达坂喜盐芽孢杆菌D-8^T在低渗冲击下的双向凝胶电泳分析

为了解革兰氏阳性中度嗜盐菌适应低渗冲击的机制,采用双向凝胶电泳技术,研究达坂喜盐芽孢杆菌(Halobacillus dabanensis)D-8T在低渗冲击下的差异蛋白表达谱。利用ImagemasterTM2D Platinum软件分析到大约650个蛋白点,大多数蛋白分子量分布在17.5~66kDa,等电点为4.0~5.9,偏酸性。在20%盐浓度中生长的D-8菌株受到0%盐浓度的低渗冲击5min及50min后34个蛋白点的表达发生改变,包括20个表达上调的点和14个点表达下调。用MALDI-TOF/MS及MASCOT软件鉴定了4个与低渗胁迫有关的蛋白,分别为热激蛋白DanK、柱状决定蛋白、青霉素结合蛋白和5-莽草酸烯醇式丙酮酸-3-磷酸合成酶。其中,热激蛋白适应低渗胁迫时表达上调为首次报道。     

达坂喜盐芽孢杆菌D-8T在低渗冲击下的双向凝胶电泳分析

为了解革兰氏阳性中度嗜盐菌适应低渗冲击的机制,采用双向凝胶电泳技术,研究达坂喜盐芽孢杆菌（Halobacillus dabanensis）D-8T在低渗冲击下的差异蛋白表达谱。利用ImagemasterTM 2D Platinum 软件分析到大约650个蛋白点,大多数蛋白分子量分布在17.5～66kDa,等电点为4.0～5.9,偏酸性。在20％盐浓度中生长的D-8菌株受到0％盐浓度的低渗冲击5min及50min后34个蛋白点的表达发生改变,包括20个表达上调的点和14个点表达下调。用MALDITOF/MS及MASCOT软件鉴定了4个与低渗胁迫有关的蛋白,分别为热激蛋白DanK、柱状决定蛋白、青霉素结合蛋白和5莽草酸烯醇式丙酮酸3磷酸合成酶。其中,热激蛋白适应低渗胁迫时表达上调为首次报道。     

信号分子AI-2合成关键基因luxS对嗜盐喜盐芽孢杆菌盐适应性的影响

【背景】群体感应信号分子AI-2是由S-核糖基高半胱氨酸裂解酶(S-ribosylhomocysteinase, LuxS)和S-腺苷高半胱氨酸核苷酶(S-adenosylhomocysteine nucleosidase, Pfs,也被称为MtnN)催化S-腺苷高半胱氨酸(S-adenosylhomocysteine, SAH)生成的4,5-二羟基-2,3-戊二酮(4,5-dihydroxy-2,3- pentanedione, DPD)自发环化形成,在细菌中广泛参与调节多种重要的生理过程,如趋化、生物发光、生物膜形成等;然而该信号分子对嗜盐喜盐芽孢杆菌盐适应性的影响尚未报道。【目的】对嗜盐喜盐芽孢杆菌(Halobacillus halophilus)群体感应信号分子AI-2进行体外合成并揭示AI-2对嗜盐喜盐芽孢杆菌盐适应性的影响。【方法】以嗜盐喜盐芽孢杆菌为研究对象,首先通过实时定量PCR分析luxS转录水平与盐浓度之间的联系;之后对LuxS、MtnN-1、MtnN-2、MtnN-3进行多序列比对分析寻找其关键氨基酸位点并通过异源表达纯化4种蛋白进行AI-2的体外合成;最后使用同源重组方法敲除luxS并通过盐胁迫、胞内相容性溶质含量检测、生物膜形成检测等试验研究luxS对嗜盐喜盐芽孢杆菌盐适应性的影响。【结果】实时定量分析表明嗜盐喜盐芽孢杆菌中luxS的转录水平随盐浓度上升而上调,而且受到Cl离子浓度的正向调控。生长曲线结合生物发光试验表明随着嗜盐喜盐芽孢杆菌的生长,培养液上清中AI-2活性在15 h达到最大,此外随着盐浓度的提高,其AI-2活性也会增强。体外酶促反应试验表明LuxS能够与MtnN-1或MtnN-2共同催化合成AI-2。利用同源重组技术成功构建了嗜盐喜盐芽孢杆菌luxS缺失突变体,在低盐条件下(0.5 mol/L NaCl)野生型、luxS缺失突变体和互补菌株的生长曲线无明显差异,而在高盐条件下(3.5 mol/L NaCl) luxS突变体的生长较为缓慢,互补菌株则能够恢复到与野生型相似的生长状况。此外,luxS的缺失会导致嗜盐喜盐芽孢杆菌在高盐条件下的盐胁迫存活率、胞内相容性溶质含量以及生物膜形成能力的下降,而互补菌株则可以恢复到接近野生型的水平。【结论】嗜盐喜盐芽孢杆菌能够合成信号分子AI-2,其luxS受到Cl离子浓度的正向调控并对其盐适应性具有重要的调控作用,为进一步研究嗜盐喜盐芽孢杆菌的盐适应性调节机制提供基础。     

转运蛋白提高凝结芽孢杆菌酸耐受性

光学纯乳酸作为可降解生物材料——聚乳酸(polylactic acid,PLA)的前体物质,正在受到广泛关注。乳酸发酵过程中酸性产物的积累会影响菌株的生长,提高菌株酸耐受性具有重要意义。本研究以乳酸生产菌株凝结芽孢杆菌(Bacillus coagulans) DSM1为出发菌株,通过对凝结芽孢杆菌DSM1及其乳酸脱氢酶双敲除菌株(DldhL1DldhL2)进行比较转录分析,筛选酸耐受相关的转运蛋白基因。对关键基因RS16330、RS06895、RS16325、RS10595、RS00500、RS07275、RS10635及RS01930进行实时定量PCR分析,发现基因RS06895、RS10595、RS00500和RS10635在发酵12 h和24 h转录水平显著增强。过表达RS10595基因的菌株,在中性(pH 6.0)条件下生长状况和发酵性能均受到抑制,但在酸性条件下(pH 4.6),其乳酸生成相比对照组显著提高。上述结果表明,RS10595基因与菌株DSM1的酸耐受性密切相关。本研究有助于进一步探究凝结芽孢杆菌酸耐受的机制,也为构建耐酸菌株提供了基础。     

地衣芽孢杆菌9945a转运蛋白YdgF1的功能鉴定

【目的】本研究对地衣芽孢杆菌(Bacillus licheniformis) 9945a菌株中ydgF1基因编码的转运蛋白进行功能鉴定。【方法】分别构建ydgF1基因过表达株9945a/pHY300-Shu-ydgF1和敲除株9945aΔydgF1,设计了以D-丙氨酸为唯一氮源的磷酸盐D-丙氨酸(phosphate D-alanine, PDA)培养基来考察菌株的生长能力,并进行细胞吸收实验。利用实时荧光定量PCR(real-time quantitative polymerase chain reaction, RT-qPCR)评价菌株9945a与9945aΔydgF1在LB培养基中不同生长时期ydgF1的相对表达量,然后对9945a与9945aΔydgF1后期的培养基平板活菌计数,计算菌落形成单位(colony-forming units, CFU)。【结果】在PDA培养基中,9945aΔydgF1的比生长速率始终低于9945a,而9945a/pHY300-Shu-ydgF1最大比生长速率为0.336h–1,是9945a/pHY300-Shu的1.98倍,并且培养15 h后9945...     

苏芸金芽孢杆菌HD-1质粒基因文库的构建

本文报告以λ系列charnn 28为载体,通过限制核酸内切酶Bam HI部分酶解获得“目的”质粒DNA片段,构建了苏芸金芽孢杆菌HD—1的质粒DNA基因文库,所得重组子值超过要求的理论值。为进一步研究苏芸金杆菌HD—1的基因结构以及基因功能奠定了基础。     

嗜盐菌中甘氨酸甜菜碱的合成途径及其生物学功能

在嗜盐菌长期的盐适应或短期的盐胁迫过程中,甘氨酸甜菜碱(又名三甲基甘氨酸,N,N,N-trimethylglycine)发挥着极为重要的作用。甘氨酸甜菜碱在嗜盐菌中的生物合成有2种途径:胆碱氧化途径和甘氨酸甲基化途径。前者以胆碱为底物,由胆碱脱氢酶(cholinedehydrogenase,BetA)和甜菜碱乙醛脱氢酶(betaine aldehyde dehydrogenase,BetB)经2次氧化生成甜菜碱;后者以甘氨酸作为底物,由甘氨酸肌醇甲基转移酶(glycine sarcosine N-methyltransferase,GSMT)和肌氨酸二甲基甘氨酸甲基转移酶(sarcosine dimethylglycine N-methyltransferase,SDMT)经3次N-甲基化生成甜菜碱。目前在JGI-IMG和EZBioCloud数据库中公布了134株嗜盐菌标准菌株的全基因组序列。其中,约56.0%的嗜盐细菌和约39.6%的嗜盐古菌拥有胆碱氧化途径所需的2个基因;约9.7%的嗜盐细菌和约0.7%的嗜盐古菌携带甲基化途径所需的2个基因。其中,8株嗜盐细菌同时拥有胆碱氧化途径和甘氨酸甲基化途径所需的全部基因。甘氨酸甜菜碱生物合成基因在典型微生物菌株或经济作物中的表达可以提高其耐盐抗逆能力,这种独特的优势已经引起科学家们强烈的兴趣,相信未来,嗜盐菌中甘氨酸甜菜碱生物合成领域内的科学理论和技术应用会有重大的突破。     

多能硫杆菌基因文库的构建以及Rubis CO基因的筛选

以pUC9质粒DNA作为载体,用PstⅠ酶切、碱性磷酸酯酶处理后,与PstⅠ部分酶切的多能硫杆菌染色体DNA3－10kbDNA片段连接,转化大肠杆菌JM83,在MacConkey培养基上筛选重组于,所得的重组子为6.5×10³,达到建库要求的理论值。进一步用光合细菌Rhodobactersphaeroides类型Ⅱ磷酸核酮糖羧化酶／加氧酶基因（rbcL－rbcS）为探针,从该库中筛选到了含有多能硫杆菌的1,5－二磷酸核酮糖羧化酶／加氧酶（RubisCO）基因片段的重组子     

盐生植物中亚滨藜甜菜碱醛脱氢酶(AcBADH)基因的表达及其启动子的分离

克隆了盐生植物中亚滨藜 (Atriplexcentralasiat icaIljin)甜菜碱醛脱氢酶 (AcBADH)cDNA ,推测的氨基酸序列与其它物种的BADH有较高的同源性。Northern杂交结果表明 ,ABA 1 0 0 μmol L和NaCl40 0mmol L处理 48h后 ,BADH的表达增加 1 0倍左右。通过筛选中亚滨藜基因组文库 ,得到了AcBADH的基因组序列。它全长 7.7kb ,包含 1 5个外显子和 1 .2kb启动子区。除了TATA box和CAAT box以外 ,在AcBADH启动子区还发现了一些与胁迫有关的其它元件 ,如GC motif、EIRE、MRE、WUN motif、ABRE和HSE。     

盐胁迫下三色苋甜菜碱及有关酶含量的变化

三色苋(Amaranthus tricolor)不同器官中的甜菜碱(GB)含量显著不同.除子叶外,根、茎和叶的GB含量和茎、叶中的胆碱单加氧酶(CMO)含量都因300 mmol/L的NaCl处理而增加.甜菜碱醛脱氢酶(BADH)的表达无论盐处理与否在所有器官中都能检测到,其含量变化不大.当种子发芽时,具备合成GB的能力,CMO含量增加;在此之前未能检测到CMO,也不能合成GB.研究结果表明三色苋响应盐胁迫而合成GB的关键酶是CMO.     

甜菜碱提高植物抗寒性的机理及其应用

甜菜碱是植物重要的渗透调节物质,在低温等逆境条件下,许多植物细胞中迅速积累甜菜碱以维持细胞的渗透平衡.对近几年来甜菜碱提高植物抗寒性的机理研究及其应用,包括甜菜碱的生物合成途径、低温胁迫下甜菜碱对植物的保护机理、甜菜碱合成酶基因的转化及外源甜菜碱在植物抗寒中的应用进行了综述.     

Synthesis of 1-octadecyl 5-betainylamino-5-deoxy-β-d-fructopyranoside hydrochloride as a new long-chain cationic sugar-based surfactant

The synthesis of a novel long-chain cationic surfactant bearing a fructopyranoside polar head functionalized at the C-5 position by a natural glycine betaine residue through an amide linkage is described.     

<Emphasis Type="Italic">Marinococcus halophilus</Emphasis> DSM 20408<Superscript>T </Superscript>encodes two transporters for compatible solutes belonging to the betaine-carnitine-choline transporter family: identification and characterization of ectoine transporter EctM and glycine betaine transporter BetM

In response to osmotic stress, the halophilic, Gram-positive bacterium Marinococcus halophilus accumulates compatible solutes either by de novo synthesis or by uptake from the medium. To characterize transport systems responsible for the uptake of compatible solutes, a plasmid-encoded gene bank of M. halophilus was transferred into the transport-deficient strain Escherichia coli MKH13, and two genes were cloned by functional complementation required for ectoine and glycine betaine transport. The ectoine transporter is encoded by an open reading frame of 1,578 bp named ectM. The gene ectM encodes a putative hydrophobic, 525-residue protein, which shares significant identity to betaine-carnetine-choline transporters (BCCTs). The transporter responsible for the uptake of glycine betaine in M. halophilus is encoded by an open reading frame of 1,482 bp called betM. The potential, hydrophobic BetM protein consists of 493 amino acid residues and belongs, like EctM, to the BCCT family. The affinity of whole cells of E. coli MKH13 for ectoine (K_s=1.6 M) and betaine (K_s=21.8 M) was determined, suggesting that EctM and BetM exhibit a high affinity for their substrates. An elevation of the salinity in the medium resulted in an increased uptake of ectoine via EctM and glycine betaine via BetM in E. coli MKH13 cells, demonstrating that both systems are osmoregulated.Communicated by W.D. Grant     

Effects of repetitive transcranial magnetic stimulation on ER stress-related genes and glutamate, γ-aminobutyric acid and glycine transporter genes in mouse brain

Repetitive transcranial magnetic stimulation (rTMS) is an emerging therapy for the treatment of psychiatric disorders. However, the mechanisms underlying the therapeutic effects of rTMS are still unclear, limiting its optimisation. Lasting effects suggest changes in disease-related genes, so we conducted gene chip and qRT-PCR analyses of genes associated with psychiatric diseases in the mouse brain at various times following 1, 20, 30 or 40 days of rTMS. Many genes were differentially expressed in the rTMS-treated mouse brain compared to sham controls, including genes encoding neurotransmitter transporters (upregulation of EAAT4, GLAST, GLT-1, GAT2, GAT4, GLYT1 and GLYT2), and endoplasmic reticulum (ER)-stress proteins (downregulation of IRE1α, IRE1β, and XBP1, upregulation of ATF6 and GRP78/Bip). Expression changes in many of these genes were also observed 10 days after the last rTMS treatment. In PC12 cells, rTMS upregulated GRP78/Bip mRNA and enhanced resistance against H₂O₂ stress. These results suggest that rTMS differentially modulates multiple genes associated with psychiatric and neurodegenerative disorders. Sustained changes in the expression of these genes may underlie the therapeutic efficacy of chronic rTMS.     

Metabolism of dimethylsulfoniopropionate and glycine betaine by a marine bacterium

Abstract The metabolism of the methylated osmolytes glycine betaine (GB) and dimethylsulfoniopropionate (DMSP) was studied in a bacterium (strain MD 14–50) isolated from a colony of the cyanobacterium Trichodesmium . MD 14–50 when grown on DMSP cleaved dimethylsulfide (DMS) from DMSP and oxidized acrylate. In contrast to DMSP, GB was metabolized by sequential N-demethylations. Low concentrations (100 μM) of DMSP or GB allowed the growth of MD 14–50 on glucose at higher salinities than in their absence. At elevated salinities, DMSP was accumulated intracellularly with less catabolism and DMS production. Thus, DMSP and GB were catabolized by different mechanisms but functioned interchangeably as osmolytes.     

Glycine betaine and polar lipid composition in halophilic archaebacteria in response to growth in different salt concentrations

Abstract Examples of halophilic archaebacteria contain low levels of between 1 and 20 mM trimethyl glycine (glycine betaine). In disrupted cell preparations, the glycine betaine is associated with the membrane fraction and is not detectable in cell supernatants. Cells of Natronococcus occultus grown in different salt concentrations show an increase in cell-associated glycine betaine along with an increase in the ratio of phosphatidyl glycerophosphate (PG) to phosphatidyl glycerol (PG) in the cell membrane.     

Influence of high salinity on biofilm formation and benzoate assimilation by <Emphasis Type=Italic>Pseudomonas aeruginosa</Emphasis>

Pseudomonas species were used in bioremediation technologies. In situ conditions, such as marine salinity, could limit the degradation of hydrocarbons and aromatic compounds by the bacteria. Biofilm ability to tolerate environmental stress could be used to increase biorestoration. In this report, we used scanning confocal laser microscopy and microtiter dish assay to analyse the impact of hyperosmotic stress on biofilm formation by Pseudomonas aeruginosa. We used benzoate as the sole carbon source and the effect of the stress on its degradation was also studied. Hyperosmotic shock inhibited the biofilm development and decreased the degradation of benzoate. The osmoprotectant glycine betaine partially restored both the biofilm formation and benzoate degradation, suggesting that it could be used as a complement in bioremediation processes.Alexis Bazire and Farès Diab contributed equally to this work.     

利用一碳化合物(C1)细菌的分离及基因组文库的构建

从污水样品中筛选到能利用甲醇的菌株B,经16SrDNA测序分析鉴定为肺炎克氏杆菌(Klebsiella pneumo-niae)。甲醛耐受能力的测试表明该菌对甲醛具有较强耐受能力,能在含有8￣15mmol/L甲醛的LB培养基上生长。Southern杂交分析说明这菌株的基因组中有甲基营养菌6-磷酸己酮糖合成酶(HPS)和6-磷酸果糖异构酶(PHI)基因的同源序列。本研究以pUC118为载体构建了基因组文库,进一步检测结果说明所构建的基因组文库质量符合要求。