arcA基因提高大肠杆菌对有机溶剂的耐受性

【目的】将来源于恶臭假单胞菌(Pseudomonas putida JUCT1)的基因arc A(编码精氨酸脱亚胺酶)整合到Escherichia coli JM109(DE3)基因组中,以提高该菌对有机溶剂的耐受性。【方法】以P.putida JUCT1的基因组为模板扩增基因arc A,并与p ET-20b(+)连接后导入E.coli JM109(DE3)中,验证该基因提高E.coli JM109(DE3)对有机溶剂的耐受性。利用Red同源重组的方法将arc A整合到E.coli JM109(DE3)基因组中。【结果】E.coli JM109(DE3)/p ET-20b(+)-arc A在添加了2.0%(体积比)环己烷、0.1%(体积比)甲苯、4.0%(体积比)萘烷和0.1%(体积比)丁醇的培养基中培养8 h后,其OD660由初始的0.2分别上升到0.8、0.9、1.8和1.3。将arc A成功整合到E.coli JM109(DE3)基因组中,获得了具有较好遗传稳定性的溶剂耐受E.coli JM109(DE3)宿主菌株。【结论】外源基因arc A能提高大肠杆菌菌株的有机溶剂耐受性,为工业化应用中耐溶剂微生物菌株的构建提供了实验依据和理论基础。     

草菇热激蛋白60基因(Vvhsp60)生物信息学分析及低温下的表达

【背景】生物受到温度胁迫时,热激蛋白被诱导并在短时间内大量产生,可以使受损的蛋白质恢复正常构象,增强生物对逆境胁迫的耐受性。【目的】初步探究草菇热激蛋白60(Vvhsp60)与低温耐受性的关系,为深入开展草菇不耐低温特性的遗传改良奠定理论基础。【方法】对Vvhsp60进行生物信息学分析,以低温敏感型草菇菌株V23及耐低温菌株VH3为实验材料,利用实时荧光定量PCR技术分析低温胁迫及热激诱导后在低温下草菇菌丝体中Vvhsp60基因的表达水平。【结果】草菇Vvhsp60编码蛋白不存在信号肽,不属于分泌蛋白,在线粒体和细胞质内发挥生物学作用,属于双向跨膜蛋白。低温处理显著提高了V23与VH3菌丝体中Vvhsp60基因的表达量,而且VH3中的表达量显著高于V23,推测Vvhsp60基因的表达量高可能有助于增强草菇对低温胁迫的耐受性。经热激处理后两菌株Vvhsp60基因的表达量显著高于各自未热激处理的对照组,表明热激处理可诱导Vvhsp60基因的表达。【结论】Vvhsp60与草菇低温耐受性相关,并且热激可以诱导Vvhsp60基因的表达。     

基于膜压力应答途径的大肠杆菌丁醇耐受性

【目的】筛选丁醇压力下Escherichia coli中参与溶剂压力应答的细胞信号传导途径,并从应答途径出发,提高E.coli丁醇耐受性。【方法】在丁醇压力下,利用RT-PCR分析大肠杆菌内膜压力应答途径中反应调节因子(response regulator,RR)的表达水平,通过Red同源重组以及一步克隆的方法分别构建外膜脂蛋白Nlp E和分子伴侣蛋白Spy的敲除菌株E.coli JM109(Δnlp E)和E.coli JM109(Δspy)及重组菌株E.coli JM109/p QE80L-nlp E和E.coli JM109/p QE80L-spy,并测定其溶剂耐受性和细胞膜疏水性。【结果】0.8%(V/V)丁醇处理10 h后,Cpx和Bae双组分压力应答途径中的cpx R和bae R基因的表达水平分别提高了8.3和3.3倍;分别在含0.6%(V/V)四氢呋喃、0.1%(V/V)甲苯和0.6%(V/V)环己烷的培养基中培养10 h后,重组菌株E.coli JM109/p QE80L-spy和E.coli JM109/p QE80L-nlp E的OD600相比对照组(OD600增长0.02-0.04)分别增长了0.13-0.17和0.05-0.13,重组菌的溶剂耐受性得到了显著提高。【结论】Cpx和Bae系统参与大肠杆菌丁醇压力应答,分子伴侣蛋白Spy的过表达能够有效提高大肠杆菌对有机溶剂的耐受性,本研究为阐明微生物有机溶剂耐受性机制提供了理论依据。     

来源于铜绿假单胞杆菌(Pseudomonas aeruginosa)所产蛋白酶PT121克隆表达及其在小肽合成上的应用

【目的】基于前期筛选到的P.aeruginosa PT121所产有机溶剂耐受性蛋白酶,本研究对该蛋白酶进行克隆表达,研究了重组蛋白的酶学性质及小肽合成上的应用。【方法】参考文献中报道的相似蛋白酶pseudolysin设计引物从菌株PT121基因组中克隆到耐有机溶剂蛋白酶PT121基因las B。构建诱导表达重组质粒p ET22b-las B,于大肠杆菌中进行表达。考察蛋白酶PT121酶学性质及小肽合成上的应用。【结果】序列分析表明las B基因编码信号肽、前肽及成熟肽3个部分,成熟肽部分含有301个氨基酸,分子量约33 k Da,属于金属蛋白酶M4家族。通过破碎条件优化,一步法制备得到较为纯净的重组蛋白酶PT121,其比活力达7700 U/mg,该酶呈现了较高的热稳定性,p H稳定性及溶剂耐受性,与野生菌P.aeruginosa PT121所产蛋白酶性质一致。在50%DMSO体系中高效催化合成了多种小肽,其中阿斯巴甜前体(Cbz-Asp-Phe-NH2)产率高达91%。【结论】蛋白酶PT121基因在大肠杆菌中克隆表达为进一步研究相关催化机理及分子改造奠定了基础。     

受低毒病毒调控的板栗疫病菌总蛋白质组学

【目的】为解析低毒病毒调控板栗疫病菌生理性状和致病性的机制,在总蛋白质组水平上寻找受病毒侵染调控的宿主蛋白及其编码基因。【方法】使用双向电泳方法对野生型菌株EP155和受低毒病毒感染的菌株EP713进行差异蛋白质组分析,同时,对差异表达蛋白质编码基因的mRNA水平进行定量分析。【结果】共找到71个因病毒侵染而发生差异表达的蛋白质点,分别属于58种不同蛋白质。以EP155为对照,表现为上调的19个,下调的52个,主要涉及能量代谢,蛋白质、核酸和碳水化合物代谢,信号传导以及压力应激和氧化还原反应。对10个受病毒调控的蛋白的编码基因进行了mRNA水平定量,其中7个基因受病毒感染后的mRNA水平变化与蛋白水平变化趋势一致,3个基因的mRNA水平变化与蛋白水平变化趋势不相符,表明低毒病毒对板栗疫病菌不同基因的调控可以发生在不同的调控层面上。【结论】低毒病毒的侵染弱化了宿主TCA循环的能量流动,调控了宿主体内的甲基化过程及真菌致病因子的表达。     

红色荧光蛋白基因标记产鼠李糖脂铜绿假单胞菌SG及其在油藏中的应用（英文）

【背景】微生物在油田注采系统中的迁移直接影响到油藏微生物群落组成及其在油田生产中的应用。然而,由于缺少特异性标记,很难将目标微生物同众多的土著微生物区分开。因此,需要构建携带特异性基因的微生物菌株。【目的】为了有效追踪定位微生物在油田注采系统中的迁移,本文构建一株红色荧光蛋白标记假单胞菌。【方法】运用染色体同源重组的方法,将带有组成型表达启动子的红色荧光蛋白编码基因(red fluorescent protein gene,rfp)插入到一株分离自油藏环境且产鼠李糖脂的铜绿假单胞菌SG染色体上编码β-内酰胺酶基因内部,获得标记菌株SG-rfp。【结果】构建的菌株SG-rfp能够在非诱导条件下表达红色荧光蛋白,而且对氨苄青霉素、卡那霉素、链霉素和庆大霉素不具有耐受性。与野生型菌株SG相比,构建的SG-rfp菌株也能够在有氧和缺氧条件下产生鼠李糖脂,在岩芯驱油实验中能够较好地提高原油采收率。此外,应用菌株SG-rfp,本文研究并证实了微生物在含油多孔介质中的迁移扩散及所受限制。【结论】本文所构建的菌株SG-rfp为深入研究微生物在油田注采系统中的迁移及微生物在油田生产中的应用提供了有力工具...     

红色荧光蛋白基因标记产鼠李糖脂铜绿假单胞菌SG及其在油藏中的应用

【背景】微生物在油田注采系统中的迁移直接影响到油藏微生物群落组成及其在油田生产中的应用。然而,由于缺少特异性标记,很难将目标微生物同众多的土著微生物区分开。因此,需要构建携带特异性基因的微生物菌株。【目的】为了有效追踪定位微生物在油田注采系统中的迁移,本文构建一株红色荧光蛋白标记假单胞菌。【方法】运用染色体同源重组的方法,将带有组成型表达启动子的红色荧光蛋白编码基因(red fluorescent protein gene,rfp)插入到一株分离自油藏环境且产鼠李糖脂的铜绿假单胞菌SG染色体上编码β-内酰胺酶基因内部,获得标记菌株SG-rfp。【结果】构建的菌株SG-rfp能够在非诱导条件下表达红色荧光蛋白,而且对氨苄青霉素、卡那霉素、链霉素和庆大霉素不具有耐受性。与野生型菌株SG相比,构建的SG-rfp菌株也能够在有氧和缺氧条件下产生鼠李糖脂,在岩芯驱油实验中能够较好地提高原油采收率。此外,应用菌株SG-rfp,本文研究并证实了微生物在含油多孔介质中的迁移扩散及所受限制。【结论】本文所构建的菌株SG-rfp为深入研究微生物在油田注采系统中的迁移及微生物在油田生产中的应用提供了有力工具。     

利用非经典分泌蛋白质实现脂肪酶A的分泌表达

【目的】以枯草芽孢杆菌脂肪酶A(Lipase A)为报告蛋白,尝试利用4种非经典分泌蛋白质及其前50个氨基酸作为分泌信号以实现其分泌表达。【方法】我们扩增了脂肪酶A的编码基因和非经典分泌蛋白质的编码序列,构建了8种针对脂肪酶A的分泌表达载体,并转化至枯草芽孢杆菌WB800菌株,通过测定重组菌株的酶活、利用蛋白质电泳和免疫印迹等技术检测脂肪酶A的分泌情况【结果】以Pdh A的氨基酸序列和Sod A、Eno的前50氨基酸序列作为分泌信号的重组菌株较好的实现了脂肪酶A的分泌表达。【结论】部分非经分泌蛋白质的编码基因或其前50个氨基酸序列能够引导脂肪酶A分泌至细胞外。     

黄色粘球菌发育阶段外膜蛋白表达的变化

【目的】黄色粘球菌是研究原核发育的一种模式生物,对其膜蛋白的研究仍然十分缺乏。【方法】利用6种预测软件,在黄色粘球菌的基因组中筛选编码外膜蛋白(OMP)的基因。根据报告基因lacZ,检测这些基因在营养性生长和发育阶段的表达。【结果】基于生物信息学分析,筛选出11个编码外膜蛋白的基因。其中2个基因(MXAN3106和MXAN3883)在发育阶段表达量上升,它们分别编码Secretin家族和Fimbrial usher protein (FUP)家族转运蛋白。其余9个基因在发育起始阶段表达量降低或保持较低水平,它们均编码TonB依赖型受体或外排蛋白。【结论】这些数据提示,黄色粘球菌由生长到发育的转换过程,伴随着膜蛋白表达的显著变化。     

华癸根瘤菌7653R中一个RND型药物外排泵的功能表型及调控鉴定

【目的】研究华癸根瘤菌7653R中MCHK_0866和MCHK_0867编码的RND家族外排泵的功能表型。【方法】对外排泵编码基因及候选调控基因在基因组上的结构进行分析。采用测定OD_(600)观察菌株生长曲线的变化。通过测定最低抑菌浓度检测菌株的药物敏感性,RT-PCR检测目的基因经特定物质处理后表达量的变化。通过细菌单杂交系统初步检测外排泵的转录调控。【结果】MCHK_0866和MCHK_0867所编码蛋白共同组成一个RND家族射流泵。缺失该外排泵后,细菌生长曲线在稳定期OD_(600)数值降低,对萘啶酸、四环素和SDS的敏感性发生变化,萘啶酸处理细菌后2个基因的表达量增加。同时,下游属于Tet R转录因子家族的基因MCHK_0869表达产物作用于MCHK_0867的启动子区域。【结论】该外排泵与萘啶酸的运输有关,缺失后自身生长受到影响,表达受到下游转录因子的调控。     

有机溶剂胁迫处理对菌株Bacillus subtilis OST23a产胞外多糖的影响

【目的】研究有机溶剂胁迫处理对菌株分泌胞外多糖的影响并确定最佳条件。【方法】利用分泌抗氧化活性胞外多糖海洋细菌Bacillus subtilis OST23a及其突变菌株UD292为出发菌株,在考察菌株有机溶剂耐受性的基础上,测定不同浓度正己烷胁迫处理不同时间后该菌株抗氧化胞外多糖产量。【结果】结果表明最佳胁迫处理浓度和时间分别为3%和6 h,此时Bacillus subtilis OST23a和菌株UD292胞外多糖分泌量分别从9.02 mg/L和43.92 mg/L显著提高到52.97 mg/L和201.81 mg/L,且胞外多糖的抗氧化性能无显著变化。Bacillus subtilis OST23a和菌株UD292连续传代试验结果表明菌株遗传性状较稳定。【结论】有机溶剂胁迫可以提高细菌分泌胞外多糖的能力,在微生物育种方面有潜在的应用。     

Role of RNA and protein synthesis and turnover in the heat-induced increase in nuclear protein

The proteins which become associated with nuclei during hyperthermic exposure were characterized by labeled amino acid incorporation. Actinomycin-D (Act-D) or cycloheximide (CHM) pretreatment was used to determine whether concurrent RNA or protein synthesis is required for hyperthermia to induce the increase in nuclear protein content. Prior to heat exposure exponentially growing HeLa cells were (i) pulse labeled for 1 h, (ii) labeled for 36 h, or (iii) labeled for 24 h followed by 17 h chase. The nuclear specific activity (CPM/microgram protein) of [3H]lysine-labeled proteins did not change under any of the labeling conditions, whereas that of [3H]leucine-containing proteins increased significantly with (i) but not with (ii) or (iii), while that of [3H]tryptophan-labeled protein increased significantly with (i) and (ii) but not with (iii). Act-D treatment 1 h prior to and during heating did not affect nuclear protein increase, while CHM-treated cells showed generally less nuclear protein content (70% of control at 60 min) but nevertheless significant nuclear protein increase upon heating (60% increase at 60 min from 0 min). These results suggest that those proteins associated with nuclei following heat exposure are nonhistones with a high turnover rate, and the process dose not require the synthesis of RNA or proteins.     

Amino acid catabolism by perfused rat hindquarter. The metabolic fates of valine.

Hindquarters from starved rats were perfused with plasma concentrations of amino acids, but without other added substrates. Release of amino acids was similar to that previously reported, but, if total amino acid changes were recorded, alanine and glutamine were not formed in excess of their occurrence in muscle proteins. In protein balance (excess insulin) there was no net formation of either alanine or glutamine, even though the branched-chain amino acids and methionine were consumed. If [U-14C]valine was present, radiolabelled 3-hydroxyisobutyrate and, to a lesser extent, 2-oxo-3-methylbutyrate accumulated and radiolabel was incorporated into citrate-cycle intermediates and metabolites closely associated with the citrate cycle (glutamine and glutamate, and, to a smaller extent, lactate and alanine). If a 2-chloro-4-methylvalerate was present to stimulate the branched-chain oxo acid dehydrogenase, flux through this step was accelerated, resulting in increased accumulation of 3-hydroxyisobutyrate, decreased accumulation of 2-oxo-3-methylbutyrate, and markedly increased incorporation of radiolabel (specific and total) into all measured metabolites formed after 3-hydroxyisobutyrate. It is concluded that: amino acid catabolism by skeletal muscle is confined to degradation of the branched-chain amino acids, methionine and those that are interconvertible with the citrate cycle; amino acid catabolism is relatively minor in supplying carbon for net synthesis of alanine and glutamine; and partial degradation products of the branched-chain amino acids are quantitatively significant substrates released from muscle for hepatic gluconeogenesis. For valine, 3-hydroxyisobutyrate appears to be quantitatively the most important intermediate released from muscle. A side path for inter-organ disposition of the branched-chain amino acids is proposed.     

Hepatic uptake of amino acids at mid-lactation in the rat.

The enzymic determination of D-3-hydroxybutyrate and acetoacetate normally involves the use of 3-hydroxybutyrate dehydrogenase (HBDH, EC 1.1.1.30) of bacterial origin. We show that HBDH from Rhodopseudomonas spheroides (BCL, grade II) contains a 3-hydroxyisobutyrate dehydrogenase (HIBDH) activity: activity with 3-hydroxyisobutyrate as substrate was greater than 10% of that with 3-hydroxybutyrate. However, HBDH could be prepared essentially free of HIBDH activity by incubation at 37 degrees C in the presence of 1 mM-CaCl2, to produce an enzyme preparation that may be used for the specific determination of 3-hydroxybutyrate. Use of the purified enzyme preparations indicated that a major product of valine metabolism in hemidiaphragms from 40 h-starved rats was 3-hydroxyisobutyrate rather than 3-hydroxybutyrate.     

The oligosaccharyltransferase complex from pig liver: cDNA cloning, expression and functional characterisation

Oligosaccharyltransferase (OST) is an oligomeric protein complex which catalyses the transfer en bloc of Glc₃-Man₉-GlcNAc₂ from Dol-PP to specific asparagine residues in the nascent polypeptide chain. In order to study the function of the pig enzyme subunits, we have cloned OST48, ribophorin I and ribophorin II and characterized these proteins after in vitro translation as well as after expression in COS-1 cells. The individual full-length cDNAs contained open reading frames (ORFs) encoding polypeptides with calculated molecular masses of 48.9[emsp4 ]kDa (OST48), 68.7[emsp4 ]kDa (ribophorin I) and 69.3[emsp4 ]kDa (ribophorin II), respectively. A Kyte and Doolittle hydrophobicity analysis revealed that OST48, ribophorin I and ribophorin II possess a type I membrane topology with the bulk of their polypeptide chains directed towards the ER-lumen. In contrast to OST48, ribophorin I and II contain, respectively, three or two potential N-glycosylation sites of the Asn-Xaa-Thr/Ser type; only one is found to function as the acceptor site in each protein.Transfection of COS-1 cells with vector constructs encoding either OST48, ribophorin I, or a ribophorin I variant tagged with a myc-peptide sequence, resulted in the over-expression of polypeptides whose molecular masses were similar to those calculated from the respective cDNA ORFs. None of these three polypeptides, or ribophorin II, were found to display OST activity when over-expressed alone. By contrast, a modest but reproducible 25% increase of activity was observed when OST48 together with ribophorin I, or OST48 and myc-tagged ribophorin I, were co-expressed, indicating that these two subunits are probably responsible for the catalytic activity in the hetero-oligomeric OST complex. The only modest over-expression of transferase activity suggests that either the dimeric enzyme complex is catalytically unstable, or that the OST48 and ribophorin I polypeptides are unable to fold properly when other subunit components of the hetero-oligomeric OST complex are lacking. OST48 as well as ribophorin I are expressed in COS-1 cells as ER-resident proteins. Whereas OST48 carries a double-lysine motif in the –3/–5 position of its cytosolic C-terminal domain, ribophorin I does not contain recognizable ER-retention information. Replacing the lysine residue in the –3 position by leucine resulted in plasma membrane expression of the OST48-Leu polypeptide, indicating that this sequence motif may be able to influence OST48 localisation. No cell surface staining was observed when OST48-Leu was co-expressed with ribophorin I. This suggests that localisation of OST48 in the ER is mediated by interaction with ribophorin I rather than by the double-lysine motif.     

Cloning and overexpression of the 3-hydroxyisobutyrate dehydrogenase gene from pseudomonas putida E23

The structural gene for NAD+-dependent 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31) from Pseudomonas putida E23 was cloned in Escherichia coli cells to obtain a large amount of the enzyme and its nucleotides were sequenced to study its structural relationship with other proteins. The gene encoded a polypeptide containing 295 amino acid residues and was in a cluster with the gene for methylmalonate semialdehyde dehydrogenase. Transformed E. coli cells overproduced 3-hydroxyisobutyrate dehydrogenase, and the recombinant enzyme was purified to homogeneity with a high yield. Lysine and asparagine residues, which are important in catalysis of the 3-hydroxyacid dehydrogenase family, are conserved in this enzyme.     

Cloning and sequence analysis of a cDNA for 3-hydroxyisobutyrate dehydrogenase. Evidence for its evolutionary relationship to other pyridine nucleotide-dependent dehydrogenases

A 1.7-kilobase pair cDNA clone encoding 3-hydroxyisobutyrate dehydrogenase has been isolated by screening a rat liver lambda gt11 library with a 17-base oligonucleotide probe which corresponds to a portion of the N-terminal amino acid sequence of rabbit liver 3-hydroxyisobutyrate dehydrogenase. The cDNA contains an open reading frame of 1038 base pairs which includes an amino acid sequence that matches the N-terminal 35 amino acid sequence of rabbit 3-hydroxyisobutyrate dehydrogenase at 33 residues. The cDNA predicts a 300-amino acid mature protein with an amino acid composition and molecular weight very similar to that of rabbit liver 3-hydroxyisobutyrate dehydrogenase. Northern blot analysis of total RNA from several rat tissues shows an mRNA of approximately 2.0 kilobase pairs in each tissue. Relative mRNA levels were: kidney greater than liver = heart greater than muscle. The amino acid sequence of 3-hydroxyisobutyrate dehydrogenase shows similarity to several other pyridine nucleotide-dependent dehydrogenases. The resemblance to malate and lactate dehydrogenases suggests that the nucleotide-binding domain is located in the N-terminal region of the protein.     

Identification of mammalian mitochondrial proteins that interact with IAPs via N-terminal IAP binding motifs

Direct IAP binding protein with low pI/second mitochondrial activator of caspases, HtrA2/Omi and GstPT/eRF3 are mammalian proteins that bind via N-terminal inhibitor of apoptosis protein (IAP) binding motifs (IBMs) to the baculoviral IAP repeat (BIR) domains of IAPs. These interactions can prevent IAPs from inhibiting caspases, or displace active caspases, thereby promoting cell death. We have identified several additional potential IAP antagonists, including glutamate dehydrogenase (GdH), Nipsnap 3 and 4, CLPX, leucine-rich pentatricopeptide repeat motif-containing protein and 3-hydroxyisobutyrate dehydrogenase. All are mitochondrial proteins from which N-terminal import sequences are removed generating N-terminal IBMs. Whereas most of these proteins have alanine at the N-terminal position, as observed for previously described antagonists, GdH has an N-terminal serine residue that is essential for X-linked IAP (XIAP) interaction. These newly described IAP binding proteins interact with XIAP mainly via BIR2, with binding eliminated or significantly reduced by a single point mutation (D214S) within this domain. Through this interaction, many are able to antagonise XIAP inhibition of caspase 3 in vitro.     

Ethanol and oleate inhibition of alpha-ketoisovalerate and 3-hydroxyisobutyrate metabolism by isolated hepatocytes.

Ethanol inhibited glucose synthesis from alpha-ketoisovalerate by isolated rat hepatocytes without significant inhibition of flux through the branched-chain alpha-ketoacid dehydrogenase complex. Accumulation of 3-hydroxyisobutyrate, an intermediate in the catabolism of alpha-ketoisovalerate, was increased by ethanol, indicating inhibition of flux at the level of 3-hydroxyisobutyrate dehydrogenase. 3-Hydroxybutyrate caused the same effects as ethanol, suggesting inhibition was a consequence of an increase in the mitochondrial NADH/NAD+ ratio. Flux through the 3-hydroxyisobutyrate dehydrogenase was more sensitive to regulation by the mitochondrial NADH/NAD+ ratio than flux through the branched-chain alpha-ketoacid dehydrogenase. Oleate also inhibited glucose synthesis from alpha-ketoisovalerate, but marked inhibition of flux through the branched-chain alpha-ketoacid dehydrogenase complex was caused by this substrate.