Cloning and characterization of genes encoded in dTDP-D-mycaminose biosynthetic pathway from a midecamycin-producing strain, Streptomyces mycarofaciens

Two subclusters from Streptomyces mycarofaciens,a midecamycin producer,were clonedand partially sequenced.One region was located at the 5' end of the mid polyketide synthase(PKS)genesand contained the genes midA,midB and midC.The other region was at the 3' end of the PKS genes andcontained midK,midI and midH.Analysis of the nucleotide sequence revealed that these genes encodedTDP-glucose synthase(midA),dTDP-glucose dehydratase(midB),aminotransferase(midC),methyltransferase(midK),glycosyltransferase(midI)and an assistant gene(midH).All of these genes areinvolved in the biosynthesis of dTDP-D-mycaminose,the first deoxysugar of midecamycin,and intransferring the mycaminose to the midecamycin aglycone in S.mycarofaciens.Similar to gene pairsdes Ⅷ/desⅦ in S.venezuelae and tylMⅢ/tylMⅡ in S.fradiae,the product of midH probablyfunctions as an auxiliary protein required by the MidI protein for efficient glycosyltransfer in midecamycinbiosynthesis.     

A ketoreductase gene fromStreptomyces mycarofaciens 1748 DNA involved in biosynthesis of a spore pigment

An efficient plasmid transformation system forS. mycarofaciens 1748 has been established. In order to determine the function of MKR gene in S.mycarofaciens 1748, the gene disruption experiment was carried out. For this purpose the plasmid pKC1139 was used. A recombinant strain with white spore appeared, in contrast to the grey-colour spore of S.myarofaciens 1748. This suggested that homologous recombination between plasmidborne MKR gene sequence and the chromosome of S.mycarofaciens 1748 had occurred. A Southern hybridization experiment using a-³²P-labelled MKR gene as probe indicated that the desired integration event had occurred in the recombinant. The result of gene disruption showed that the alteration of this gene in the chromosome of S.mycarofaciens 1748 made sporulating colonies remain white instead of taking on the typical grey colour of sporulating wild type colonies, suggesting that MKR gene is involved in the biosynthesis of a spore pigment. The recombinant strain was incubated with fermentation medium optimised for midecamycin production. A TLC assay showed that the recombinant strain produced midecamycin in quantities comparable to that ofS. mycarofaciens 1748. A pCN8B12 was a clone from genomic library of midecamycin producing strain which contained a 28-kb DNA insert. The 28-kb DNA fragment contained act I-homologous and act III-homologous regions. he PKS (act I-homologous) and MKR (act III-homologous) genes that define spore pigment of midecamycin producing strain were Jocalized by restriction endonuclease digestion with pCN8B12, indicating that they are separated by about 10 kb DNA. The polyketide synthase gene cluster of simila; organization has not been reported yet.     

利用强启动功能片段提高麦迪霉素4”，-羟基丙酰化酶基因的表达

利用PCR技术将本室克隆到的强启动功能片段取代麦迪霉素丙酰化酶基因(mpt)的启动子或与mpt基因自身启动子串连,获得含mPt重组质粒pCHFPE3和pCHFPE2。用含有这两个质粒的Streptomyces lividans TK24对螺旋霉素进行微生物转化,结果表明,与含有原启动子的mpt.S.lividans TK24(p.WFPE)相比,丙酰螺旋霉素的组分比例分别提高了89.02％和58.53％。含重组质粒pCHFPE2的螺旋霉素产生菌S.spiramyceticus发酵产物中丙酰螺旋霉素的组分也有较大辐度的提高。说明利用该强启动功能片段可以提高麦迪霉素丙酰化酶基因的表达。     

麦迪霉素产生菌酮基还原酶基因的研究

将麦迪霉素产生菌基因文库中与放线紫红索酮基还原酶基因actⅢ有同源性的4·0kb DNA片段克隆到质粒载体pWHM3中,构成重组质粒pCB4。将质粒pCB4转入酮基还原酶基因缺陷菌株——加利利链霉菌ATCC3167l中,得到转化子。转化子发酵产物经TLC和HPLC分析证明是阿克拉菌酮,与加利利链霉菌原株ATCC31133的产物相同,说明麦迪霉素产生菌酮基还原酶基因互补了加利利链霉菌ATCC31671中缺陷的酮基还原酶基因,使其恢复了产生阿克拉菌酮的能力。4．Okb DNA片段插入方向相反的重组质粒pCBR4在加利利链霉菌ATCC31671中发酵产物经TLC分析证明也是阿克拉菌酮,这说明4．0kbDNA片段中麦迪霉素产生菌酮基还原酶基因具有自身的启动子。对4．0kb DNA片段进行了限制酶酶切分析,建立了其酶切图谱。以actⅢ基因为探针,经分子杂交以及亚克隆和DNA转化实验,将麦迪霉索产生菌酮基还原酶基因定位于BssH Ⅱ—BamH Ⅰ 1．3kb DNA片段上。对1．3kb DNA片段核苷酸序列分析结果表明：此1．3kbDNA片段中含有一个独立的ORF,起始密码ATG,终止密码TAG,含783bp;在起始密码上游有GGAGG5个核苷酸SD序列;此ORF编码260个氨基酸,与actⅢ基因编码的261个氨基酸相似性为77．4%,相同性为66．7％,对麦迪霉素产生苗酮基还原酶基因的可能作用进行了讨论。     

A ketoreductase gene from Streptomyces mycarofaciens 1748 DNA involved in biosynthesis of a spore pigment

An efficient plasmid transformation system forS. mycarofaciens 1748 has been established. In order to determine the function of MKR gene in S.mycarofaciens 1748, the gene disruption experiment was carried out. For this purpose the plasmid pKC1139 was used. A recombinant strain with white spore appeared, in contrast to the grey-colour spore of S.myarofaciens 1748. This suggested that homologous recombination between plasmidborne MKR gene sequence and the chromosome of S.mycarofaciens 1748 had occurred. A Southern hybridization experiment using a-³²P-labelled MKR gene as probe indicated that the desired integration event had occurred in the recombinant. The result of gene disruption showed that the alteration of this gene in the chromosome of S.mycarofaciens 1748 made sporulating colonies remain white instead of taking on the typical grey colour of sporulating wild type colonies, suggesting that MKR gene is involved in the biosynthesis of a spore pigment. The recombinant strain was incubated with fermentation medium optimised for midecamycin production. A TLC assay showed that the recombinant strain produced midecamycin in quantities comparable to that ofS. mycarofaciens 1748. A pCN8B12 was a clone from genomic library of midecamycin producing strain which contained a 28-kb DNA insert. The 28-kb DNA fragment contained act I-homologous and act III-homologous regions. he PKS (act I-homologous) and MKR (act III-homologous) genes that define spore pigment of midecamycin producing strain were Jocalized by restriction endonuclease digestion with pCN8B12, indicating that they are separated by about 10 kb DNA. The polyketide synthase gene cluster of simila; organization has not been reported yet. Project supported by the National Natural Science Foundation of China.     

麦迪霉素4"-O-丙酰基转移酶(mpt)基因结构的研究

对含有麦迪霉素4"-O-丙酰基转移酶(mpt)基因的BamHI-BamHI 8.0kb的DNA片段进行限制性酶切分析,绘制出了含有21个酶切位点的限制性酶切图谱。以含有碳霉素异戊酰基转移酶基因(CarE)的2.4kb DNA片段为探针,经Southern blot分子杂交,将mpt定位于一个EcoRI-EcoRI-Pstl 3.0kb的DNA片段上,将该片段克隆至大肠杆菌/链霉菌穿梭质粒载体pWHM3上,获得重组质粒pWFPE。含有pWFPE的螺旋霉素产生菌产二素链霉菌(S.ambofaciens)及变铅青链霉菌(J.lividans)TK24均可将内源产生的或外源加入的螺旋霉素酰化为4"-O-丙酰螺旋霉素。对EcoRI-EcoRI-PstI 3.0kbDNA片段上mpt基因进行序列分析,在该片段上有一个开放阅读框架,它以ATG为起始密码子,以TGA为终止密码子,与其序列对应的编码产物含有388个氨基酸。Mpt基因的G+C mol％为68.0,密码子第三位上G+C mol％为91.5。Mpt基因编码的氨基酸序列与CarE基因编码的氨基酸序列的相同性为67.6％,相似性为86.4％。在起始密码子上游6bp处存在…     

Determination of midecamycin by capillary zone electrophoresis with electrochemical detection

Capillary zone electrophoresis was employed for the determination of midecamycin using an end-column amperometric detection with a carbon fiber micro-disk bundle electrode at a constant potential of +1.15 V vs. saturated calomel electrode. The optimum conditions of separation and detection are 1.00x10(-3) mol l(-1) Na(2)HPO(4)-3.49x10(-4) mol l(-1) NaOH (pH 11.4) for the buffer solution, 20 kV for the separation voltage, 5 kV and 5 s for the injection voltage and the injection time, respectively. The limit of detection is 5.0x10(-7) mol l(-1) or 0.41 fmol (S/N=3). The linear range of the calibration curve is 1.00x10(-6)-1.00x10(-3) mol l(-1). The relative standard deviation is 1.4% for the migration time and 4.9% for the electrophoretic peak current. The method could be applied to the determination of midecamycin in human urine. In this case, a separation voltage of 14 kV was used.     

麦迪霉素产生菌生米卡链霉菌1748变株68的特性研究

A stable mutant No. 68 was obtained by treatment of S. mycarofaciens 1748 spores at high temperature. The electromicroscopic examination has shown that the mutant No. 68 and parent strain 1748 both have the spore chains of the spiratype. The spores of both strain are cylindrical in shape. The only difference is that the spores of the mutant No. 68 are of smooth surface, but the 1748 are of thorny. The physiological characteristics of both strains are also very similar with slight differences in utilization of few carbon sources and in cultural characters in few medium. Feeding experiment has shown that the mutant No. 68 was blocked in the formation of the macrolide lactone in the midecamycin biosynthetic pathway. This suggested that the mutant No. 68 might be a polyketide synthase genes deficient mutant. The ability of the mutant No. 68 to convert spiramycin into 4"-propionylspiramycin indicated that the mutant No. 68 contained the midecamycin 4"-propionyltransferase and could be used for microbial bioconversion of spiramycin into 4"-propionylspiramycin.     

Drug sensitivity of Ureaplasma urealyticum,persisting in patients with chronic inflammatory diseases of the urogenital tract

A total of 65 U. urealyticum cultures isolated from patients with chronic inflammatory diseases of the urogenital tract after their prolonged persistence of in the human body, were studied for sensitivity to medicinal preparations of different groups: tetracyclines (tetracycline and doxycycline), macrolids (erythomycin, clarithromycin, midecamycin, josamycin), quinolon (pefloxacin), amino glycoside (gentamicin), lincoamides (lincomycin, clindamycin). The majority of isolated U. urealyticun were highly sensitive to josamycin, clacide, doxycycline (89.2, 84.6, 76/9% respectively), and somewhat lesser number of these organisms were highly sensitive to midecamycin and pefloxacin (51.3 and 44.4% respectively). Among U. urealyticum strains circulating in the Moscow region some strains which persisted in patients with chronic inflammatory diseases of the urogenital tract for a long time were found to be resistant to erythromycin (23.1%), tetracycline (19.5%), and in very rare cases (1.6%) they were found to multiple drug resistance to all preparations under study. In view of the varying sensitivity of the clinical isolates of U. urealyticum to medicines and the presence of resistant forms in their population, the sensitivity of the isolated U. urealyticum should be determined in vitro prior to drug therapy.     

Deletion of mdmB impairs mitochondrial distribution and morphology in Aspergillus nidulans

Mitochondria form a dynamic network of interconnected tubes in the cells of Saccharomyces cerevisiae or filamentous fungi such as Aspergillus nidulans, Neurospora crassa, or Podospora anserina. The dynamics depends on the separation of mitochondrial fragments, their movement throughout the cell, and their subsequent fusion with the other parts of the organelle. Interestingly, the microtubule network is required for the distribution in N. crassa and S. pombe, while S. cerevisiae and A. nidulans appear to use the actin cytoskeleton. We studied a homologue of S. cerevisiae Mdm10 in A. nidulans, and named it MdmB. The open reading frame is disrupted by two introns, one of which is conserved in mdm10 of P. anserina. The MdmB protein consists of 428 amino acids with a predicted molecular mass of 46.5 kDa. MdmB shares 26% identical amino acids to Mdm10 from S. cerevisiae, 35% to N. crassa, and 32% to the P. anserina homologue. A MdmB-GFP fusion protein co-localized evenly distributed along mitochondria. Extraction of the protein was only possible after treatment with a non-ionic and an ionic detergent (1% Triton X-100; 0.5% SDS) suggesting that MdmB was tightly bound to the mitochondrial membrane fraction. Deletion of the gene in A. nidulans affected mitochondrial morphology and distribution at 20 degrees C but not at 37 degrees C. mdmB deletion cells contained two populations of mitochondria at lower temperature, the normal tubular network plus some giant, non-motile mitochondria.     

Engineered biosynthesis of 16-membered macrolides that require methoxymalonyl-ACP precursors in Streptomyces fradiae

Development of host microorganisms for heterologous expression of polyketide synthases (PKS) that possess the intrinsic capacity to overproduce polyketides with a broad spectrum of precursors supports the current demand for new tools to create novel chemical structures by combinatorial engineering of modular and other classes of PKS. Streptomyces fradiae is an ideal host for development of generic polyketide-overproducing strains because it contains three of the most common precursors—malonyl-CoA, methylmalonyl-CoA and ethylmalonyl-CoA—used by modular PKS, and is a host that is amenable to genetic manipulation. We have expanded the utility of an overproducing S. fradiae strain for engineered biosynthesis of polyketides by engineering a biosynthetic pathway for methoxymalonyl-ACP, a fourth precursor used by many 16-membered macrolide PKS. This was achieved by introducing a set of five genes, fkbG–K from Streptomyces hygroscopicus, putatively encoding the methoxymalonyl-ACP biosynthetic pathway, into the S. fradiae chromosome. Heterologous expression of the midecamycin PKS genes in this strain resulted in 1 g/l production of a midecamycin analog. These results confirm the ability to engineer unusual precursor pathways to support high levels of polyketide production, and validate the use of S. fradiae for overproduction of 16-membered macrolides derived from heterologous PKS that require a broad range of precursors.     

Immunochemical similarity of P-450 HFLa, a form of cytochrome P-450 in human fetal livers, to a form of rat liver cytochrome P-450 inducible by macrolide antibiotics

A protein immunochemically related to P-450 HFLa, a form of cytochrome P-450 purified from human fetal livers, was detected in rat liver microsomes. The content of the immunoreactive protein in rat liver microsomes was increased by treatments with phenobarbital, pregnenolone 16 alpha-carbonitrile (PCN), erythromycin, erythromycin estolate, and oleandomycin but not with 3-methylcholanthrene, imidazole, ethanol, isosafrole, josamycin, midecamycin, or miocamycin. The activity of erythromycin N-demethylase correlated with the content of the immunoreactive protein in rat liver microsomes (r = 0.72). In addition, anti-P-450 HFLa IgG inhibited erythromycin N-demethylase in liver microsomes from erythromycin- or oleandomycin-pretreated rats. Furthermore, the content of the immunoreactive protein highly correlated with that of P-450 PB-1, which is distinct from Waxman's terminology, and is one of the forms of PCN-inducible cytochrome P-450s (r = 0.95). From these results and the results reported so far, it seems possible that P-450 HFLa is one of the forms of cytochrome P-450 inducible by glucocorticoids.     

我国华南沿海海水养殖鱼类病原菌美人鱼发光杆菌(Photobacterium damselae)分离株的毒力基因和耐药性分析

[目的] 为探讨我国华南沿海海水养殖鱼类病原菌美人鱼发光杆菌的非典型毒力基因和耐药性的时空变化,解析影响其毒力和耐药性变化的可能环境因素,为该菌所引起的病害防控提供建议。[方法] 本研究以分离自我国广东和海南沿海患病海水鱼的35株美人鱼发光杆菌为研究对象,利用普通PCR扩增技术,分析5个非典型毒力基因在菌株中的分布情况,并采用纸片扩散法（K-B）分析菌株对15种抗生素的耐药性。[结果] 19株菌含有1–2个被检测的非典型毒力基因,尤其是hlyA和vvh的检出率均高于20%。35株菌多重耐药指数为0.00–0.67,表现出27种耐药谱,多重耐药率（菌株耐抗生素种类>3）达到60.00%,尤其对万古霉素、阿莫西林、麦迪霉素和利福平的耐药率均高于50%,但对庆大霉素、诺氟沙星、环丙沙星、氯霉素和氟苯尼考的耐药率均低于10%。非典型毒力基因含量和耐药性,呈现一定的随年份增加而增强以及海南>广东的时空差异,尤其是耐药性中的谱型丰富度、多重耐药率、某一菌株的最多耐药数量以及多重耐药指数,海南（分别是1.00,69.23%,10,0.32）均大于广东（分别是0.82,54.55%,9,0.25）。[结论] 美人鱼发光菌的非典型毒力基因可能是通过水平基因转移获得,海南与广东区域美人鱼发光菌毒力基因与耐药的差异主要受到温度和抗生素使用的影响。     

麦迪霉素产生菌具有启动功能的DNA片段的克隆和分析

利用启动子探针质粒载体pIJ486从麦迪霉素产生菌总DNA中克隆得到了一段具有启动功能的DNA片段.通过限制性酶酶切分析,测定插入DNA片段大小为2.3kb.又利用载体pIJ486和pIJ487的新霉素抗性结构基因上游有多酶切点方向相反的性质,分析了插入片段在两个不同方向上的启动能力.结果表明,在两个方向上均有启动功能,但强弱相差六倍.其中在XbaI-HindIII方向上具有较强的启动能力,在变铅青链霉菌中新霉素抗性水平可达20mg/ml以上.进一步对插入片段的三个BamHI小片段进行分析的结果表明,较强启动子区域集中在BamHI-BamHI 0.79kb DNA片段上.     

Thermosensitive omsA mutation of Escherichia coli that causes thermoregulated release of periplasmic proteins.

A mutant of Escherichia coli with a thermosensitive defect, possibly in the outer membrane (omsA mutant), was isolated from E. coli K-12 by mutagenization and selection for thermosensitivity and beta-lactam supersensitivity of growth. The mutant also showed very high sensitivity to other antibiotics, such as macarbomycin, midecamycin, rifampin, and bacitracin. The mutation was recessive to the wild type and was mapped at about 4 min on the E. coli chromosome between fhuA and metD. The mutation caused rapid release into the medium of periplasmic enzymes such as RTEM penicillinase but practically no cytoplasmic enzyme when cells grown at 30 degrees C were transferred to 37 or 42 degrees C. Electron microscopic observations showed many large double-layered vesicles attached to the surface of cells incubated at 42 degrees C. We conclude that the mutant had a mutation that caused a temperature-dependent defect in the outer membrane structure or its assembly (named an oms mutation). The omsA mutant may be useful for production of periplasmic proteins, which it releases into the culture medium on shift up of temperature.     

2010年上海市食源性沙门菌菌型分布和药敏分析以及快速检测方法的建立

目的：通过对2010年上海市市售食品中食源性沙门菌污染状况、菌型分布及药敏试验进行分析,初步确定该地区食源性沙门菌的菌型分布及耐药性,为防治因沙门菌感染引起的食源性疾病提供科学依据,并摸索建立了针对沙门菌的快速检测方法。方法：对农贸市场和超市的5类840份市售食品进行沙门菌分离鉴定及耐药性分析,并根据沙门菌的侵袭蛋白A（invA）基因序列设计保守引物,特异性快速检测沙门菌。结果：本次市售食品沙门菌阳性率检出率为4．29％,共36株沙门菌,生禽畜肉所占比例高达91％。主要血型为鼠伤寒沙门菌,德尔卑沙门菌和都柏林沙门菌。36株沙门菌药敏分析显示：所有菌株对头孢类等抗生紊敏感较高,但对氨苄西林、麦迪霉素、环丙沙辛、呋喃妥因等存在普遍较多耐药菌株,并在此基础上通过PCR法成功地特异性检测出沙门菌。结论：上海市市售食品沙门菌污染以生禽畜肉为主,对抗菌药物的耐药性较高。目前治疗市售食品中食源性沙门菌引起的食源性疾病应首选头孢内等抗生素。另外PCR快速检测方法也操作简单,特异性强,灵敏度高,对食品中沙门菌污染能起到快速检测和监控     

Quantitative determination of erythromycylamine in human plasma by liquid chromatography-mass spectrometry and its application in a bioequivalence study of dirithromycin

A sensitive, rapid liquid chromatographic-electrospray ionization mass spectrometric method for determination of erythromycylamine in human plasma was developed and validated. Erythromycylamine in plasma (0.2 mL) was extracted with ethyl acetate, the organic phase was transferred to another clear 1.5 mL Eppendorf tube and evaporated to dryness under gentle nitrogen stream at 45 degrees C, and the residue was dissolved in 100 microL of mobile phase. The samples were separated using a Thermo Hypersil HyPURITY C18 reversed-phase column (150 mm x 2.1 mm I.D., 5 microm). A mobile phase containing 10 mM of ammonium acetate (pH = 6.4)-acetonitrile-methanol (50:10:40, v/v/v) was used isocratically eluting at a flow rate of 0.2 mL/min. Erythromycylamine and its internal standard (IS), midecamycin, were measured by electrospray ion source in positive selective ion monitoring mode. The method demonstrated that good linearity ranged from 4.5 to 720 ng/mL with r = 0.9997. The limit of quantification for erythromycylamine in plasma was 4.5 ng/mL with good accuracy and precision. The mean extraction recovery of the method was higher than 75.1% and 72.7% for erythromycylamine and IS, respectively. The intra-day and inter-day precision ranged from 5.2% to 6.4% and 5.6-9.3% (relative standard deviation, RSD), respectively. The established method has been successfully applied to a bioequivalence study of two dirithromycin formulations for 18 healthy volunteers.     

毛头鬼伞真菌具有抗烟草花叶病毒活性的y3基因的克隆及对烟草的转化

【目的】蛋白质Y3具有抗烟草花叶病毒(TMV)活性并由y3基因编码。本文的目的是从真菌毛头鬼伞(Coprinus comatus)中克隆y3基因全长并在植物体中展现其对TMV的抑制活性。【方法】我们利用试剂盒5′-Full RACE Core Set(TaKaRa)扩增了y3基因cDNA5′-端未知序列,通过RT-PCR获得了全长序列,并把该全长序列与CaMV 35 S启动子和NOS终止子一起插入多克隆位点(MCS)构建了植物表达载体pCAMBIA1301-y3,用于农杆菌介导的烟草转化。【结果】y3基因全长534碱基对,包含1个开放阅读框(ORF),编码一条含130个氨基酸残基的肽链(GenBank检索号:GQ859168;EMBL:FN546262)。其cDNA序列和由它推到的氨基酸序列均与已发表的y3基因部分片段有高度相似性(94%)。Northern杂交分析证实了y3基因在转基因烟草中得到表达。接种TMV的转基因植株表现出抗TMV的活性。【结论】我们克隆了y3基因全长并得到了转基因植株。在转基因植株中,由于y3基因的表达改善了植株的抗病毒活性。y3基因的克隆和表达无疑为该基因的进一步研究奠定了基础。     

EphA3基因稳定表达细胞模型的建立与分析

目的：建立稳定表达外源EphA3基因的小鼠成纤维细胞株模型,初步探讨EphA3基因表达对肿瘤发生、发展的影响。方法：通过脂质体介导的方法,将真核表达载体pcDNA3.1（-）/myc-his-EphA3转染NIH3T3细胞,用Western印迹确定外源EphA3基因表达;通过MTT实验、软琼脂集落形成实验,观察EphA3基因表达对NIH3T3细胞生物学特性的影响。结果：建立了稳定转染EphA3基因的NIH3T3细胞株;EphA3基因表达的小鼠成纤维NIH3T3细胞生长速度没有明显变化,但在软琼脂上锚着非依赖生长的能力加强。结论：建立了稳定表达外源EphA3基因的NIH3T3细胞株,EphA3基因稳定表达具有诱导正常NIH3T3细胞发生恶性转化的重要生物功能。