Expression of the Hypersensitive Response-assisting Protein in <Emphasis Type="Italic">Arabidopsis</Emphasis> Results in Harpin-dependent Hypersensitive Cell Death in Response to <Emphasis Type="Italic">Erwinia carotovora</Emphasis>

Active defense mechanisms of plants against pathogens often include a rapid plant cell death known as the hypersensitive cell death (HCD). Hypersensitive response-assisting protein (HRAP) isolated from sweet pepper intensifies the harpin_Pss-mediated HCD. Here we demonstrate that constitutive expression of the hrap gene in Arabidopsis results in an enhanced disease resistance towards soft rot pathogen, E. carotovora subsp. carotovora. This resistance was due to the induction of HCD since different HCD markers viz. Athsr3, Athsr4, ion leakage, H₂O₂ and protein kinase were induced. One of the elicitor harpin proteins, HrpN, from Erwinia carotovora subsp. carotovora was able to induce a stronger HCD in hrap-Arabidopsis than non-transgenic controls. To elucidate the role of HrpN, we used E. carotovora subsp. carotovora defective in HrpN production. The hrpN⁻ mutant did not induce disease resistance or HCD markers in hrap-Arabidopsis. These results imply that the disease resistance of hrap-Arabidopsis against a virulent pathogen is harpin dependent.     

Structure and regulation of the Erwinia carotovora subspecies carotovora SCC3193 cellulase gene celV1 and the role of cellulase in phytopathogenicity

The ceIV1 gene encoding a secreted cellulase (CelV1) of Erwinia carotovora subsp. carotovora SCC3193 was cloned and its nucleotide sequence determined. The gene contains an open reading frame of 1511 by and codes for an exported protein of 504 amino acids. The predicted amino acid sequence of Ce1V1 was highly similar to that of CeIV of another E. c. subsp. carotovora strain SCRI193 but completely different from the previously characterized cellulase, CelS, of the strain SCC3193. Gene fusions to the lacZ reporter were employed to characterize the regulation of celV1 and celS. Both genes are coordinately induced in a growth phase-dependent manner and are catabolite repressed. Expression of celV1 but not celS was stimulated by plant extracts. The celS gene was expressed at a much lower level than celV1 under all conditions tested. Inactivation of the celV1 gene in E. c. subsp. carotovora strain SCC3193 by marker exchange showed that celV1 encodes the major cellulase of strain SCC3193, as the resulting mutant strain SCC6001 was devoid of cellulase activity. Ce1Vl mutants exhibited reduced virulence suggesting that CelV1, although not absolutely required for pathogenicity, enhances the ability of strain SCC3193 to macerate plant tissue. Inactivation of the celS gene in the celV1 mutant did not lead to any further decrease in virulence.     

Isolation and characterisation of transposon-induced mutants of Erwinia carotovora subsp. atroseptica exhibiting reduced virulence

Summary The blackleg pathogen Erwinia carotovora subsp. atroseptica (Eca) causes an economically important disease of potatoes. We selected a genetically amenable Eca strain for the genetic analysis of virulence. Tn5 mutagenesis was used to generate nine mutants which exhibited reduced virulence (Rvi^-) of strain SCRI1043. Following physiological characterisation, mutants were divided into three classes: (1) auxotrophs; (2) extracellular enzyme mutants; and (3) a growth rate mutant. The isolation of these Rvi^- mutants has allowed us to consider some factors that affect Eca virulence.     

Characterization of a genomic clone of Erwinia carotovora subsp. carotovora TG3 encoding a pectolytic enzyme of apparent molecular weight 78 kDa

Summary A clone containing the gene encoding a pectolytic enzyme of Erwinia carotovora subsp. carotovora was selected as the one that showed maceration on a solid medium containing sodium polypectate. The gene was located on a 3.2-kb DNA fragment flanked by a BglII site and a Hin-dIII site. Via mini-Mudlac mutagenesis, a possible promoter site was located within the gene between the BglII site and the EcoRI site. The mRNA transcribed from the promoter was directed from the BglII site toward the EcoRI site, determined from the orientation of the inserted mini-Mudlac. The probable gene product was identified as a 78 kDa protein. The enzyme activity of the Escherichia coli clone was detected mainly in the periplasmic space. Potato tuber slices were not macerated by the E. coli clone and synthesis of the enzyme in E. coli was not regulated by the enzyme substrate, sodium polypectate.     

携带Paenibacillus polymyxa WLY78固氮基因簇(nif)的重组大肠杆菌Escherichia coli78-7转录组分析

[目的]来自Paenibacillus polymyxa WLY78的固氮基因簇（nifBHDKEfNXhesAnifV）可以转化入Escherichia coli中表达并使重组大肠杆菌合成有固氮活性的固氮酶。本文拟通过对重组大肠杆菌E.coli 78-7的转录组分析以提高其固氮能力。[方法]对固氮条件（无氧无NH₄⁺）和非固氮条件（空气和100 mmol/L NH₄⁺）培养的重组大肠杆菌E.coli 78-7进行转录组分析。[结果]nif基因在两种培养条件下显著表达,说明在重组大肠杆菌中可规避原菌中氧气和NH₄⁺对nif基因的负调控。对于固氮过程必需的非nif基因,如参与钼、硫、铁元素转运的mod、cys和feoAB,这些基因在两种培养条件下表达水平有差异。而参与铁硫簇合成的suf和isc基因簇在两条件下表达水平差异巨大。此外,参与氮代谢的基因在固氮条件下显著上调。[结论]重组大肠杆菌中与固氮相关的非nif基因在该菌的固氮过程中具有较大影响,本文对在异源宿主中调高固氮酶活性研究具有重要意义。     

In vitro translation of type A Clostridium botulinum neurotoxin heavy chain and analysis of its binding to rat synaptosomes

Botulinum neurotoxins (BoNTs) are highly potent toxins that inhibit neurotransmitter release from peripheral cholinergic synapses. BoNTs consist of a toxifying light chain (LC; 50 kDa) and a binding/translocating heavy chain (HC; 100 kDa) linked through a disulfide bond. A DNA fragment encoding type A Clostridium botulinum heavy chain (BoNT/A HC) was amplified by polymerase chain reaction and cloned into an E. coli PET-15b vector. In vitro translated [³⁵S]BoNT/A HC was identified by anti-BoNT/A polyclonal antibodies, and was used to investigate the binding of the toxin to rat synaptosomes. The binding of [³⁵S]BoNT/A HC to synaptosomes was abolished by 500-fold excess of cold BoNT/A, and by incubation with trypsin. Treatment of BoNT/A HC with anti-BoNT/A or G_T1b blocked its binding to synaptosomes. The radioactive BoNT/A HC recognized three proteins corresponding to a molecular mass of 150 (P150), 120 (P120), and 75 (P75) kDa in rat and bovine synaptosomal preparations. These results represent the first successful expression of functional full-length BoNT heavy chain.     

Regulation of nif gene expression in Enterobacter agglomerans: nucleotide sequence of the nifLA operon and influence of temperature and ammonium on its transcription

The nucleotide sequence of a plasmid-borne 3.9 kb XhoI-SmaI fragment comprising the 3-region of the nifM gene, the nifL and nifA genes and the 5-region of nifB gene of Enterobacter agglomerans was determined. The genes were identified by their homology to the corresponding nif genes of Klebsiella pneumoniae. A typical ⁵⁴-dependent promoter and a consensus NtrC-binding motif were identified upstream of nifL. The predicted amino acid sequence of NifL showed close similarities to NifL of K. pneumoniae and Azotobacter vinelandii. However, no histidine residue was found to correspond to histidine-304 of A. vinelandii NifL, which had been proposed to be required for the repressor activity of NifL. The NifA sequence with a putative DNA binding motif (Q(X₃) A (X₃) G (X₅)I) and an ATP binding site in the C-terminal and central domains, respectively, resembles that of other known NifA proteins. The function of the nifL and nifA genes was demonstrated in vivo using a binary plasmid system by their ability to activate a nifH promoter-lacZ fusion at different temperatures and concentrations of NH ₄ ⁺ . Maximal promoter activity occurred at 25°C, and it appears that the sensitivity of NifA to elevated temperatures is independent of NifL. The expression of nifL inhibited promoter activity in the presence of NifA when the initial NH ₄ ⁺ concentration in the medium exceeded 4 mM.Communicated by H. Böhme     

大肠杆菌胞内半胱氨酸响应型启动子的筛选表征

【目的】半胱氨酸是一种重要的含硫氨基酸,广泛应用于化妆品、药品、食品等行业,微生物发酵法合成半胱氨酸已成为当前研究的热点。基于比较转录组学分析等技术,筛选并表征大肠杆菌(Escherichia coli)胞内对半胱氨酸浓度变化显著响应的启动子。【方法】在Escherichia coli W3110培养基中外源添加不同终浓度的半胱氨酸,通过比较转录组学分析筛选转录水平显著响应半胱氨酸浓度变化的基因,融合目标基因的启动子片段与荧光报告基因egfp构建启动子文库,进一步测定不同半胱氨酸浓度条件下,含有不同启动子重组菌的绿色荧光蛋白(greenfluorescent protein, GFP)荧光强度。【结果】筛选并挖掘了随着半胱氨酸浓度提高而转录水平显著提升的27个基因,并将基因的潜在启动子片段与荧光报告基因egfp融合构建启动子文库,筛选获得对半胱氨酸变化具备特异性响应的启动子P_E2。最后,对P_E2启动子-35区间隔区域AAAT进行随机突变,最终获得在1-7g/L半胱氨酸浓度范围内特异性响应性能显著提高的启动子P_E2-33。...     

The wild-type allele of tonB in Escherichia coli is dominant over the tonB1 allele, encoding TonBQ160K, which suppresses the btuB451 mutation

The entire coding sequence of the tonB gene, except for nine codons at the 3 end, was deleted from the chromosome of Escherichia coli. Introduction of the btuB451 suppressor mutant tonB1 into the chromosome of such a tonB deletion strain showed that the tonB1 allele was active as a suppressor in a single copy at 37° C and 42° C but not at 28° C. No temperature dependence was seen when FepA- or FhuA-dependent activities of the tonB1 gene product (TonB_Q160K) were tested. The btuB451 suppressor activity of tonB1 was inhibited by the simultaneous presence within the cells of the tonB ⁺ allele on a multicopy plasmid. This represents the first case of dominance among different tonB alleles. Inhibition of suppression was abolished by overexpression of the btuB451-encoded receptor protein. Competition for binding of TonB⁺ and TonB_Q150K to ExbB was excluded as the cause of dominance. Based on our data we conclude that competition for binding of TonB ⁺ and TonB_Q160K to the btuB451 gene product is the reason for the observed dominance. The implications of these findings for the mechanism of btuB451 suppression by tonB1 are discussed.     

Cleavage of di- and tripeptides by Prevotella ruminicola

The final step in the conversion of protein to amino acids by the common Gram-negative rumen bacterium, Prevotella (formerly Bacteroides) ruminicola , is the cleavage of di- and tripeptides. Dipeptidase and tripeptidase activities were predominantly cytoplasmic, and toluene treatment increased the rate of Ala₂ and Ala₃ hydrolysis by whole cells, suggesting that transport limited the rate of hydrolysis of extracellular di- and tripeptides. The hydrolysis of Ala₂ and Ala₃ by whole cells was not affected by protonophores, ionophores or dicyclohexylcarbodiimide, but Ala₂ hydrolysis by EDTA-treated cells was inhibited by the Ca²⁺/H⁺ ionophore, tetronasin. Ala₃ hydrolysis was not affected by protonophores or ionophores in EDTA-treated cells. The dipeptidase of strain M384 was inhibited > 99% by 1,10-phenanthroline and 39% by EDTA but not other protease inhibitors, consistent with the enzyme being a metalloprotease. Tripeptidase was insensitive to protease inhibitors, except for a 33% inhibition by EDTA. Cleavage of tripeptides occurred at the bond adjacent to the N-terminal amino acid. Distinct di-, tri- and oligopeptidase peaks were obtained by anion-exchange liquid chromatography of disrupted cells. Banding patterns on native PAGE using activity staining also indicated that P. ruminicola M384 had separate single dipeptidase and tripeptidase enzymes which hydrolysed a range of peptides. The dipeptidase of strain M384 was different from other strains of P. ruminicola: strains GA33 and B₁4 had activities which ran at the same R_f; strain GA33 had another band of lower activity; strain 23 had two bands different from those of the other strains. The tripeptidases ran at the same R_f for the different strains. Dipeptidase activity of all strains was inhibited by 1,10-phenanthroline on gels. Gel permeation chromatography indicated that the M_r of the dipeptidases from strains M384 and B₁4 were 115 000 and 114 500 respectively, and 112 500 and 121 500 for the corresponding tripeptidases. Thus the metabolism of small peptides by P. ruminicola involves separate permeases and intracellular peptidases for di- and tripeptides.     

Changes in properties of phytopathogenic bacteria effected by plasmid pRD1

Transfer of plasmid pRD1 from Escherichia coli K12J62-1 to phytopathogenic bacteria, Agrobacterium tumefaciens, Xanthomonas beticola and Erwinia carotovora subsp. carotovora caused changes in conjugant properties not determined by the plasmid and the emergence of the properties not present in the parent strains. Clones have been obtained with intermediate properties between donor and recipient, including those with altered or lost virulence. In transconjugants of A. tumefaciens virulence increased. In transconjugants of X. beticola and E. carotovora subsp. carotovora highly virulent as well as avirulent forms have been observed. The loss of virulence in X. beticola correlated with the Nif^* phenotype. Plasmid pRD1 also affected the biochemical properties of the new hosts.     

Comparative study of Cr(VI) uptake and reduction in industrial effluent by Ochrobactrum intermedium and Brevibacterium sp.

Two chromium-resistant bacterial strains, CrT-1 and CrT-13, tolerant up to 40mg K₂CrO₄ ml^–1 on nutrient agar, 25mgml^–1 in nutrient broth, and up to 10mgml^–1 in acetate-minimal media, were identified as Ochrobactrum intermedium and Brevibacterium sp., respectively, on the basis of 16S rRNA gene sequencing. Uptake of chromate was greater in living cells than in heat-killed on dried cells. CrT-1 reduced 82%, 28% and 16% of Cr(VI) at 100, 500, and 1000gml^–1 after 24h while CrT-13 reduced 41%, 14% and 9%. Other heavy metals at low concentrations did not affect these reductions. At 150 and 300gml^–1 in an industrial effluent sample Cr(VI) was reduced by 87% and 71%, respectively, with CrT-1 and by 68% and 47% with CrT-13.Revisions requested 17 May 2004; Revisions received 2 July 2004     

Competitive nodulation blocking of Afghanistan pea is determined by nodDABC and nodFE alleles in Rhizobium leguminosarum

Summary One well-defined competitive interaction amongst rhizobia is that between compatible and non-compatible strains of Rhizobium leguminosarum with respect to the nodulation of some primitive pea genotypes. The Middle Eastern pea cv Afghanistan is nodulated effectively can R. leguminosarum TOM, but its capacity to nodulate can be blocked if a mixed inoculation is made with R. leguminosarum PF₂. This PF₂ phenotype (Cnb) is encoded by its symbiotic plasmid and cosmid clones thereof. We found that Cnb is also encoded by the well-characterized Sym plasmid pRL1JI of R. leguminosarum strain 248. We have isolated and characterized a 6.9 kb HindIII fragment of pSymPF₂ which confers the Cnb⁺ phentoype on other (Cnb^–) rhizobia. A Tn5 site-directed Cnb^– mutant was constructed by homogenotization and was also found to be Nod^– on the European pea cv Rondo. DNA hybridization and complementation analysis indicated that the 6.9 kb Cnb⁺ fragment contained the nodD, nodABC and nodFE operons. Analysis of the Cnb phenotype of nod::Tn5 alleles of pRL1JI showed that mutations of nodC, nodD or nodE all abolished Cnb activity whereas mutants in nodI and nodJ reduced activity to 50% of the wild-type level.     

美洲黑杨凋落叶分解初期对小白菜生长的影响

采用盆栽试验,研究了美洲黑杨(Populus deltoides)凋落叶分解初期对受体植物小白菜(Brassica chinensis)生长和生理的影响。试验设置0、30、60和90 g/盆4个凋落叶施用水平(分别记作CK、L30、L60和L90)。同时,为检验凋落叶施入是否对土壤通气透水性产生明显影响进而影响受体植物的生长,用蒸煮后的凋落叶设置平行空白试验,即30、60、90 g/盆3个蒸著后的凋落叶处理(分别记作Z30、Z60和Z90)。将各处理的凋落叶分别与7 kg土壤混合,播种小白菜。在播种后50、80 d测定小白菜株高和生理指标。结果表明:1)高量(L90)凋落叶下小白菜的高生长和鲜重于50 d时被显著抑制,80 d时长势恢复正常;2)80 d时各处理净光合速率(Pn)与CK水平相当,色素含量略低于CK;3)50、80 d时,低(L30)、中(L60)量处理的超氧化物歧化酶(SOD)活性无明显变化,高量处理下SOD活性升高;4)各处理丙二醛(MDA)含量在50、80 d时与CK均无显著差异。总的来看,杨树各凋落叶量处理对小白菜的影响表现为:低、中量促进,高量抑制,而经蒸煮后的凋落叶处理间差异不显著。表明,低、中量杨树凋落叶在土壤中分解对小白菜生长及生理代谢的影响主要表现为促进作用,而施入高量凋落叶的初期,化感抑制作用明显。     

皂荚提取物的杀螺活性

[目的]研究皂荚生物农药活性,开发利用皂荚资源,发展环境友好的绿色植物源农药。[方法]采用室内生测和田间试验研究皂荚壳乙醇提取物的杀螺活性。[结果]皂荚提取物对福寿螺有显著的毒杀活性,对幼螺和成螺72 h的LC₅₀分别为40.56、109.83 mg·L^-1。田间试验表明,皂荚提取物对福寿螺有较好的防效,施用40 g·m^-2的皂荚提取物处理7 d后卵块减少率为100.00%（成螺失去产卵的能力）,防效为（99.12±1.26）%。[结论]皂荚提取物对福寿螺较好的生物防治效果,是一种潜在的生物杀螺剂。     

大型海藻龙须菜抽提液对褶皱臂尾轮虫生命表参数的影响

大型海藻富含多种活性物质,具有抗衰老等生物活性;轮虫是良好的潜在抗衰老研究模式生物。本研究以褶皱臂尾轮虫(Brachionus plicatilis)作为实验对象,研究了不同浓度的大型海藻龙须菜抽提液(0,250,500,750,1000 mg/L)和不同浓度的食物(蛋白核小球藻和普通小球藻)对褶皱臂尾轮虫生命表参数的影响。结果表明:与对照组相比,食物浓度为1.0×10~6个/mL蛋白核小球藻时,不同浓度龙须菜抽提液对轮虫产卵数、平均寿命、净生长率以及世代时间有显著促进效应(P0.05);轮虫平均产卵数及寿命在龙须菜抽提液浓度750 mg/L处达到最高,分别为16只和13.9d(P0.05)。食物浓度为2.0×10~6个/mL普通小球藻时,轮虫平均产卵数和寿命在抽提液浓度为500 mg/L处达到最高,分别为16只和13.6d(P0.05),轮虫平均寿命和净生长率均有显著提高(P0.05)。相同龙须菜抽提液浓度下,食物浓度为1.0×10~6个/mL蛋白核小球藻下轮虫的净生长率、世代时间均显著高于食物浓度为2.0×10~6个/mL蛋白核小球藻培养的轮虫(P0.05);食物浓度为2.0×10~6个/mL时,普通小球藻培养轮虫的净生长率和世代时间均显著高于蛋白核小球藻实验组(P0.05)。交互作用分析显示,龙须菜抽提液与小球藻的交互作用对褶皱臂尾轮虫的内禀增长率有显著影响(P0.05)。研究结果表明,大型海藻龙须菜抽提液对褶皱臂尾轮虫的生长与生殖有促进作用,延长轮虫寿命。     

外源激素对马尾松花芽分化及内源物质的影响

为探讨马尾松球花形成与植物激素水平的关系,该研究对贵州省都匀无性系种子园11年生马尾松进行不同浓度的 IAA、IBA、GA3、BAP等植物激素处理,采用考马斯亮蓝G-250染色法、蒽酮法分别对不同浓度不同激素处理后的枝条上针叶中的可溶性蛋白质和可溶性糖含量变化进行测定,并在第二年开花时对试验枝条的开花情况进行了调查。结果表明：在8－11月份,进行500 mg·L-1的BAP 处理有利于马尾松雌球花和雄球花的形成,100 mg·L-1的GA3处理有利于马尾松雌球花的形成;而GA3250 mg·L-1和GA3500 mg·L-1处理有利于马尾松雄球花的形成,IAA 250 mg·L-1对马尾松雌雄球花同枝的数量有提高作用。在10－11月份,对马尾松进行500 mg·L-1的BAP、IAA、GA3处理后,马尾松针叶内蛋白质含量变化有显著影响;在10月份时,进行BAP 100 mg·L-1处理后,其可溶性糖含量及可溶性蛋白含量均可达到极显著水平。而在8月份与10月份时,分别进行IBA 100 mg·L-1与IBA 250 mg·L-1处理后,其可溶性蛋白含量与其对照差异处于极显著水平;在11月份时,进行GA3100 mg·L-1处理后,其可溶性蛋白含量与其对照差异处于极显著水平;而在11月份时,进行IBA 500 mg·L-1处理后,其可溶性蛋白含量与对照差异处于显著水平。     

Acid phosphatase activity in hemolymph of the migratory grasshopper,Melanoplus sanguinipes, duringBeauveria bassiana infection

The distribution and abundance of acid phosphatase (AP) in hemolymph (HL), plasma (PM) and hemocyte lysate supernatant (HLS) of the migratory grasshopper,Melanoplus sanguinipes infected withBeauveria bassiana (strain GK 2016) has been examined. AP activity was determined at intervals from 30 min to 60 h postinjection of 2 μl of 1×10⁸ conidia/ml per grasshopper. The enzyme was detected with the substrate β-glycerophosphate in sodium acetate acetic acid buffer form hemocytes (HC) and withp-nitrophenol phosphate sodium salt for HL, PM and HLS. In results of experiment 1 proportion of HC showing AP activity increased 1–2 h, then returned to normal after 4 h. However, inB. bassiana-injected grasshoppers, a second increase was noted 24 h later which was not seen in the Tween-80-injected insects. Uninjected controls showed no change with time in the proportion of HC with AP activity. Studies were also made of the distribution of AP activity in the HL, PM, and HLS. AP activity in HL appeared to vary with the sex of the grasshoppers. Females showed increase in AP activity in HL 18–24 h after injection withB. bassiana, whereas males only showed an increase 1 h after injection. Assay of HLS showed that the level of AP activity did not change significantly throughout the experiment. Changes in AP activity in PM, in bothB. bassiana — and Tween-80-injected insects (both sexes) paralleled those of the HL, indicating that the enzyme is released from the HC. The observations are discussed in terms of the possible role of AP in the immune response ofM. sanguinipes.     

Occurrence of bacteriophage T4 receptor in Erwinia carotovora

Summary We have tested for the presence of the receptor for the Escherichia coli phage T4 in different isolates of the plant pathogenic enterobacteria Erwinia carotora subsp. carotovora and subsp. atroseptica. Several of the isolates appeared to contain a functional T4 receptor as shown by phage adsorption and phage-induced lysis of the bacteria. Two of the isolates could even sustain lytic growth of T4. In addition, we show that the transducing derivative of T4, T4GT7, can be employed to transfer plasmids from E. coli to E. carotovora thus opening up new possibilities for genetic analysis of Erwinia.