally genes from one cluster code for cytoplasmic enzymes of a metabolic pathway specific for the breakdown of galactose to intermediates of glycolysis.     

Extracellular proteases from Xanthomonas campestris pv. campestris, the black rot pathogen.

Two proteases (PRT1 and PRT2) were fractionated from culture supernatants of wild-type Xanthomonas campestris pv. campestris by cation-exchange chromatography on SP-5PW. Inhibitor experiments showed that PRT 1 was a serine protease which required calcium ions for activity or stability or both and that PRT 2 was a zinc-requiring metalloprotease. PRT 1 and PRT 2 showed different patterns of degradation of beta-casein. The two proteases comprised almost all of the extracellular proteolytic activity of the wild type. A protease-deficient mutant which lacked both PRT 1 and PRT 2 showed considerable loss of virulence in pathogenicity tests when bacteria were introduced into mature turnip leaves through cut vein endings. This suggests that PRT 1 and PRT 2 have a role in black rot pathogenesis.     

Transformation of Xanthomonas campestris pathovar campestris with plasmid DNA

Procedures for the introduction of plasmid DNA into Gram-negative bacteria have been adapted and optimized to permit transformation of the plant pathogen Xanthomonas campestris pathovar campestris with the cloning vector pKT230 and other broad-host-range plasmids. The technique involves CaCl2-induced competence and heat shock and is similar to that routinely used for Escherichia coli. Wild-type X. c. campestris strains appear to restrict incoming unmodified DNA, so that plasmid DNA for transformation must be prepared from X. c. campestris (into which it has previously been introduced by conjugation). To overcome this disadvantage a restriction-deficient mutant has been isolated.     

Extracellular proteases from Xanthomonas campestris pv. campestris, the black rot pathogen

Two proteases (PRT1 and PRT2) were fractionated from culture supernatants of wild-type Xanthomonas campestris pv. campestris by cation-exchange chromatography on SP-5PW. Inhibitor experiments showed that PRT 1 was a serine protease which required calcium ions for activity or stability or both and that PRT 2 was a zinc-requiring metalloprotease. PRT 1 and PRT 2 showed different patterns of degradation of beta-casein. The two proteases comprised almost all of the extracellular proteolytic activity of the wild type. A protease-deficient mutant which lacked both PRT 1 and PRT 2 showed considerable loss of virulence in pathogenicity tests when bacteria were introduced into mature turnip leaves through cut vein endings. This suggests that PRT 1 and PRT 2 have a role in black rot pathogenesis.     

Secretion of heterologous cyclodextrin glycosyltransferase of Bacillus sp. E1 from Escherichia coli

Summary To characterize the molecular properties of CGTase from alkalophilic Bacillus sp. E1 (BCGTE1), a genomic clone for a CGTase was isolated. Expression of recombinant BCGTE1 in E. coli was analyzed by immunoblotting. It showed that the nascent recombinant BCGTE1 expressed was 87 kDa but it was processed into the mature enzyme of 81 kDa. With the process it was secreted predominantly into the culture medium via periplasmic space. This feature is different from other Bacillus CGTases expressed in E. coli, which were present mostly in the periplasmic space.     

Comprehensive analysis of the extracellular proteins from Xanthomonas campestris pv. campestris B100

The extracellular proteome of Xanthomonas campestris pv. campestris (Xcc) cultivated in minimal medium was isolated from the cell-free culture supernatant and separated by two-dimensional gel electrophoresis. This technique resolved 97 clearly visible protein spots, which were excised, digested with trypsin and identified on the basis of their peptide mass fingerprints generated by matrix assisted laser desorption/ionisation-time of flight-mass spectrometry. Using this approach 87 different proteins could be distinguished. The Signal P software predicted putative signal peptides for 53% of the extracellular proteins. These proteins are probably transported over the inner membrane and are localized in the periplasm, the outer membrane or secreted into the extracellular space. Among the secreted proteins are 11 degradative enzymes, which are involved in pathogenesis of Xcc. The proteins without obvious secretion signals are known to serve functions in the cytosol. How the cytosolic proteins are delivered to the extracellular space remains unclear.     

Isolation of a Plasmid from Xanthomonas campestris pv. vignicola

A modified method is described for isolating high yields of plasmids without chromosome contamination from Xanthomonas campestris pv. vignicola (X. c. pv. vignicola), the causal agent of blight disease in Vigna species and also in Phaseolus vulgaris. Applying this method a plasmid of X, c. pv. vignicola was detected representing an estimated molecular weight of 95 megadaltons. Heat curing of the strain revealed that the detected plasmid had no effecton virulence but seemed to influence colony morphology.     

Studies on xanthan production from Xanthomonas campestris

Xanthan is an heteropolysaccharide produced by Xanthomonascampestris. Xanthan gum fermentation by a local isolate of X. campestris using different carbon sources was studied. The production of polysaccharide was influenced by the carbon source used. The production of the xanthan was 15.654 g/l with synthetic medium. Production of xanthan at various temperatures ranging between 25v°C and 40v°C was studied. The growth and production was maximum between 25-30v°C. Xanthan production was maximum at pH 7.0-7.5.     

Sequence and molecular analysis of the rpoA cluster genes from Xanthomonas campestris pv. campestris

The Xanthomonas campestris rpsM (S13)-rpsK (S11)-rpsD (S4)-rpoA (alpha)-rplQ (L17) cluster, encoding RNA polymerase alpha-subunit and four ribosomal proteins, reside in a 3164-bp DNA region. The N-terminal sequence of the authentic alpha-protein determined chemically matches that predicted from the nucleotide sequence. rplQ is monocistronic, instead of being co-transcribed with the other genes as in Escherichia coli. Antiserum against the His-tagged alpha-protein cross-reacted with the E. coli alpha-protein.     

Isolation and characterization of a porin-like outer membrane protein from Xanthomonas campestris pv. campestris

Xanthomonas campestris pv. campestris, a plant-associated pathogenic bacterium, is the causal agent of foliar spots and blights in crucifers. The major outer membrane protein, Omp37, of 37 kDa, has been identified, purified to homogeneity, and its characterization has also been carried out. Native Omp37 behaved as a trimer, as revealed by gel filtration and SDS-PAGE. FTIR measurements revealed a high beta-structure content. The pore-forming ability of the purified Omp37 was studied by the liposome swelling assay. Omp37, to our knowledge, is the first porin that has been isolated from Xanthomonas. This study clearly demonstrates that Omp37 is related to the family of trimeric bacterial porins.     

Biological role of xanthomonadin pigments in Xanthomonas campestris pv. campestris

Previous studies have indicated that the yellow pigments (xanthomonadins) produced by phytopathogenic Xanthomonas bacteria are unimportant during pathogenesis but may be important for protection against photobiological damage. We used a Xanthomonas campestris pv. campestris parent strain, single-site transposon insertion mutant strains, and chromosomally restored mutant strains to define the biological role of xanthomonadins. Although xanthomonadin mutant strains were comparable to the parent strain for survival when exposed to UV light; after their exposure to the photosensitizer toluidine blue and visible light, survival was greatly reduced. Chromosomally restored mutant strains were completely restored for survival in these conditions. Likewise, epiphytic survival of a xanthomonadin mutant strain was greatly reduced in conditions of high light intensity, whereas a chromosomally restored mutant strain was comparable to the parent strain for epiphytic survival. These results are discussed with respect to previous results, and a model for epiphytic survival of X. campestris pv. campestris is presented.     

大肠杆菌转录后调控蛋白CsrA的C末端具有抑制十字花科黑腐病菌胞外蛋白酶活性的功能

CsrA(在有些细菌例如十字花科黒腐病菌中也称为RsmA,统称CsrA/RsmA)是一类在细菌中广泛分布而且氨基酸序列非常保守的RNA结合蛋白。研究表明,CsrA/RsmA作为转录后全局调控因子参与了包括细胞碳代谢、次生代谢、运动性、生物被膜形成以及动植物病原菌的致病过程等许多细胞过程的调控。CsrA/RsmA在不同的细菌中的生物学功能各有异同。在大肠杆菌中,CsrA的主要功能是控制细胞的碳代谢、运动性和生物被膜形成;而在十字花科黑腐病菌(Xanthomonas campestris pathovar campestris,Xcc)中,CsrA的同源蛋白RsmA的主要功能除了控制碳代谢、运动性和生物被膜形成外,还控制致病性和胞外酶的产生。大肠杆菌的CsrA(CsrAE.coli)是否具有XccRsmA(RsmAXcc)的功能?为了回答这个问题,我们用大肠杆菌的csrA基因(csrAE.coli)互补Xcc的rsmA基因(rsmAXcc)的缺失突变体DM2506。结果显示,csrAE.coli能够互补DM2506的所有表型,说明CsrAE.coli具有RsmAXcc的全部功能。更重要的是,我们发现,无论是rsmAXcc突变体还是野生型菌株,导入表达CsrAE.coli的质粒后均导致其致胞外蛋白酶活性的严重下降,证明CsrAE.coli具有抑制Xcc胞外蛋白酶活性的作用。进一步的实验证实,CsrAE.coli的C末端第53～61位氨基酸残基具有抑制Xcc胞外蛋白酶活性的功能。     

Monoclonal antibodies reacting with the exopolysaccharide xanthan from Xanthomonas campestris

We have prepared murine hybridomas secreting monoclonal antibodies against the exopopolysaccharide xanthan from Xanthomonas campestris pv. campestris 646 after fusing NSO myeloma cells and spleen cells from BALB/c mice immunized with xanthan. Four hybridomas, secreting antibodies designated A6 (IgM kappa), B3 (IgM kappa), D1 (IgM kappa), and D3 (IgG2A kappa), were selected for further studies. All antibodies reacted with a range of different xanthans. Competition studies using variants of the exopopolysaccharide as competitors suggested that specificity was mainly against the side-chain. One of the antibodies (B3) appeared to require the fully acylated side-chain with the pyruvylated terminal mannose as the immunodominant part. The three others were assumed to be directed against the nonsubstituted trisaccharide with the inner mannose-glucuronic acid being immunodominant. None of the antibodies reacted with cellulose (the xanthan backbone). Using immunoblotting techniques on nitrocellulose paper both a mixture of monoclonal antibodies, and also polyclonal ascitic fluid, could detect xanthan quantities of approximately 0.1 microgram.     

Efficacy of four semi-selective media for recovery of Xanthomonas campestris pv. campestris from tropical soils

R. FUKUI, R. ARIAS AND R. ALVAREZ. 1994. Four semi-selective media, CS20 ABN, aesculin—trehalose (ET), Fieldhouse—Sasser (FS), and starch—methionine (SM), were compared for efficacy in recovering Xanthomonas campestris pv. campestris from artificially and naturally infested soils. Recoveries of X. c. campestris from soils infested with relatively large populations were similar on the four media. The FS and ET media exhibited higher selectivity against background saprophytes, whereas enumeration of X. c. campestris on CS20 ABN or SM was often hampered by the overgrowth of background saprophytes. Among three starch-containing media (CS20 ABN, ET and FS media), the zones of starch hydrolysis, characteristic of colonies of X. c. campestris, were most distinctive for FS medium. This allowed easier identification of the target colonies among numerous non-target colonies in tests with soil containing smaller numbers of X. c. campestris. Although the starch zone was also distinctive on CS20 ABN, this medium was not as effective as FS because the starch zones were so large that neighbouring zones fused with each other and many saprophytes formed colonies within the zones. Overall, FS was most suitable for soil studies in terms of the consistent recovery of the pathogen, the selectivity against saprophytes, and the differentiation from non-target organisms.     

野生型油菜黄单胞菌的过氧化氢释放特性

采用外源过氧化氢和油菜组织处理油菜黄单胞菌野生型(XpW)和烷基过氧化物还原酶亚基C突变型(Xp1),检测了各体系中过氧化氢的释放情况,并测定了油菜黄单胞菌野生型的最大过氧化氢耐受浓度范围,以明确植物-病原菌互作过程中是否具有病原细菌源的过氧化氢产生.结果显示:(1)过氧化氢处理3.5 h后,对外源过氧化氢的清除率相对于0.5 h时XpW为100%,Xp1为-26%,说明野生型对培养体系中过氧化氢的清除率高于突变型;油菜组织处理后,体系中产生和积累的过氧化氢情况是:XpW为3.5 h时比0.5 h时高2.105倍,Xp1为3.5 h时比0.5 h时低25.2%,说明野生型培养体系中产生和积累的过氧化氢量高于突变型.(2)野生型的最大过氧化氢耐受浓度范围为1.715 08×105～2.450 11×105 μmol/L,远远高于油菜组织处理XpW菌液时体系中最大检测到的过氧化氢浓度,说明本实验中油菜组织处理XpW菌液过程中产生的过氧化氢的浓度不能够杀灭XpW.由此推测,油菜组织处理体系中野生型油菜黄单胞菌能够产生部分的过氧化氢,其过氧化氢的产生与烷基过氧化物还原酶亚基C相关,支持植物-病原细菌相互作用过程中可能有细菌源的过氧化氢产生的观点.     

Bactericidal activity of glycinecin A, a bacteriocin derived from Xanthomonas campestris pv. glycines, on phytopathogenic Xanthomonas campestris pv. vesicatoria cells

The ability of glycinecin A, a bacteriocin derived from Xanthomonas campestris pv. glycines 8ra, to kill closely related bacteria has been demonstrated previously by our group. In the present study, we aimed at determining the glycinecin A-induced cause of death. Treatment with glycinecin A caused slow dissipation of membrane potential and rapid depletion of the pH gradient. Glycinecin A treatment also induced leakage of potassium ions from X. campestris pv. vesicatoria YK93-4 cells and killed sensitive bacterial cells in a dose-dependent manner. Sensitive cells were killed within 2 h of incubation, most likely due to the potassium ion efflux caused by glycinecin A. These results suggest that the bactericidal mechanism of action of glycinecin A is correlated with the permeability of membranes to hydroxyl and potassium ions, leading to the lethal activity of the bacteriocin on the target bacteria.