The secretion of lecithinase of Pseudomonas alcaligenes S2 was via type Ⅱ secretion pathway

Strain S2 is a lecithin （or phosphatidylcholine）- solubilizing bacterium, which was isolated from the rice rhizosphere in rural areas of Beijing, China. On the basis of a polyphasic study involving phenotypic tests, physiological and biochemical tests, 16S rDNA sequence analysis, G＋C content determination and DNA-DNA hybridizations analysis, strain S2 was identified as Pseudomonas alcaligenes. R alcaligenes S2 was mutagenized with Tn5 and four mutants showing decreased or increased solubilizing ability of lecithin were isolated based on the halo size around colonies on the solid plate supplemented with egg yolk. To characterize the genes of R alcaligenes S2 involved in solubilization of lecithin, the EcoR I fragments of the chromosomes from the four mutant strains carrying a single transposon were cloned, and the DNA sequences flanking the Tn5 were determined. The Tn5 insertion sites in the mutants M808, M1329 and M1400, showing decreased solubilizing ability of lecithin, were found to be located in the xcpS, xcpX and xcpW , respectively, whose products XcpS, XcpX and XcpW were the components of type Ⅱ secretion pathway. Complementation of xcpS, xcpX and xcpW could restore the corresponding mutants M808, M1329 and M1400 to solubilize lecithin. The data suggested that mutation in one of these xcp genes would lead to the absence of mature lecithinase secretion into the extracellular medium. The data also indicated that the secretion of lecithin-hydrolyzing enzyme of R alcaligenes was via type Ⅱ secretion pathway. In the mutant M20 showing increasing lecithin-hydrolyzing activity, the interrupted gene showed 86% identity with chpA of Pseudomonas aeruginosa PAO1, whose product plays an important role in controlling twitching motility of the bacterial ceils.     

Tn5转座子插入p95基因不影响AcMNPV的复制

从Tn5转座子介导的AcMNPV随机插入突变体库中,分离到一株复制正常的突变体AcApra41.突变定位发现Tn5转座子插入了病毒p95基因中.为了排除AcApra41中还有其他突变,利用同源重组法构建了p95基因定点插入突变的重组病毒AcGFP-P95in.PCR确认p95基因中插入了Tn5转座子;Western blot也证实AcApra41和AcGFP-P95in感染的细胞中,P95蛋白的分子量都因为插入突变而变小,由野生型的95 ku变为 55 ku.病毒复制动态曲线和荧光显微镜观察证实带有该插入突变的病毒能够在Sf9细胞中正常复制,并表达极晚期基因.这一结果表明完整的P95蛋白对病毒复制是非必须的.     

趋磁螺菌AMB-l磁小体合成相关基因的克隆及功能分析

为了更清楚的了解趋磁螺菌产磁小体的合成机理和调节途径,用Tn5转座子诱变的方法筛选得到了2株磁小体合成降低的突变株,并克隆了突变株中被插入失活的基因,分别为编码ABC型Fe3+转移系统中的离子结合蛋白的amb3385基因和功能未知的amb3672基因.互补实验表明携带amb3385和amb3672基因的广宿主载体可以不同程度地恢复突变株中磁小体的合成,证明了D.Schüler关于磁小体合成假说的第一个步骤,即Fe3+从胞外向经由Fe3+转运蛋白运输至了胞内.由于amb3672基因比对时未发现特殊相似基因,其功能尚需进一步研究.     

Regulation of eight avr genes by hrpG and hrpX in Xanthomonas campestris pv. campestris and their role in pathogenicity

Eight putative avirulence genes in Xanthomonas campestris pv. campestris (Xcc) strain 8004 were characterized by Tn5gusA5 mutagenesis and gene expression analysis. The virulence test of mutants on Chinese radish showed that all mutants in individual avr genes except avrBs2 mutant were not significantly different from the wild type in virulence. The avrBs2 mutant showed reduced virulence and bacterial growth in planta. Gene expression analysis using β-glucuronidase as reporter indicated that avrBs1.1，avrBs1，avrXccB，avrXccC，avrXccE1 were regulated by hrpG, whereas avrXccA1, avrXccA2 and avrBs2 were not. RT-PCR analysis showed that all hrpG-regulated genes except avrBs1 were also regulated by hrpX. In addition, it was demonstrated that avrBs1　 was responsible for elicitation of a type III dependent hypersensitive reaction (HR) on nonhost plant pepper ECW-10R, and wild type Xcc 8004 was unable to cause HR on pepper ECW-20R.     

Isolation and characterization of a <Emphasis Type="Italic">Sinorhizobium fredii</Emphasis> mutant that cannot utilize proline as the sole carbon and nitrogen source

Sinorhizobium fredii strain HN01 can use proline as the sole carbon and nitrogen source. A mutant strain GXHN100 unable to catabolize proline was screened from 6000 Tn5gusA5 random insertional mutants of S.fredii strain HN01. Sequencing analysis showed that an open reading frame, named pmrA (proline metabolic relative), was inserted by the Tn5gusA5. A positive clone, namedp GXHN100 which containing 3.3kb foreign DNA fragment of S.fredii strain HN01, was isolated from a partial gene library of S.fredii HN01 by colony in situ hybridization. Sequence analysis showed that pGXHN100 contained the entire pmrA gene. The 3.3kb DNA fragment of pGXHN100 was cloned into a broad-host-range cosmid vector pLAFR3 to form plasmid pGXHN200 which was subsequently introduced into GXHN100 to form a complemented strain GXHN200. Plant test showed that GXHN100 was effective and no obvious changes in nitrogenase activity comparing with parental strain. But GXHN100 nodulated 2 days later on soybean and its nodulation efficiency and competitiveness were decreased.The complemented strain GXHN200 restored the nodulation efficiency and competitiveness of GXHN100 to the wild type.     

铜绿假单孢菌yfa操纵子中α2-M基因与抵抗力的关系

目的研究铜绿假单孢菌yfa-α2M基因与其抵抗力的关系。方法通过基因敲除的方法,得到铜绿假单孢菌的突变株PAO1Δyfa-α2M。在不同的底物存在的条件下,比较突变株和野生型菌株的生长曲线。结果突变株PAO1Δyfa-α2M对Polymixin的抗性明显降低。结论基因yfa-α2M可能与铜绿假单胞菌的抗性有关。     

氰戊菊酯对Tn细胞系P450及蛋白质谱表达的影响

研究氰戊菊酯对粉蚊夜蛾(Trichoplusia ni, Tn)细胞系细胞色素P450(P450s)的诱导作用及蛋白质谱表达的影响.用MTT法和台盼兰法观察不同浓度氰戊菊酯处理Tn细胞12 h后细胞的存活率;光谱测定法测定P450s含量;以12.5 μmol/L氰戊菊酯处理Tn细胞12 h,通过二维液相色谱分离技术分析处理前后Tn细胞系蛋白质谱的变化.结果显示：当氰戊菊酯处理浓度高于17.5 μmol/L时,Tn细胞形态发生明显变化,且细胞死亡率显著高于对照组(P<0.01).P450s的含量有随氰戊菊酯浓度增加而升高的趋势,并在氰戊菊酯浓度为12.5 μmol/L时达到最高(8.562 nmol/mg protein);P450s的含量亦有随处理时间延长而升高的趋势,且在12 h时达到最高(5.28±1.14 nmol/mg protein).通过二维液相色谱分离技术得到124个差异组分,其中42个组分上调,82个组分下调.结果表明：氰戊菊酯对P450s确有诱导作用,且氰戊菊酯诱导前后Tn细胞中蛋白的变化可能与P450s诱导途径中某些受体的变化有关.     

基于转座子策略提高链霉菌中landomycin E产量的研究

【目的】对影响放线菌链霉菌Streptomyces globisporus产landomycin E(laE)的代谢网络进行研究,以提高次生代谢物的产量。【方法】通过构建含强启动子和抗性标记的转座子Tn7为基础的转座子,整合至S.globisporus的染色体产生突变库,筛选高产量的突变株并对其代谢网络进行研究分析。【结果】利用构建好的Tn7-转座子连续转化链霉菌S.globisporus,经过数轮的突变和筛选,得到6株产量有较大改变的突变株,对整合位点的亚克隆和测序结果表明,该位点整合导致编码类似细菌的某些调节因子如TetR和GntR家族的蛋白的基因失活。【结论】所构建的经过修饰的微型Tn7-转座子不仅带有抗性标记且有启动子,可插入链霉菌染色体产生突变,进而提高次生代谢物laE的产量,同时也证明,转座子基载体可应用于非模式菌链霉菌。     

Type VI secretion delivers bacteriolytic effectors to target cells

Peptidoglycan is the major structural constituent of the bacterial cell wall, forming a meshwork outside the cytoplasmic membrane that maintains cell shape and prevents lysis. In Gram-negative bacteria, peptidoglycan is located in the periplasm, where it is protected from exogenous lytic enzymes by the outer membrane. Here we show that the type VI secretion system of Pseudomonas aeruginosa breaches this barrier to deliver two effector proteins, Tse1 and Tse3, to the periplasm of recipient cells. In this compartment, the effectors hydrolyse peptidoglycan, thereby providing a fitness advantage for P. aeruginosa cells in competition with other bacteria. To protect itself from lysis by Tse1 and Tse3, P. aeruginosa uses specific periplasmically localized immunity proteins. The requirement for these immunity proteins depends on intercellular self-intoxication through an active type VI secretion system, indicating a mechanism for export whereby effectors do not access donor cell periplasm in transit.     

北京棒杆菌天冬氨酸激酶M372I-T379S双突变体的构建及其酶学性质

为构建高酶活力天冬氨酸激酶(aspartokinase, AK), 并削弱或解除Lys(lysine)反馈抑制作用突变体, 通过定点突变和高通量筛选技术构建突变体M372I,T379S和M372I-T379S, 对野生型（WT）和突变体分别进行诱导表达、 纯化及酶学性质表征. 结果表明： 突变体M372I,T379S和M372I-T379S AK与WTAK相比, V_max分别提高了13.77,15.02,15.60倍, K_m和n值均降低； 最适pH值分别升高为8.0,8.5,8.5, 且半衰期分别延长了1.0,0.9,2.3 h； M372I-T379S AK最适温度为30 ℃, 比WT AK高2 ℃； 当浓度为1~10 mmol/L时, 突变体均削弱或部分解除了抑制剂Lys的反馈抑制作用.     

芳烃污染胁迫下产碱假单胞菌的蛋白质表达

微生物降解是环境中芳烃类污染物处理的重要手段与方法，研究降解菌暴露在污染物下蛋白差异表达的情况，可以揭示微生物利用有机物做为碳源与能源的途径与方法．本项目以蛋白质二维电泳研究结果为基础，对产碱假单胞菌在污染物胁迫下表达的蛋白质点进行N端测序分析，对诱导性的同工酶进行了克隆与测序分析．结果显示，产碱假单胞菌在污染物胁迫下能表达与细菌的趋化性密切相关的蛋白，在研究的菌株中存在同工不同源的酶系统．     

Signal sequence mutations disrupt feedback between secretion of an exported protein and its synthesis in E. coli

Recent studies in a eukaryotic system indicate that a block in secretion can lead to a block in the translation of secretory proteins. This feedback on protein synthesis is thought to be a result of an interaction of the signal recognition particle with the signal sequences of nascent proteins. Genetic studies in the prokaryote Escherichia coli suggest that a complex secretion machinery and a similar feedback mechanism exist. In addition, mutations affecting two genes, secA and secC, thought to encode components of the bacterial secretion machinery, selectively interfere with the synthesis of exported proteins. This selective interference with translation may be a result of recognition by the secretion machinery of signal sequences. If so, alteration of the signal sequence of a particular protein by mutation should eliminate the block in synthesis for that protein. We show here that signal sequence mutants for an exported protein, maltose binding protein, prevent the block in synthesis of this protein in a secA mutant.     

Protein P37 of mycoplasma hyorhinis induces secretion of TNF-α from human peripheral blood mononuclear cells

High mycoplasmal infection ratio in gastric cancer tissues suggests a possible association between my-coplasma infection and tumorigenesis.Because TNF-α plays an important role in carcinogenesis caused by microbes in-fection and P37 is a major immunogen of mycoplasma hy-orhinis(M.hyor.),investigating whether P37 could induce expression and secretion of TNF-α will be very significant fo elucidate the possible molecular mechanism of gastric car-cinogenesis involved with M.hyor.At the present study,we cloned full gene of p37 by PCR and mutated the 7 codes of TGA into TGG firstly,then expressed the P37 protein suc-cessfully with pGEX-4T-1 vector in E.coli,which was veri-fied with Western bolt.By RT-PCR and sensitive L929 cell toxic assay,we found that P37 protein could induce expres-sion and secretion of TNF-α from human peripheral blood mononuclear cells,and the inducing activity of P37 could be dramatically blocked by McAb PD4.These results suggest that the induction of TNF-α secretion by P37 probably plays an importan role in diseases caused by M.hyor.infection and needs to be further investigated.     

茄青枯假单胞菌T2015致病相关基因突变体的构建

分离自花生植株的茄青枯假单胞菌(Ralstonia solanacearum)T2015的Tn5-lacZ插入突变体T2135是极性突变体.采用现代分子生物学实验技术,首先构建了同一操纵子中ORF2的表达质粒pGX6191,并进行生物学验证,然后采用三亲本结合技术导入极性突变体T2135,得到茄青枯假单胞菌T2015中与致病相关基因ORF3的突变体T2135/R.     

北京棒杆菌天冬氨酸激酶M372I-T379S双突变体的构建及其酶学性质

为构建高酶活力天冬氨酸激酶(aspartokinase, AK), 并削弱或解除Lys(lysine)反馈抑制作用突变体, 通过定点突变和高通量筛选技术构建突变体M372I,T379S和M372I-T379S, 对野生型（WT）和突变体分别进行诱导表达、 纯化及酶学性质表征. 结果表明： 突变体M372I,T379S和M372I-T379S AK与WTAK相比, V_max分别提高了13.77,15.02,15.60倍, K_m和n值均降低； 最适pH值分别升高为8.0,8.5,8.5, 且半衰期分别延长了1.0,0.9,2.3 h； M372I-T379S AK最适温度为30 ℃, 比WT AK高2 ℃； 当浓度为1~10 mmol/L时, 突变体均削弱或部分解除了抑制剂Lys的反馈抑制作用.     

茄假单胞菌寄主花生特异性毒性基因的定位

通过对从茄假单胞菌中克隆的pGX1252进行亚克隆分析,发现含pGX1252右边4.5kbKpnI/EcoRI片段的亚克隆pGX1404仍象pGX1252一样能使对花生不致病菌株T2014在花生上致病.用转座子Tn5-loc对pGX1404进行了饱和插入诱变,一共分离得到6个在pGX1404上不同位置的独立插入突变,其中离pGX1404右末端约1.4kb、2.2kg的两个Tn5-loc插入使pGX1404丧失了扩大T2014寄主范围的能力,证实hsv基因位于pGX1404的右边.     

The effect of pmpR on the type III secretion system in Pseudomonas aeruginosa

The type III secretion system(T3SS) plays important roles in Pseudomonas aeruginosa pathogenicity.Previously,we reported that the uncharacterized protein PmpR could regulate pqsR,an important regulator in the quorum-sensing system,by directly binding to its promoter region.As the T3SS is controlled by the quorum-sensing system,here,we investigated the relationship between PmpR and the T3SS.Our data showed that expression of the T3SS genes exoS,exoY,exoT,and exsD was dramatically increased in a pmpR-deletion mutant compared with that in the wild-type P.aeruginosa strain PAO1.Data from DNA mobility assays indicated that PmpR affects the T3SS indirectly.It is unlikely that PmpR controls the T3SS via the Pseudomonas quinolone signal(PQS) because the PQS negatively regulates the T3SS,while pmpR negatively regulates the PQS.The effect of PmpR on the T3SS seems to be independent of the PQS;further investigation is required to uncover the underlying regulatory pathways.