植物病原黄单胞菌退出群体感应生理状态的分子机制和生物学意义

黄单胞菌是一类引起多种作物病害的病原细菌总称.它们利用自身产生的DSF(Diffusible signaling factor)-家族群体感应(quorum sensing,QS)信号分子感应群体密度,调控致病相关基因的表达.当黄单胞菌培养达到对数生长后期时,培养体系中DSF信号分子浓度迅速降低,呈现一种典型的群体感应...     

青草沙水库中一株铜绿微囊藻抑藻细菌的作用研究

【目的】为了研究青草沙水库中土著微生物对藻类生长的抑制作用,从水库水体中筛选出对藻类有抑制作用的细菌并研究其对铜绿微囊藻的抑制效果。【方法】通过对水库水体中的细菌进行划线分离和筛选,挑选出一株对铜绿微囊藻生长有较好抑制作用的菌株CL。考察其对铜绿微囊藻的抑制效果及不同培养时间和菌液浓度对抑藻效果的影响,并对菌株进行16S rDNA序列分析。【结果】实验菌液浓度为4.5×108CFU/mL 8.4×108CFU/mL时,细菌对铜绿微囊藻的抑制率可达45.4%。抑藻效果随培养时间先增后降,在静置培养第6天抑藻效果达到最大。该菌经过16S rDNA序列分析,属于黄单胞菌科的寡养单胞菌。【结论】从青草沙水库中筛选出了对铜绿微囊藻有抑制作用的土著细菌寡养单胞菌,对青草沙水体铜绿微囊藻的控制具有一定的潜在应用价值。     

拟南芥CEO1基因在氯化镉诱导的氧化胁迫中的作用

以拟南芥ceo1、突变体为材料,研究CEO1（clone eight-one）在镉胁迫条件下作用的结果表明,与野生型植株相比,150μmol·L^-1的CdCl2处理10d后,拟南芥ceo1突变体表现为植株生长矮小,叶片卷曲发黄,根系短小。镉处理后,拟南芥突变体幼苗叶中H2O2的积累较多;镉处理1h后的突变体中抗坏血酸过氧化物酶（APX）活性明显上升,至2h时又开始下降,而镉处理2h后,野生型APX活性才开始增加。镉处理2h后的野生型的谷胱甘肽还原酶（GR）显著增加,而突变体无明显变化。两种类型拟南芥的超氧化物歧化酶（SOD）与过氧化氢酶（CAT）的活性没有明显差异。     

细菌除草剂黄单胞菌反枝苋致病菌的筛选

从杂草反枝苋根际土壤中分离到大量的根际细菌,利用谷氨酰胺合成酶抑制剂模型和蛋白核小球藻筛选模型进行快速、高效的初筛,并结合温室盆栽复筛,筛选出一株具有较强除草活性的细菌野油菜黄单胞菌反枝苋致病变种。温室盆栽试验表明,该黄单胞菌对反枝苋、荠菜等双子叶杂草具有较强的抑制作用。      

24-脱氢胆固醇还原酶抗氧化应激作用的功能结构域的鉴定

24-脱氢胆固醇还原酶（DHCR24）具有抗氧化应激引起的细胞凋亡的作用,但其机制尚不清楚。先前的实验数据显示在细胞水平DHCR24可能具有清除过氧化氢的作用。在生物信息学方法预测的基础上,构建了以pIVEX2.4a为载体的人DHCR24基因及其两种功能域缺失的突变型重组表达载体pIVEX2.4a-DHCR24（野生型）、pIVEX2.4a-ORD（预测氧化还原功能域保留型）和pIVEX2.4a-CTR（碳端保留型）,通过体外快速翻译表达系统RTS500进行体外大量表达,经提纯得到各种DHCR24纯化蛋白。Fluoro CatalaseTM荧光过氧化氢酶体外检测实验首次证明野生型DHCR24具有较强的过氧化氢清除能力,且DHCR24-ORD突变型具有与野生型同等强度的过氧化氢清除活性,而DHCR24-CTR突变型则失去了此能力,提示DHCR24的氧化还原功能域在其清除过氧化氢作用方面起着重要作用。为了进一步研究其抗氧化应激作用的生理学意义,利用双重免疫荧光染色法对DHCR24在细胞内的定位情况进行了调查。 结果表明,DHCR24定位于内质网,提示其内质网的定位可能与其抗氧化应激作用有关。     

抗生素对于对虾苗池水体细菌区系的影响

以细菌16S rDNA片段作为分子标记,采用PCR结合DGGE（Denaturing gradient gel electrophoresis）的方法构建了细菌区系指纹图谱,研究了对虾苗池使用硫酸链霉素、土霉素和氨苄青霉素3种抗生素后水体细菌区系的变化.结果表明：在120 h试验期内,与对照组相比,苗池水体分别经0.5 mg·L^-1的硫酸链霉素、土霉素和氨苄青霉素处理后,细菌区系均产生了显著变化;对照组水体0～30 h时段的细菌DGGE条带聚为一组,56～120 h取样时的条带聚为一组;而3个处理组水体0～56 h时段的细菌DGGE条带聚为一组,72～120 h聚为一组.对DGGE图谱典型条带进行测序,经BLAST-N比对,表明对虾苗池水体细菌多样性丰富,包括可培养的细菌（主要包括亚硫酸盐杆菌、红假单胞菌、美人鱼发光杆菌、聚球菌、海洋放线菌、黄杆菌、丝状光合细菌、粘细菌、哈氏弧菌）和某些不可培养的其他海洋细菌等.其中,单胞菌、发光杆菌、放线菌、黄杆菌、粘细菌和2种不可培养的其他海洋细菌不受抗生素的影响;而硫细菌、丝状光合细菌和另外8种不可培养的其他海洋细菌则因抗生素种类不同产生了不同的时空变化.     

野油菜黄单胞菌中烯脂酰ACP还原酶的功能鉴定

烯脂酰ACP还原酶是细菌脂肪酸合成的关键酶之一.本研究通过生物信息学分析发现,野油菜黄单胞菌Xanthomonas campestris(Xcc)8004基因组中XC_0119(Xccfab V)注释为反-2-烯脂酰Co A还原酶基因.但其编码产物与铜绿假单胞菌的烯脂酰ACP还原酶Fab V具有较高的同源性,并含有相同的催化活性中心Tyr-(Xaa)8-Lys序列.用携带Xccfab V的质粒载体互补大肠杆菌fab I温度敏感突变株JP1111,转化子能在42℃生长,表明Xccfab V能遗传互补大肠杆菌fab I突变.体外重建脂肪酸合成反应表明,Xcc Fab V能催化不同链长的烯脂酰ACP还原为脂酰ACP,且催化活性不受三氯森抑制.遗传学研究表明,Xccfab V是必需基因,不能获得Xccfab V基因敲除突变株.将携带大肠杆菌fab I的外源质粒导入野生菌后,可敲除染色体上的fab V基因,获得的替换突变株生长特性和脂肪酸组成未发生显著变化,但替换突变株对三氯森敏感.上述结果证实,野油菜黄单胞菌fab V是必需基因,编码烯脂酰ACP还原酶,参与脂肪酸从头合成反应,且Fab V是Xcc对三氯森耐受的根本原因.     

溶解肠杆菌的交流电电解刺激过程

以葡萄糖为惟一碳源,研究了溶解肠杆菌(Enterobacter dissolvens)在外加电场交流电的刺激下细胞的生长和代谢的过程.研究表明,在低电流10 mA刺激下,交流电刺激下的细菌没有明显的刺激效应;当采用100 mA的电流通电24 h,交流电刺激下的细菌出现了明显的刺激效应,当通电12 h后,菌液葡萄糖的降解率和脱氢酶活性分别为对照参比的1.4倍和2.17倍,细胞浓度呈现明显的增长.分析原因可能是由于电极产物中没有过氧化氢的产生.     

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。     

多孔板-MTT比色法测定植物抗菌成分对细菌的抑制活性

多孔板-MTT比色法测定植物抗菌成分对细菌抑制活性的步骤为：每孔加入浓度为10^6cfu／mL的供试菌液90灿,然后加入不同浓度的药液10μL,28℃暗培养24h后,每孔中加入5mg／mL的MTT溶液10μL,继续培养4h后加入10％二甲基亚砜100μL,振荡30min,在570nm处测定溶液的吸光值。采用以上方法,测定植物抗菌成分蓝桉醇对辣椒斑点病黄单胞菌（Xanthomonas vesicatoria）和枯草芽孢杆菌（Bacillus subtilis）的半抑制浓度（IC50）分别为0．158和0．395mg／mL,小檗碱对溶血葡萄球菌（Staphylococcus haemolyticus）的IC50为0．587mg／mL。结果表明,多孔板-MTT比色法能快速、微量地测定植物成分对细菌的抑制活性。     

Mesosome formation is accompanied by hydrogen peroxide accumulation in bacteria during the rifampicin effect

Ultrastructural alteration and hydrogen peroxide localization were examined in Xanthomonas campestris pv. phaseoli during rifampicin effect using transmission electron microscopy. Bacterial cells were treated with rifampicin and then were examined by electron microscopy to observe the changes of ultrastructure or hydrogen peroxide accumulation in living cells that took place before lysis. Intriguingly, rifampicin treatment led to presence of an additional location of hydrogen peroxide accumulation within the cells. There was an association between the frequency and size of the additional location of hydrogen peroxide accumulation and the concentration of rifampicin. Furthermore, an additional ultrastructure, mesosomes, was also present in cells during rifampicin effect. The frequency and size of mesosome increased with the increasing concentration of rifampicin. Result of multiple linear regression showed that the size of mesosome plays as a key factor in the quantity of excess hydrogen peroxide accumulation in cells during rifampicin effect. Linear correlation was confirmed between quantity of excess hydrogen peroxide accumulation and the size of mesosome in cells during rifampicin effect. This finding intensely indicated that mesosomes are just the additional location of hydrogen peroxide accumulation in cells under cellular injury caused by rifampicin treatment. The mesosome formation is always accompanied by excess hydrogen peroxide accumulation in X. campestris pv. phaseoli during rifampicin effect.     

转录调控因子OxyRxoo对水稻白叶枯病菌H2O2降解途径的调控作用

摘要：【目的】为了阐明水稻白叶枯病菌(Xanthomonas oryzae pv. oryzae, 简称Xoo)转录调控因子OxyRxoo对过氧化氢(H2O2) 降解途径的调控作用。【方法】本研究对推导的H2O2识别调控基因oxyRxoo进行了基因克隆、序列分析、缺失突变和互补试验及其相关表型的鉴定。【结果】克隆的oxyRxoo基因序列与其它几种病原黄单胞菌的同源序列高度保守。OxyRxoo是LysR家族成员之一,具有PBPb结构域和DNA结合保守结构域(HTH)。用标记交换法构建了△oxyRxoo基因缺失突变体。与野生型菌株PXO99A相比,尽管△oxyRxoo在离体培养条件下的生长无明显变化,但H2O2抗性显著地降低,过氧化物酶(CAT)活性明显下降,基因互补可以使之恢复; 过氧化物酶基因表达下调, oxyRxoo自身表达显著上调。【结论】OxyRxoo作为一个重要转录调控因子,调控了Xoo的 H2O2降解途径。     

An investigation into copper catalyzed D-penicillamine oxidation and subsequent hydrogen peroxide generation

D-Penicillamine is a potent copper (Cu) chelating agent. D-Pen reduces Cu(II) to Cu(I) in the process of chelation while at the same time being oxidized to D-penicillamine disulfide. It has been proposed that hydrogen peroxide is generated during this process. However, definitive experimental proof that hydrogen peroxide is generated remains lacking. Thus, the major aims of these studies were to confirm and quantitatively assess the in vitro production of hydrogen peroxide during copper catalyzed D-penicillamine oxidation. The potential cytotoxic effect of hydrogen peroxide generation was also investigated in vitro against MCF-7 human breast cancer cells. Cell cytotoxicity resulting from the incubation of D-penicillamine with copper was compared to that of D-penicillamine, copper and hydrogen peroxide. The mechanism of copper catalyzed D-penicillamine oxidation and simultaneous hydrogen peroxide production was investigated as a function of time, concentration of cupric sulfate or ferric chloride, temperature, pH, anaerobic condition and chelators such as ethylenediaminetetraacetic acid and bathocuproinedisulfonic acid. A simple, sensitive and rapid HPLC assay was developed to simultaneously detect D-penicillamine, its major oxidation product D-penicillamine disulfide, and hydrogen peroxide in a single run. Hydrogen peroxide was shown to be generated in a concentration dependent manner as a result of D-penicillamine oxidation in the presence of cupric sulfate. Chelators such as ethylenediaminetetraacetic acid and bathocuproinedisulfonic acid were able to inhibit D-penicillamine oxidation. The incubation of MCF-7 human breast cancer cells with D-penicillamine plus cupric sulfate resulted in the production of reactive oxygen species within the cell and cytotoxicity that was comparable to free hydrogen peroxide.     

Rapid suppression of multidrug resistance of leukemic cells by oxidative srtess

The effect of a short-time (1 h) oxidative stress on multidrug resistance (MDR) of murine leukemic P388VR cells has been investigated. We studied the production of reactive oxygen species (ROS) in cells depending on the composition of medium and the concentration of cells and hydrogen peroxide, as well as the effect of hydrogen peroxide on MDR of cells. MDR was determined from the transport of calcein acetoxymethyl ester out of the cells and from a change in cell sensitivity to vincristine. The amount of ROS arising in cells was determined using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH₂-DA). It was shown that the rate of ROS formation in cells decreases after the addition of serum to the medium and with an increase of the cell number. By the action of hydrogen peroxide, the amount of ROS increases directly with its concentration. Oxidative stress generated by 30–300 μM hydrogen peroxide decreases the MDR of the cells. The effect of hydrogen peroxide increases with the treatment duration and concentration of hydrogen peroxide. MDR determined by the criterion of the efflux of calcein ester from cells is completely suppressed after 1-h exposure to 300 μM hydrogen peroxide. At a concentration of hydrogen peroxide of 60 μM and treatment duration of 1 h, the sensitivity of P388VR cells to vincristine increases to reach the sensitivity of the wild-type P388 cells. Rapid (about 1 h) suppression of MDR is caused by inhibition of the activity of transport proteins. MDR decrease induced by oxidative stress can be used in therapy of tumors resistant to anticancer drugs.     

Kinetics and mechanisms of catalase in peroxisomes of the mitochondrial fraction

1. The primary intermediate of catalase and hydrogen peroxide was identified and investigated in peroxisome-rich mitochondrial fractions of rat liver. On the basis of kinetic constants determined in vitro, it is possible to calculate with reasonable precision the molecular statistics of catalase action in the peroxisomes. 2. The endogenous hydrogen peroxide generation is adequate to sustain a concentration of the catalase intermediate (p(m)/e) of 60-70% of the hydrogen peroxide saturation value. Total amount of catalase corresponds to 0.12-0.15nmol of haem iron/mg of protein. In State 1 the rate of hydrogen peroxide generation corresponds to 0.9nmol/min per mg of protein or 5% of the mitochondrial respiratory rate in State 4. 3. Partial saturation of the catalase intermediate with hydrogen peroxide (p(m)/e) in the mitochondrial fraction suggests its significant peroxidatic activity towards its endogenous hydrogen donor. A variation of this value (p(m)/e) from 0.3 in State 4 to 0 under anaerobic conditions is observed. 4. For a particular preparation the hydrogen peroxide generation rate in the substrate-supplemented State 4 corresponds to 0.17s(-1) (eqn. 6), the hydrogen peroxide concentration to 2.5nm and the hydrogen-donor concentration (in terms of ethanol) to 0.12mm. The reaction is 70% peroxidatic and 30% catalatic. 5. A co-ordinated production of both oxidizing and reducing substrates for catalase in the mitochondrial fraction is suggested by a 2.2-fold increase of hydrogen peroxide generation and a threefold increase in hydrogen-donor generation in the State 1 to State 4 transition. 6. Additional hydrogen peroxide generation provided by the urate oxidase system of peroxisomes (8-12nmol of uric acid oxidized/min per mg of protein) permits saturation of the catalase with hydrogen peroxide to haem occupancy of 40% compared with values of 36% for a purified rat liver catalase ofk(1)=1.7x10(7)m(-1).s(-1) and k'(4)=2.6x10(7)m(-1). s(-1)(Chance, Greenstein & Roughton, 1952). 7. The turnover of the catalase ethyl hydrogen peroxide intermediate (k'(3)) in the peroxisomes is initially very rapid since endogenous hydrogen peroxide acts as a hydrogen donor. k'(3) decreases fivefold in the uncoupled state of the mitochondria.     

Ferriprotoporphyrin IX and cell lysis: a protective role for hydrogen peroxide

Two potentially lytic substances, ferriprotoporphyrin IX (FP) and hydrogen peroxide, may coexist and partially detoxify each other in sickle cells and in erythrocytes infected with malaria parasites. Since hydrogen peroxide can decompose FP, its effect on hemolysis induced by FP and by the complex of FP with chloroquine was investigated. Human erythrocytes suspended at a concentration of 0.5% in a 50 microM solution of FP underwent approximately 42% hemolysis during the course of 2 hours. Twenty-five micromolar chloroquine potentiated hemolysis to 99%, and preincubation of 50 microM FP with 25 microM hydrogen peroxide for 5 minutes reduced hemolysis to 4%. Mixing either FP or hydrogen peroxide first with chloroquine abolished the effect of hydrogen peroxide. Detoxification of FP by hydrogen peroxide may be an important protective mechanism in certain hemolytic anemias, and inhibition of detoxification could account for the effectiveness of chloroquine in malaria.     

Glycerophosphate-dependent peroxide production by brown fat mitochondria from newborn rats

Glycerophosphate (GP)-dependent, ferricyanide-induced hydrogen peroxide production was studied in brown adipose tissue mitochondria from newborn rats. Relations between the rate of hydrogen peroxide production and total amount of hydrogen peroxide produced at different GP and ferricyanide concentrations were determined. It was found that the rate of hydrogen peroxide production increases with increasing GP concentration and decreases with increasing ferricyanide concentration. Total amount of hydrogen peroxide produced increases with increasing ferricyanide concentration, however, not proportionally, and the efficiency of this process (oxygen/ferricyanide ratio) strongly declines. Data presented provide further information on the character and kinetics of hydrogen peroxide production by mammalian mitochondrial glycerophosphate dehydrogenase.     

Plant resistance inductors and active forms of oxygen. II. The influence of chitooligosaccharides on the production of hydrogen peroxide with the involvement of oxalate oxidase in common cultures of wheat calluses and bunt pathogen

We have studied the influence of various concentrations of chitooligosaccharides (CO) on hydrogen peroxide (H2O2) generation, with the involvement of oxalate oxidase (OO), in rhizoids and in zones of hard brand pathogen penetration, as well as on oxalate oxidase activity in wheat calluses. In the control group, diaminobenzidin (DAB)-staining was typical of 30% of peripheral rhizoid cells, which provided their resistance during infection. In the zone of fungus penetration, the appearance of DAB-stained parenchyma-like cells was observed. Simultaneously, the activity OO cytoplasmic fraction increased, whereas OO activity in its ion-bound fraction was suppressed. Low concentrations of medication induced rhizoid formation, increased the number of DAB-stained cells in the pathogen penetration zone, and induced OO activity in cytoplasmic and ionically cell wall-bound fractions. On the contrary, a high concentration of CO (100 mg/ml) suppressed rhizoid formation, Oo activity and the fungus growth. The discovered correlation between enzymatic activation under CO influence, the high level of protective response during infection, and the intensity of rhizoid formation may suggest the community of protective and morphogenetic mechanisms of reactions in plant cells in respect to hydrogen peroxide production.     

Construction and Physiological Analysis of a Xanthomonas Mutant To Examine the Role of the oxyR Gene in Oxidant-Induced Protection against Peroxide Killing

We constructed and characterized a Xanthomonas campestris pv. phaseoli oxyR mutant. The mutant was hypersensitive to H₂O₂ and menadione killing and had reduced aerobic plating efficiency. The oxidants’ induction of the catalase and ahpC genes was also abolished in the mutant. Analysis of the adaptive responses showed that hydrogen peroxide-induced protection against hydrogen peroxide was lost, while menadione-induced protection against hydrogen peroxide was retained in the oxyR mutant. These results show that X. campestris pv. phaseoli oxyR is essential to peroxide adaptation and revealed the existence of a novel superoxide-inducible peroxide protection system that is independent of OxyR.     

Hydrogen Peroxide and Sodium Perborate Bleaching of Pulp from Olive Tree Residues

This paper will consider the influence of the operating conditions used in the hydrogen peroxide bleaching (concentration 1–5 % and process time 30–210 min) and sodium perborate bleaching (sodium perborate concentration 1–5 %, hydrogen peroxide 0–2% and process time 60–180 min) of olive wood trimmings pulp on the yield, kappa index and viscosity of the resulting pulp, and on strength related properties of paper sheets (stretch index and burst index) in order to determine the best bleaching conditions of this pulp. Medium to low hydrogen peroxide concentrations (1–3 %) and a high operation time (210 min) were desired in the bleaching of pulp. A high sodium perborate concentration and hydrogen peroxide concentration (5 % and 2 % respectively) and medium to low operation time (60–120 min) were desired for the sodium perborate bleaching. A comparison of both bleaching agents, under similar or under optimum operating conditions, revealed that sodium perborate bleaching results in lower brightness, a higher kappa index and also higher viscosity than hydrogen peroxide bleaching. Moreover, both provided similar stretch index and burst index values for sodium perborate bleaching with respect to hydrogen peroxide bleaching.