脱色希瓦氏菌(Shewanella decolorationis)S12T的脱色特性

从印染废水活性污泥中分离到一株高效染料脱色菌,经鉴定该菌株为希瓦氏菌属的一个新种,命名为脱色希瓦氏菌(Shewanelladecolorationis)S12T。该菌株在偶氮染料浓度为50mg/L的培养基中培养4h后,染料去除率达到96%,对偶氮染料的最高脱色浓度达到2000mg/L。在浓度为500mg/L的偶氮染料平板上生长4d后,可观察到明显的脱色圈。全波长光谱扫描的结果表明希瓦氏菌S12T以生物降解的方式对偶氮染料进行脱色。希瓦氏菌S12T的脱色酶为组成型的胞内酶。     

脱色希瓦氏菌(Shewanella decolorationis)S12T的脱色特性*

从印染废水活性污泥中分离到一株高效染料脱色菌,经鉴定该菌株为希瓦氏菌属的一个新种,命名为脱色希瓦氏菌(Shewanelladecolorationis)S12^T。该菌株在偶氮染料浓度为50mg/L的培养基中培养4h后,染料去除率达到96%,对偶氮染料的最高脱色浓度达到2000mg/L。在浓度为500mg/L的偶氮染料平板上生长4d后,可观察到明显的脱色圈。全波长光谱扫描的结果表明希瓦氏菌S12^T以生物降解的方式对偶氮染料进行脱色。希瓦氏菌S12相似文献   

用于植物病原细菌标记的pBB-GFP载体构建及应用北大核心CSCD

扩增青枯劳尔氏菌RipAK基因启动子序列,与lacZ基因融合得到p HM1:P_(RiPAK)LacZ。携带pHM1:P_(RiPAK)LacZ的青枯劳尔氏菌在营养丰富和基本培养基中都有LacZ活性,表明RipAK启动子可以推动lacZ基因的表达。为构建用于标记植物病原细菌的绿色荧光表达载体,把RipAK启动子和gfp基因克隆到质粒pBBR1MCS-5,使得gfp基因在RipAK启动子的驱动下表达;构建的表达载体pBB-GFP在大肠杆菌中即可表达绿色荧光蛋白。pBB-GFP载体能有效标记青枯劳尔氏菌、番茄细菌性斑点病菌和柑橘溃疡病菌,在荧光显微镜下观察到3种植物病原细菌呈短杆状,青枯劳尔氏菌还可形成多个菌体串联的线状结构。荧光标记对3种病原菌在寄主植物上的致病力没有影响,将标记菌株分别滴加在寄主植物叶片的创伤处,可观察到大量的绿色荧光聚集。本研究构建的pBB-GFP载体能用于多种植物病原细菌的绿色荧光标记,标记后的病原细菌在液体培养及侵染寄主植物过程中都能观察到荧光。     

用于植物病原细菌标记的pBB-GFP载体构建及应用

扩增青枯劳尔氏菌RipAK基因启动子序列,与lacZ基因融合得到p HM1:P_(RiPAK)LacZ。携带pHM1:P_(RiPAK)LacZ的青枯劳尔氏菌在营养丰富和基本培养基中都有LacZ活性,表明RipAK启动子可以推动lacZ基因的表达。为构建用于标记植物病原细菌的绿色荧光表达载体,把RipAK启动子和gfp基因克隆到质粒pBBR1MCS-5,使得gfp基因在RipAK启动子的驱动下表达;构建的表达载体pBB-GFP在大肠杆菌中即可表达绿色荧光蛋白。pBB-GFP载体能有效标记青枯劳尔氏菌、番茄细菌性斑点病菌和柑橘溃疡病菌,在荧光显微镜下观察到3种植物病原细菌呈短杆状,青枯劳尔氏菌还可形成多个菌体串联的线状结构。荧光标记对3种病原菌在寄主植物上的致病力没有影响,将标记菌株分别滴加在寄主植物叶片的创伤处,可观察到大量的绿色荧光聚集。本研究构建的pBB-GFP载体能用于多种植物病原细菌的绿色荧光标记,标记后的病原细菌在液体培养及侵染寄主植物过程中都能观察到荧光。     

Pseudomonas plecoglossicida NyZ12基因无痕敲除方法的建立

旨在建立一种适合环己胺降解菌NyZ12基因无痕敲除的可靠方法。通过overlapping PCR技术将目的基因上下游同源臂融合并克隆到自杀载体pEX18km上,将重组质粒转化到大肠杆菌S17pir中,再通过接合转移到假单胞菌NyZ12菌株内,经pEX18km质粒上sacB基因的反向筛选得到突变株并通过PCR方法和测序鉴定。结果显示,成功构建了假单胞菌NyZ12菌株orf4637的基因突变株(NyZ12Δ4637)。通过自杀载体同源重组可以成功获得敲除的无痕突变株,且突变株基因组上没有任何抗性筛选标记残留,为环己胺降解菌NyZ12基因功能研究提供了可靠的基因敲除技术。     

染料脱色菌的分子分类和有关基因与其脱色特性的关系

从印染废水处理系统中分离到12株具有不同脱色特性的细菌,比较分析这12株细菌的质粒携带情况、分子分类地位和黄素还原酶基因(fre)的拷贝数与其脱色特性之间的关系。结果发现,所分离到的这12株菌分别归属于希瓦氏菌属、克雷伯氏菌属、假单胞菌属、气单胞菌属和苍白杆菌属。具有较广谱脱色能力的细菌在系统进化树上基本聚为一类,分别归属于希瓦氏菌属和气单胞菌属,其质粒携带率也较低。fre基因在不同菌属中的基因型存在很大的差异,仅仅采用一套引物无法对不同菌属中的fre基因进行有效的扩增。     

Tn10介导的Bit cryIVA基因在荧光假单胞菌染色体中的整合及表达

苏云金杆菌以色列亚种的杀虫晶体蛋白基因cryIVA被亚克隆到自杀型转座子质粒载体pLOF／Km的TN10中,构建了转座子质粒PLF97A。通过电转化／转座作用,cryIVA随Tn10转座并整合到荧光假单胞菌FP．DE2染色体中,构建了工程菌株FP．DE202。Southernblotting验证了cryIVA在FP．DE202中整合在不同的位点。Westernblotting证明了cryIVA在F．P．DE202中得到了表达,其产物对双翅目害虫韭菜迟眼蕈蚊的3龄幼虫有较强的毒杀效果。     

Mcc在脱色希瓦氏菌S12电极呼吸中的作用

【目的】研究脱色希瓦氏菌S12周质空间c型细胞色素Mcc的功能,进一步探索和补充微生物胞外电子传递过程的机制。【方法】借助自杀质粒敲除mcc基因,通过细胞浓度测定和激光共聚焦显微镜比较分析突变株和野生株之间的浮游细胞和生物膜的生长情况,并比较分析二者在微生物燃料电池电极还原、铁还原和胞外偶氮染料还原过程中的功能。【结果】Mcc缺失对铁还原和偶氮还原没有影响,但却造成电极呼吸活性下降34.1%;与野生株相比,mcc突变株的好氧生长和厌氧浮游细胞生长无明显影响,但却显著抑制了电极表面生物膜的形成。【结论】Mcc是希瓦氏菌S12电极呼吸过程中周质空间电子传递的重要组分之一,缺失会显著抑制其电极呼吸效率以及生物膜的形成。     

生防链霉菌SSD49的绿色荧光蛋白标记及其在毛白杨组培苗中的定殖

杨树溃疡病是中国杨树人工林重大生物灾害之一,采用生物防治的方法控制杨树溃疡病是持续有效的手段。为了获取能够高效定殖并对杨树溃疡病菌有良好生防效果的拮抗菌株,通过引入强启动子(erm Ep)构建了高表达绿色荧光蛋白(Green fluorescent protein,GFP)的重组质粒,进而通过接合实验将该质粒导入到链霉菌生防菌株SSD49,构建了绿色荧光蛋白标记菌株SSD49-p IJ8660Ep,利用荧光显微镜研究该生防菌在毛白杨组培苗中的定殖情况。结果表明,SSD49标记绿色荧光蛋白后没有影响其对杨树溃疡病病原菌的抑菌活性,标记菌株能够定殖于毛白杨组培苗的茎和叶中。成功将杨树溃疡病生防菌株SSD49进行了绿色荧光蛋白标记,并且该菌株在毛白杨组培苗中有一定的定殖能力。     

呋喃丹降解菌CDS-1的双标记菌株的构建

用Sau3AI消化呋喃丹降解菌Sphingomonassp.CDS-1的基因组DNA,将所得DNA片段与BamHⅠ酶切的启动子探针载体pRobe-GFP酶连后转化E.coliDH5α感受态细胞,在选择性平板上培养,从大约1×104个菌落中筛选到50个含启动子片段的阳性克隆。挑选其中一个发光强度最强的阳性克隆F7,将它的重组质粒pF7用EcoRⅠ和HindⅢ双酶切后得到包含Sphingomonassp.CDS-1启动子和gfp基因的DNA片段,将该片段克隆到广宿主载体pPZP201上,得到pPZP201-gfp质粒。将pPZP201-gfp通过三亲接合转移至Sphingomonassp.CDS-1中得到GFP标记菌株CDS-gfp,经荧光显微镜观察,gfp基因在CDS-gfp中表达量很高。对标记菌株进行连续传代10次(48h/次),发现pPZP201-gfp依然存在,而且发光明显。通过NotⅠ酶切位点把linA基因连接到pUT/mini-Tn5上构建新的转座子载体pUT/mini-Tn5-linA。以pRK600为辅助质粒将pUT/mini-Tn5-linA引入到CDS-1中,linA基因通过转座作用,插入到CDS-gfp的染色体中,得到双标记菌株CDS-GFP-LinA。该菌株是一株能同时降解γ-六六六和呋喃丹的基因工程菌,本研究的结果为研究Sphingomonassp.CDS-1的生态学行为奠定了基础。     

脱色希瓦氏菌S12 非特异性偶氮还原酶基因表达及特性

摘要：【目的】对脱色希瓦氏菌S12 (Shewanella decolorationis S12)的acpD基因(登录号EF198254)及其表达活性进行研究。【方法】采用DNAMAN软件对该基因进行序列分析。利用PCR技术克隆含原有启动子的目的基因,与pGM-T载体连接后转化仅有微弱偶氮还原活性的大肠杆菌TOP10(Escherichia coli TOP10)中进行表达。通过分光光度法测定偶氮染料的还原活性。【结果】序列分析表明,该基因编码198个氨基酸残基组成的多肽,与希瓦氏菌ANA-3(Shewa     

Identification of an uptake hydrogenase for hydrogen-dependent dissimilatory azoreduction by <Emphasis Type="Italic">Shewanella decolorationis</Emphasis> S12

Shewanella decolorationis S12, a representative dissimilatory azo-reducing bacterium of Shewanella genus, can grow by coupling the oxidation of hydrogen to the reduction of azo compounds as the sole electron acceptor, indicating that an uptake hydrogenase is an important component for electron transfer for azoreduction. For searching to the uptake hydrogenase in the genome of S. decolorationis, two operons, hyd and hya, were cloned and sequenced, which encode periplasmically oriented Fe-only hydrogenase and a Ni-Fe hydrogenase, respectively, according to the homologous comparison with other bacterial hydrogenases. In order to assess the roles of these two enzymes in hydrogen-dependent azoreduction and growth, hyd- and hya-deficient mutants were generated by gene replacement. Hya was found to be required for hydrogen-dependent reduction of azo compound by resting cell suspensions and to be essential for growth with hydrogen as electron donor and azo compound as electron acceptor. Hyd, in contrast, was not. These findings suggest that Hya is an essential respiratory hydrogenase of dissimilatory azoreduction in S. decolorationis.     

脱色希瓦氏菌(Shewanella decolorationis) S12还原不同电子受体的厌氧发酵罐培养方法

脱色希瓦氏菌Shewanella decolorationisS12在厌氧环境下能够使用多种电子受体进行厌氧呼吸。为了取得足够的细胞量用于膜蛋白质组学等科学研究的需要,本研究选取无机小分子(硝酸钠)、金属离子(柠檬酸铁)和有机大分子(偶氮染料苋菜红)作为电子受体,在使用确定成分的无机盐培养基条件下,使用不同浓度的电子供体和碳源对S12进行厌氧条件下静置和发酵罐的优化培养,采用连续补充电子受体的培养方式,确认了电子供体和碳源的合适浓度,建立了S12厌氧发酵罐培养方法。相比传统的静置厌氧培养,厌氧发酵罐培养方法在保证了严格厌氧条件下高效率还原电子受体的同时,还极大的提高了细胞生长密度。连续补充电子受体的厌氧发酵罐培养的S12最大细胞密度最大分别可达到静置厌氧培养细胞密度的325,304,369倍,而生长时间也比静置厌氧培养分别缩短了26.5%,17.6%,7.5%。这为需要大量细胞和蛋白的细菌厌氧呼吸生长实验建立了可行方法,对于进行兼性厌氧呼吸的微生物的大规模厌氧培养具有借鉴意义。     

Humic substances act as electron acceptor and redox mediator for microbial dissimilatory azoreduction by Shewanella decolorationis S12

The potential for humic substances to serve as terminal electron acceptors in microbial respiration and the effects of humic substances on microbial azoreduction were investigated. The dissimilatory azoreducing microorganism Shewanella decolorationis S12 was able to conserve energy to support growth from electron transport to humics coupled to the oxidation of various organic substances or H2. Batch experiments suggested that when the concentration of anthraquinone-2-sulfonate (AQS), a humics analog, was lower than 3 mmol/l, azoreduction of strain S12 was accelerated under anaerobic condition. However, there was obvious inhibition to azoreduction when the concentration of the AQS was higher than 5 mmol/l. Another humics analog, anthraquinone-2-sulfonate (AQDS), could still prominently accelerate azoreduction, even when the concentration was up to 12 mmol/l, but the rate of acceleration gradually decreased with the increasing concentration of the AQDS. Toxic experiments revealed that AQS can inhibit growth of strain S12 if the concentration past a critical one, but AQDS had no effect on the metabolism and growth of strain S12 although the concentration was up to 20 mmol/l. These results demonstrated that a low concentration of humic substances not only could serve as the terminal electron acceptors for conserving energy for growth, but also act as redox mediator shuttling electrons for the anaerobic azoreduction by S. decolorationis S12. However, a high concentration of humic substances could inhibit the bacterial azoreduction, resulting on the one hand from the toxic effect on cell metabolism and growth, and on the other hand from competion with azo dyes for electrons as electron acceptor.     

微生物燃料电池中脱色希瓦氏菌S12的产电特性研究

为了确定脱色希瓦氏菌S12的电化学活性, 采用循环伏安法(cyclic voltammograms, CV)对厌氧培养的菌株S12进行曲线扫描, 所得曲线表明S12具有一定的电化学活性, 可以用来进行产电实验。研究了不同电子供体和供体浓度对菌株S12产电的影响, 结果表明, 以浓度为10 mmol/L 的不同有机酸(甲酸钠、乳酸钠和丙酮酸钠)分别作为电子供体时, 乳酸钠产电量最大, 其最大功率密度Pmax为21.93 mW/m2, 增加乳酸钠的浓度, 菌株S12的产电量也相应增加, 当乳酸钠的浓度为20 mmol/L时, 所产生的最大功率密度达55.72 mW/m2。     

微生物燃料电池中脱色希瓦氏菌S12的产电特性研究

为了确定脱色希瓦氏菌S12的电化学活性,采用循环伏安法(cyclic voltammograms, CV)对厌氧培养的菌株S12进行曲线扫描,所得曲线表明S12具有一定的电化学活性,可以用来进行产电实验.研究了不同电子供体和供体浓度对菌株S12产电的影响,结果表明,以浓度为10mmol/L的不同有机酸(甲酸钠、乳酸钠和丙酮酸钠)分别作为电子供体时,乳酸钠产电量最大,其最大功率密度Pmax为21.93mW/m2增加乳酸钠的浓度,菌株S12的产电量也相应增加,当乳酸钠的浓度为20mmol/L时,所产生的最大功率密度达55.72 mW/m2.     

Comparison of high pressure-induced dissociation of single-stranded DNA-binding protein (SSB) from high pressure-sensitive and high pressure-adapted marine Shewanella species

The effects of hydrostatic pressure on protein quaternary structure were compared for recombinant single-stranded DNA-binding protein (SSB) derived from piezosensitive, piezotolerant, and obligately piezophilic ("pressure-loving") marine Shewanella strains. The pressure-induced dissociation of the oligomeric SSB proteins was investigated using fluorescence anisotropy. The SSBs all exhibited striking similarity in the pressure-dependent behavior of the fluorescence intensity and emission spectrum as well as in their dissociation constants at atmospheric pressure. The free energies of subunit association into tetramers for all SSBs were between -27 and -30 kcal mol(-1). However, SSB from the piezosensitive Shewanella strain S. hanedai was more sensitive to pressure than that of the SSB proteins from the piezotolerant or piezophilic bacteria. The volume change of association obtained from the pressure dependence of dissociation at a fixed protein concentration (Delta V(p)) for SSB from S. hanedai was 394-402 ml mol(-1). The Delta V(p) values for SSB from the deeper-living Shewanellas were smaller and ranged from 253 to 307 ml mol(-1). Differences between the primary structures of the SSB proteins that could correlate with differences in sensitivity to pressure-induced dissociation were examined.     

一株海洋产电菌Shewanella sp. S2的筛选和产电分析

以厦门白城海域的潮间带表面沉积物为菌种来源筛选得到一株具有电催化活性的菌株S2,该菌株的16S rRNA和gyrB基因发育树与Shewanella oneidensis MR-1同支,相似性分别为98.5%和87%,葡萄糖、木糖、半乳糖等碳源利用及最佳生长的NaCl浓度与S.oneidensis MR-1有显著差别,因此初步鉴定为Shewanella属菌株,命名为Shewanella sp.S2。初步研究了菌株S2产电活性,在以乳酸作为碳源产电时,电压最高为150mV,相应的电流密度为66.1mA/m2。