红球菌R04苯甲酸转运相关膜蛋白RHOGL009301的生理功能

【目的】探究红球菌(Rhodococcus sp.)R04膜蛋白RHOGL009301的生理功能和突变菌株的代谢特性,确定该膜蛋白的生理功能与苯甲酸转运的关系。【方法】将RHOGL009301基因与绿色荧光蛋白基因在Rhodococcus erythropolis进行融合表达,Delta Vision观察该基因蛋白产物的定位。通过基因同源重组敲除RHOGL009301基因,并对比野生型菌株和缺陷型菌株在不同碳源培养下的生长情况。HPLC测定红球菌R04野生型菌株和缺陷型菌株代谢联苯和苯甲酸时细胞内外代谢物,分析不同生长条件下代谢物的浓度变化。【结果】RHOGL009301基因与绿色荧光蛋白基因在Rhodococcus erythropolis中实现共表达,并定位在细胞膜上。获得了RHOGL009301基因的缺陷型菌株R04ΔMP,与野生型菌株相比,缺陷型菌株在联苯和苯甲酸培养条件下的生物量明显降低,生长速度减慢。HPLC分析表明RHOGL009301基因的缺失抑制了苯甲酸的转运。【结论】膜蛋白RHOGL009301是苯甲酸代谢和转运相关的蛋白,基于序列同源性分析,该膜蛋白是一种新型的苯甲酸转运蛋白。     

红球菌R04细胞的不对称分裂及其在联苯胁迫下的分裂抑制

【目的】研究红球菌R04细胞的分裂方式及联苯对其形态和细胞分裂的影响。【方法】以一株多氯联苯降解菌株(Rhodococcus sp.R04)为研究对象,利用荧光显微镜、扫描电子显微镜及透射电子显微镜分析红球菌R04在不同培养条件下的细胞分裂。【结果】红球菌R04细胞表现出对称分裂(约占30%)和不对称分裂(约占70%)两种分裂方式,且培养条件不影响不对称分裂细胞所占的比例。细胞分裂过程中,隔膜主要分布于细胞长度的30%–50%。在联苯的分解代谢过程中,红球菌R04细胞的生长分裂会受到联苯的抑制,但不影响红球菌R04细胞的分裂方式,在联苯胁迫下,细胞形成丝状化,表现出异常分裂,随着培养时间的延长,在细胞生长指数后期至转换期,细胞能够进行正常分裂。【结论】环境异生型化合物联苯/多氯联苯对其降解菌株——红球菌R04细胞的生长和分裂有较强影响,但是并不影响其分裂方式。     

N端缺失的锰过氧化氢酶生理生化特性

【目的】在红球菌(Rhodococcus sp.)R04中发现了一种高表达,N端缺失的锰过氧化氢酶(Mn-CAT),为了明确其在活性氧(Reactive oxygen species,ROS)清除与多氯联苯(Polychlorinated biphenyls,PCBs)代谢中所起的作用,本文对其生理生化特性进行了研究。【方法】利用DNAMAN对Rhodococcus sp.R04与Rhodococcus sp.R1101Mn-CAT的核酸和蛋白序列进行比对。化学合成和PCR搭桥法获取Mn-CAT全长基因。分别构建了原核表达载体p ETm3c-Mn-CAT,p ETm3c-Mn CAT-C,转入大肠杆菌(Escherichia coli)BL21,得到重组菌p ETm3c-Mn-CAT/BL21,p ETm3c-Mn CAT-C/BL21。工程菌诱导表达后,粗酶液经Q-sepharose和硫铵沉淀进行纯化。构建了锰过氧化氢酶C端(Mn CAT-C)基因的敲除载体p K18mobsac B-ΔMn CAT-C,电击法转入Rhodococcus sp.R04。荧光极化测定ROS的含量,HPLC分析多氯联苯的降解率。【结果】与Rhodococcus sp.R1101Mn-CAT基因序列相比,Rhodococcus sp.R04Mn-CAT缺少N端(R1101的Mn-CAT序列长度为915bp,R04的Mn CAT-C序列长度为468bp)。获得了纯度较高的Mn CAT-C,SDS-PAGE分析表明分子量约为23 k Da。以H2O2为底物时,Mn CAT-CKm比Mn-CATKm大,约为0.02357mol/L。通过基因同源重组的方式,得到菌株R04的Mn CAT-C敲除菌株,与野生菌株相比,敲除菌株体内ROS浓度显著增高,生长速率和多氯联苯降解速率明显下降。【结论】发现了一种N端缺失的锰过氧化氢酶,该酶具有原酶的大部分活性,且可以清除体内的ROS。Mn CAT-C基因的缺失影响了菌株的生长速率和多氯联苯的降解速率。     

联苯培养条件下红球菌R04转录表达和苯甲酸代谢途径解析

摘要:【目的】研究不同碳源,特别是联苯条件下红球菌的细胞转录应答,以挖掘与多氯联苯(PCBs)转运、代谢及其调控相关的基因,为进一步全面理解PCB微生物降解的分子机制奠定基础。【方法】以一株多氯联苯降解菌红球菌(Rhodococcus sp.R04)为材料,分别提取不同碳源(乙醇、葡萄糖和联苯)培养条件下菌体的总RNA,反转录合成cDNA。采用高通量测序法分别对这三种样品进行转录组测序,分析测序数据得出全基因组表达模式,并对不同条件下的基因表达进行差示分析,进而对联苯代谢网络和红球菌中其他基因的转录调节和代谢应答反应做出相关性分析。Q-RT-PCR分析不同碳源培养条件下的基因表达情况。【结果】测序结果表明,与葡萄糖和乙醇相比,联苯培养条件下明显上调(log2 Ratio 1)基因个数分别为375和332个。与葡萄糖相比,联苯培养条件下,相关基因上调表达量与Q-RT-PCR实验结果基本一致。功能分类获得细胞组分、分子功能和生物学过程三大类别160多个细小分支的差示表达基因,部分基因参与联苯代谢转录调控、联苯转运、抗氧化应激反应以及信号传导通路系统等多种生理过程。参与联苯上游代谢途径的众多同工酶基因中,只有bphC2和bphD1在联苯中大量上调表达,其余同工酶在联苯中基本量不变或下调表达。转录组注释及差示分析推测,红球菌R04中苯甲酸的代谢主要是通过儿茶酚邻位途径、间位途径以及原儿茶酸途径三条代谢途径完成。【结论】与葡萄糖和乙醇相比,红球菌R04在联苯培养条件下基因表达差异明显,这为我们进一步解析多氯联苯代谢特征和代谢调控提供理论依据。     

卡西霉素生物合成基因簇中调控基因calR1的功能

【背景】卡西霉素(calcimycin)是重要的离子载体抗生素,其生物合成基因簇已从教酒链霉菌NRRL3882的基因组DNA中成功克隆,但基因簇内的部分生物合成基因及调控基因的功能有待研究。【目的】研究卡西霉素产生菌教酒链霉菌NRRL3882中编码TylR家族同源转录调控蛋白的calR1基因的功能。【方法】通过PCR-targeting的方法,构建calR1基因敲除突变株及回补菌株,对突变菌株及回补菌株进行发酵,通过HPLC分析其代谢产物。利用荧光定量PCR检测ΔcalR1突变菌株和野生菌株的生物合成基因转录水平。【结果】calR1基因敲除突变株丧失产生卡西霉素的能力,但仍有中间产物噻唑霉素的积累,回补菌株中卡西霉素的产量有一定程度的恢复。RT-qPCR结果表明,卡西霉素合成相关的一些重要基因calC、calG、calU3等基因的表达量明显改变。【结论】TylR家族转录调控基因calR1是卡西霉素生物合成的调控基因。     

H-NS蛋白对副溶血弧菌hcp1的转录调控

【目的】研究调控子H-NS对副溶血弧菌T6SS1结构蛋白基因hcp1的转录调控机制。【方法】利用Western blot检测Hcp1蛋白在野生株(WT)和hns基因敲除株(Δhns)中表达水平的差异。提取WT和Δhns的总RNA,采用实时定量RT-PCR的方法验证H-NS对hcp1的转录调控关系。进而采用引物延伸实验研究hcp1的转录起始位点,并根据产物的丰度判断H-NS对hcp1的调控关系。PCR扩增hcp1的整个启动子区DNA序列,并纯化His-H-NS蛋白,通过凝胶阻滞实验(EMSA)验证His-H-NS对hcp1启动子区是否具有直接的结合作用。【结果】Western blot和实时定量RT-PCR结果显示H-NS能抑制hcp1的表达;引物延伸结果显示hcp1只有一个转录起始位点T(–62)(翻译起始位点为+1),且其转录活性是H-NS和σ54依赖性的;EMSA实验表明H-NS对hcp1的启动子区具有直接的结合作用。【结论】H-NS能直接结合到hcp1启动子区而抑制其转录表达。     

苏云金芽胞杆菌Sigma H对芽胞形成的影响

【目的】检测苏云金芽胞杆菌HD73中的转录调控因子Sigma H(σ~H)对spo0A基因转录的调控作用;异源表达纯化Sigma H蛋白,验证其对spo0A基因启动子的直接结合;检测sigH基因的缺失对苏云金芽胞杆菌HD73芽胞形成和晶体蛋白产生的影响。【方法】通过测定spo0A基因启动子指导的β-半乳糖苷酶活性评价spo0A基因在苏云金芽胞杆菌HD73野生型和sigH缺失突变体中的转录水平;通过PCR扩增苏云金芽胞杆菌HD73的sigH基因并插入到表达载体pET21b上,将质粒转入到表达菌株BL21(DE3)中,得到重组菌株BL21 (pETsigH);利用镍柱亲和纯化和阴离子交换纯化得到纯化的Sigma H蛋白;通过凝胶迁移实验(electrophoretic mobility shift assay,EMSA)验证Sigma H蛋白与spo0A基因启动子的直接结合;通过显微镜观察、活芽胞计数的方法对突变株HDΔsigH进行表型特征分析。【结果】sigH缺失后,spo0A基因转录活性降低;在大肠杆菌中正确表达并纯化出大小约为28kDa的Sigma H-His蛋白;EMSA结果表明纯化后的Sigma H-His蛋白可与spo0A基因启动子结合;镜检和活芽胞计数结果表明突变株HDΔsigH无法产生芽胞和蛋白晶体。【结论】Sigma H蛋白通过与spo0A基因启动子结合直接调控spo0A基因的表达且sigH基因的缺失阻断了苏云金芽胞杆菌中芽胞和晶体蛋白的产生。     

苏云金芽胞杆菌rocE基因的转录调控

【目的】rocE基因编码精氨酸降解途径中的精氨酸通透酶,通过分析苏云金芽胞杆菌(Bacillus thuringiensis,Bt) rocE基因的转录活性,明确rocE基因的转录调控机制。【方法】通过RT-PCR确定rocE基因所在基因簇的转录单元;β-半乳糖苷酶活性测定分析rocE基因启动子(ProcE)的转录活性;采用同源重组技术敲除BtHD73菌株的rocE基因;通过融合His标签的方法在大肠杆菌中表达纯化RocR蛋白的HTH结构域;通过凝胶阻滞实验明确RocR与rocE基因启动子的结合作用。【结果】在M9培养基中,精氨酸可诱导ProcE的转录活性;在SSM培养基和精氨酸诱导培养基中,与出发菌株HD73相比,ProcE在sigL (编码Sigma54因子)突变体和rocR突变体中的转录活性显著下降。RocR-HTH蛋白与ProcE有结合作用。rocE基因的缺失对菌体生长和Cry1Ac蛋白产量无显著影响。rocE缺失突变体的芽胞形成率为65.5%,HD73出发菌株为85.7%,显著性分析结果表明差异显著(P0.05)。【结论】rocE基因的转录活性受Sigma54的控制,并受RocR正调控。rocE基因的缺失影响菌株的芽胞形成率。     

卡西霉素生物合成调控基因calR2的功能

【背景】卡西霉素(Calcimycin)是由教酒链霉菌NRRL3882产生的吡咯聚醚类抗生素,结构独特且具有广泛的生物活性,但其生物合成调控机制尚不清楚。【目的】研究卡西霉素生物合成基因簇上编码LuxR家族同源蛋白的潜在调控基因calR2的功能。【方法】通过PCR-targeting的方法对卡西霉素基因簇上的calR2基因进行中断,HPLC对突变株及回补菌株的代谢产物进行分析。利用荧光定量RT-PCR分析ΔcalR2突变菌株和野生菌株的基因转录水平差异。【结果】calR2基因中断的突变株不能产生卡西霉素,回补菌株则恢复产生卡西霉素的能力。RT-PCR结果表明卡西霉素生物合成的一些重要骨架基因在ΔcalR2突变株中的转录水平明显降低。【结论】LuxR家族转录调控基因calR2在卡西霉素生物合成过程中起正调控作用。     

白假丝酵母CFL1基因通过转录调控参与氧化压力应答

【背景】CFL1基因是白假丝酵母高铁还原酶基因,介导胞外铁离子的还原,在白假丝酵母胞内铁稳态的维持方面发挥着重要作用。【目的】研究CFL1基因调节氧化压力应答的分子机制。【方法】采用液体培养及巨噬细胞模型,测定CFL1缺失对氧化压力耐受性和杀伤巨噬细胞能力的影响;使用羟基自由基清除剂二甲基亚砜(DMSO)分析其对缓解氧化压力敏感性的影响;采用实时荧光定量PCR分析CFL1缺失对氧化压力应答基因表达的影响;采用过氧化氢酶(CAT)活性测定方法研究CFL1缺失对CAT1基因表达的影响;通过构建WT-CAT1-GFP和cfl1Δ/Δ-CAT1-GFP菌株分析过氧化氢酶基因过表达对cfl1Δ/Δ氧化压力敏感性的影响。【结果】白假丝酵母CFL1基因的缺失会造成杀伤巨噬细胞能力的减弱,氧化压力应答基因表达的下降。过氧化氢酶基因的过表达则能恢复与野生型几乎一致的氧化压力水平。【结论】CFL1基因通过转录调控参与白假丝酵母氧化压力应答过程。     

Metabolism of hydroxybiphenyl and chloro-hydroxybiphenyl by biphenyl/chlorobiphenyl degradingPseudomonas testosteroni,strain B-356

Summary A biphenyl (BP) and chlorobiphenyl (CBP) metabolizingPseudomonas testosteroni, strain B-356 was also capable of utilizing 2-, 3-, and 4-hydroxybiphenyl. Data presented here suggest that utilization of biphenyl and mono-subtituted biphenyls involves the enzymes of the same pathway. Chloro-hydroxybiphenyls were also metabolized by strain B-356. The unsubstituted ring is first hydroxylated in position 2 and 3 and then cleaved in ameta 1, and 2, position to ultimately generate the benzoic acid derivatives. Since strain B-356 was capable of utilizing benzoic acid and mono-hydroxybenzoic acids, the utilization of biphenyl, 2-, 3-, and 4-hydroxybiphenyl is complete at non-toxic concentrations of the substrates. Chlorobenzoic acids and chloro-hydroxybenzoic acids were not metabolized further by this strain. Studies usingPseudomonas putida, strain KT2440 carrying cloned BP/CBP genes from strain B-356 provided further evidence for the presence of a common pathway for the metabolism of the above compounds inP. testosteroni, strain B-356. Suggestions are made on significance of the broad substrate specificity of the enzymes of biphenyl/chlorobiphenyl pathway in regard to their possible origin and in relation to PCB mixture degradation.     

Burkholderia xenovorans LB400联苯双加氧酶及突变体对DDTs的降解

DDTs（dichlorodiphenyltrichloroethane,1,1,1-三氯-2,2-双氯苯基乙烷）是一种典型的持久性有机污染物,曾在疟疾防治和农业除虫方面被广泛应用。虽然包括我国在内的很多国家已经禁止使用DDTs,但目前对环境中DDTs的检测发现它仍然广泛存在且具有新的输入源。DDTs的持续存在对近海生态系统和人类健康具有一定危害,因此它所造成的环境污染问题仍然值得关注。由于Rieske型芳香羟化双加氧酶能够起始多种持久性污染物的降解,过去的几十年里一直是芳香化合物降解领域的焦点。[目的] 为探讨联苯双加氧酶对DDTs的降解特性及机制,本研究选取了食异生素伯克霍尔德氏菌LB400（Burkholderia xenovorans）联苯双加氧酶及突变体对p,p''-DDT和o,p''-DDT的降解过程进行研究。[方法] 以BphAE_LB400为亲本,通过两步定点突变将283位的丝氨酸突变为蛋氨酸,获得突变体BphAE_S283M。通过比较亲本酶与突变体对DDTs的催化性能,模拟突变蛋白结构和分子对接等方法,探究其降解特性及机制。[结果] BphAE_LB400和突变体BphAE_S283M都无法降解对位的p,p''-DDT,但突变体BphAE_S283M可以代谢o,p''-DDT并产生2个立体异构体。对接p,p''-DDT的BphAE_LB400和BphAE_S283M的结构分析表明,BphAE_LB400和BphAE_S283M中p,p''-DDT的反应环均不与原晶体结构中的联苯反应环重合。而对接o,p''-DDT的BphAE_S283M的结构分析表明o,p''-DDT的反应环与晶体结构中的联苯反应环距离很近,且2、3位的碳原子与单核铁原子催化中心的距离在0.5 nm以内,此外,BphAE_S283M的催化腔表面积和体积比BphAE_LB400更大,这很可能有助于BphAE_S283M与o,p''-DDT的结合。[结论] 283位氨基酸是影响BphAE_LB400对DDTs的催化代谢能力的关键氨基酸残基,它可以通过调节反应碳原子与催化中心的距离以及催化腔的大小来影响底物特异性。本次研究进一步阐明了283位氨基酸残基的影响机理,为更有效修复DDTs污染提供理论依据和技术支持。     

Extensive biodegradation of highly chlorinated biphenyl and Aroclor 1242 by Pseudomonas aeruginosa TMU56 isolated from contaminated soils

A bacterial strain, designated TMU56, was isolated from soil that had been contaminated with electrical transformer fluid (Askarel) for over 35 years. The isolate was identified as Pseudomonas aeruginosa using its 16S rDNA sequence. This strain was found to grow on monochlorobiphenyls (CBs), including 2-chlorobenzoic acid and 4-chlorobenzoic acid. It was also found to grow on 2,4-, 2,5-, 2,2′-, and 4,4′-diCB, as well as on a wide range of other xenobiotic compounds. This is the first reported representative of the genus Pseudomonas that is capable of growing on 2,4,4′-triCB, 2,2′,5,5′-tetraCB and 2,2′,4,4′,5,5′-hexaCB as sole carbon sources. Washed benzoate-grown cells were able to degrade 89% and 56% of 2,4-diCB and 2,2′,4,4′,5,5′-hexaCB, respectively. Gas chromatography analysis of individual congeners in Aroclor 1242 (200 ppm) following a 4-day incubation showed 73.3% degradation of PCBs without the need for biphenyl as an inducer. The strain exhibited no noticeable specificity for the percentage of congener transformation or degree of chlorination.     

Variation in polychlorinated biphenyl levels in common carp from a primarily agricultural drainage

Concentrations of polychlorinated biphenyls (PCBs) in common carp, Cyprinus carpio, from the Des Moines River, Iowa, were assessed for variability related to sampling location, sampling period, fish age, and fat content. Concentrations were highest at a location near the City of Des Moines; they were substantially lower in 1981 than in 1980. Age and fat content had little influence on PCB concentrations in carp. Overall concentrations were some of the lowest recorded in the United States and Canada in recent times.The Unit is jointly supported by Iowa State University, the Iowa State Conservation Commission, and the U.S. Fish and Wildlife Service.Journal Paper No. 10754 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 2465. Financed by a grant from the U.S. Department of Defense Army Corps of Engineers and made available through the Engineering Research Institute, Iowa State University.     

Flux control analysis for biphenyl metabolism by Rhodococcus pyridinovorans R04

The flux control coefficients of the four enzymes involved in the upper pathway of biphenyl degradation were determined from transient metabolite concentrations. The first enzyme was indicated as the major rate-limiting step of the pathway with a control coefficient of 0.48. The flux control coefficients of the other three enzymes were 0.03, 0.23 and 0.27, respectively. This is the first experimental evidence of the control step in the pathway of biphenyl degradation using metabolic control analysis.     

Oxidation of biphenyl by the Cyanobacterium,Oscillatoria sp., strain JCM

The oxidation of biphenyl by Cyanobacterium, Oscillatoria sp., strain JCM was studied. The organism grown photoautotrophically in the presence of biphenyl oxidized biphenyl to form 4-hydroxybiphenyl. The structure of the metabolite was elucidated by ultraviolet and mass spectra and shown to be identical to authentic 4-hydroxybiphenyl. In addition this metabolite had properties indentical to 4-hydroxybiphenyl when analyzed by thin-layer and high-pressure liquid chromatography. Experiments with [¹⁴C]-biphenyl showed that over a 24 h period the organism oxidized 2.9% of the added biphenyl to ethyl acetate-soluble products.Abbreviations tlc thin-layer chromatography - hplc high pressure liquid chromatography     

In vivo genotoxicity of ortho-phenylphenol, biphenyl, and thiabendazole detected in multiple mouse organs by the alkaline single cell gel electrophoresis assay

In Japan, ortho-phenylphenol (OPP), biphenyl (BP), and thiabendazole (2-(4'-thiazolyl)benzimidazole, TBZ) are commonly used as a postharvest treatment to preserve imported citrus fruits during transport and storage. We used a modification of the alkaline single cell gel electrophoresis (SCG) (Comet) assay to test the in vivo genotoxicity of those agents in mouse stomach, liver, kidney, bladder, lung, brain, and bone marrow. CD-1 male mice were sacrificed 3, 8, and 24 h after oral administration of the test compounds. OPP (2000 mg/kg) induced DNA damage in the stomach, liver, kidney, bladder, and lung, BP (2000 mg/kg) and TBZ (200 mg/kg) induced DNA damage in all the organs studied. For OPP, increased DNA damage peaked at 3–8 h and tended to decrease at 24 h. For BP, on the contrary, increased DNA migration peaked at 24 h. That delay may have been due to the fact that OPP is metabolized by cytochrome 450 and prostaglandin H synthase to phenylbenzoquinone (PBQ), a DNA binding metabolite, and BP is metabolized to PBQ via OPP and m-phenylphenol. The positive response to TBZ, an aneugen, supports the in vivo DNA-damaging action of TBZ.     

Induction of glutathione-S-transferase in the Northern quahog Mercenaria mercenaria after exposure to the polychlorinated biphenyl (PCB) mixture Aroclor 1248

The glutathione-S-transferases (GSTs) from the Northern quahog (Mercenaria mercenaria) were examined after an injection of a polychlorinated biphenyl (PCB) mixture, Aroclor 1248, to a concentration of ~50 ppm. Enzymatic analysis indicated a fourfold increase in the GST activity of quahogs injected with PCBs compared with that of the control. An electrophoretic analysis of the GST from the PCB-exposed quahogs showed a 1.5-fold increase in the concentration over that of the control. Purification of the GST on a glutathione affinity column yielded a glutathione binding protein, in addition to the GSTs. However, the amount of the glutathione binding protein in the PCB-injected quahogs was found to decrease by ~50% in comparison to the glutathione binding protein in the control quahogs.     

Characterization of two biphenyl dioxygenases for biphenyl/PCB degradation in A PCB degrader, Rhodococcus sp. strain RHA1

Rhodococcus sp. RHA1 induces two biphenyl dioxygenases, the BphA and EtbA/EbdA dioxygenases, during growth on biphenyl. Their subunit genes were expressed in R. erythropolis IAM1399 to investigate the involvement of each subunit gene in their activity and their substrate preferences. The recombinant expressing ebdA1A2A3etbA4 and that expressing bphA1A2A3A4 exhibited 4-chlorobiphenyl (4-CB) transformation activity, suggesting that these gene sets are responsible for the EtbA/EbdA and BphA dioxygenases respectively. When bphA4 and etbA4 were swapped to construct the recombinants expressing ebdA1A2A3bphA4 and bphA1A2A3etbA4 respectively, compatibility between BphA4 and EtbA4 was suggested by their 4-CB transformation activities. When bphA3 and ebdA3 were swapped, incompatibility between BphA3 and EbdA3 was suggested. BphA and EtbA/EbdA dioxygenases exhibited the highest transformation activity toward biphenyl and naphthalene respectively, and also attacked dibenzofuran and dibenzo-p-dioxin. The wide substrate preference of EtbA/EbdA dioxygenase suggested that it plays a more important role in polychlorinated biphenyl (PCB) degradation than does BphA dioxygenase.