利用Tn5定位诱变筛选紫云英根瘤菌Exo-变种

利用Tn5定位诱变方法,对质粒Pjb-B5进行Tn5插入诱变,得到10个Tn5在5.9kbB5外源片段上有不同插入位点的质粒TN1-1,TN1-12,TN2-2,TN2-3,TN3-1,TN4-1,TN9-|1,TN10-1,TN13-1,TN14-1。将Tn1-1等分别转移到已经含有不相容质粒Pph1ji的紫云英根瘤菌107菌株中,使之发生同源变换。通过抗性选择及表型鉴别,筛选到3株菌落表型干燥(Muc^-)的酸性胞外多糖(EPS)合成缺陷菌株(Exo^-)107(TN2-2),107(TN10-1),107(TN13-1)\.Southern杂交分析证明这3株变种的Tn5插入确实是同源交换而不是转座产生,表明经过适当改良的Tn5定位诱变法可以应用于紫云英根瘤菌Exo-变种的筛选。     

利用Tn5gusA5对大豆慢生根瘤菌lrp基因的诱变

通过对重组质粒ｐＧＸＮ３００中的 ２．３ｋｂ ＥｃｏＲＩ片段测序分析发现,其上有一完整的ｌｒｐ基因和部分 ｐｕｔＡ基因,与 Ｋｉｎｇ ＮＤ等［１］报道的 Ｂ．ｊａｐｏｎｉｃｕｍ的ｌｒｐ基因ＤＮＡ序列有 ８８％同源性。利用 Ｔｎ５ ｇｕｓＡ５定位 诱变方法,对质粒ｐＧＸＮ３００进行插入诱变,得到２．３ｋｂ　ＥｃｏＲＩ片段上有Ｔｎ５ｇｕｓＡ５插入位点的质粒ｐＧＸＮ３００－ Ｔ３８,将ｐＧＸＮ３００－Ｔ３８转移到大豆馒生根瘤苗（Ｂ．ｊａｐｏｎｉｃｕｍ）ＧＸ２０１中,得到的ＧＸ２０１转移接合子与不相容 质粒ｐＰＨ１ＪＩ发生同源双交换。通过抗性及ｇｕｓＡ活性检测,筛选到一ｌｒｐ基因突变株。Ｓｏｕｔｈｅｍ杂交分析证 明这突变株的 Ｔｎ５ ｇｕｓＡ５插入确实是同源交换而不是转座产生,表明 Ｔｎ５ ｇｕｓＡ５ 诱变可以应用于大豆慢生根 瘤菌中的突变林筛选。     

紫云英根瘤菌Exo^—变种的生理遗传及胞外多糖组分分析

从质粒ｐＪＢ１１－Ｂ６的８．５ｋｂ野生型ＤＮＡ片段中克隆到５．８ｋｂ和２．６ｋｂ的ＤＮＡ片段,其中５．８ｋｂ的片段互补紫云英根瘤菌１０７菌株的胞外多糖（ＥＰＳ）缺陷型变种ＮＡ０６和ＮＡ１２,２,２．６ｋｂ的片段只能互补变种ＮＡ１２。回接实验和电镜切片显示,这种个变种的根瘤与野生型显著不同,无固氮活性,根瘤内基本不含类菌体;它们的Ｅｘｏ＾＋回复子的根瘤与野生型根瘤相似,多数细胞胞含有类菌体,但仍无固     

Tn5—Mob转座系统对紫云英根瘤菌SR72的诱变和诱动作用

通过接合作用将携带有转座子 Tn5—Mob 的“自杀”性载体质粒 pSUP5011引入紫云英根瘤菌 SR72,得到卡那霉素抗性(Km~r)菌落的频率为6.99×10~(-6),测得受体菌的 Km~r 自发突变频率<10~(-8)。从对1071个 Km~r 突变体进行的植物砂培结瘤试验中筛选出结瘤不固氮(Nod~ ,Fix~-)突变株17个,不结瘤(Nod~-)突变株4个。另外,还从近3000个 Km~r 突变体中选出腺苷营养缺陷型突变株3个。通过 Tn5探针进行的菌落原位杂交试验证明:这21个共生固氮突变株中均含有 Tn5序列,进一步通过接合作用将协助质粒 RP4—4(Tc~r)引入 Nod~ ,Fix~ 突变株,获得了含有 Tn5—Mob 和 RP4—4的新突变株 SR72ZR(Km~r,Tc~r),但试图通过它们的协同作用将SR72中的大质粒诱动转移到根癌农杆菌 A136的试验未获成功.     

质粒exoR‘恢复紫云英根瘤菌胞外多糖缺陷型变种有效结瘤能力

以ＰＭＮ２作为载体质粒,应用体内克隆技术,从紫云英根瘤菌胞外多糖缺陷型变种ＮＡ－１１中,分离到ｘｅｏＲ＇质粒,该杂合质粒带有Ｔｎ５及其插入位点两侧的根瘤菌中,不仅可纠正紫云英根瘤菌Ｅｘｏ＾－变种的胞外多糖合成缺陷,也能恢复该变种使宿主植物根部结瘤的能力。     

维生素C生产用菌系2980产酸菌基因转移的筛选模型及转座子Tn5诱变

维生素C的生产目前国内广泛采用由产酸菌(Gluconobacter oxydans)与伴生菌(Bacillus megaterium)组成的2980菌系,在2980中G.oxydans单独生长传代困难,其生长和产酸需要B.megaterium参与。以Bacillus subtilis Ki-2-132(pUB110)作为伴生菌与原2980的G.oxydans组合,获得稳定产酸的新菌系。在此基础上建立了适合我国混菌发酵产酸菌外源基因(Kanr)转移的筛选模型。同时报道了以携带有自杀性载体P1：：Tn5的大肠杆菌E.coli W3110为供体菌对G.oxydans进行Tn5诱变的条件和结果。     

紫云英根瘤菌结瘤基因的定位研究

以根瘤菌nod ABC基因的结构同源性为依据,对紫云英根瘸菌结瘤基因(nod)进行了定位。结果表明,紫云英根瘤菌菌株7653R的nod基因同时存在于小质粒pR37653Ra和大质粒pRa7653Rb上;S52的nod基因位于小质粒pRa52a上;SR72的nod基因单拷贝位于小质粒pRa72a上,含有部分nod ABC基因的BamHI、HiadIII和Pstl酶切片段分别为22.4kb、11．9kb和2．2kb,EcoRl酶切片段为4．6kb和3．0kb;HR104的nod基因也是单拷贝,仅存在于小质粒PRal04a中,含有部分nod ABC基因的 BaraHI、EcoRL和Pstl酶切片段分别为 22．4kb、9．1kb和2．2kb。     

应用Tn5—1uxAB—sacB基因盒对根瘤菌巨型质粒进行定向标记,缺失或消除

根瘤菌是农业生产实际中应用最多的微生物肥料。随着遗传工程技术的发展和农业生产实际的需求,遗传改良根瘤菌势必要进入田间应用（l）。如何对根瘤菌在田间的应用情况如占瘤率、与土著根瘤菌的竞争生存情况等进行监测,我们采用了发光酶基因工XXAB（2）对根瘤菌实行标记。考虑到发光酶基因在根瘤菌基因组中的不同标记位置,对根瘤菌的应用情况会产生影响,特别是对标记基因的活性强弱和标记基因的稳定性造成影响,从而影响监测结果和随后的安全性评价。因此我们首先对含发光酶基因的载体进行了改造（3）,以利于对根瘤菌的染色体和巨型…     

Tn5-Mob系统诱导根瘤菌属之间耐盐和共生性状的转移

本实验通过质粒pSUP501l及其辅助质粒RP4将Tn5-Mob随机插入苜蓿根瘤菌(Rhizo-bium meliloti 042B)的基因组中,得到86株接合子SR。随机选取4株SR,通过辅助质粒R68．45的三亲本杂交,将它们的DNA片段引入慢生型大豆根瘤菌(Bradyrhizobium,japo—nicum USDA 110),获得106株接合子BSR。大部分BSR菌株获得了生长快速的特性和耐盐性,一般能耐0．3—0．5m01／L Nacl,其中有些菌株能产生黑色素。将9o株BSR回接大豆和苜蓿植株,发现47株能在大豆和苜蓿植株结瘤,但在苜蓿卜无固氮活性;26株只能在大豆植株结瘤固氮;13株只能在苜蓿植株结瘤而不固氮;4株在大豆和菖蓿植株均不结瘤。其中,获得了4株耐盐性和固氮酶活性强的接合子。     

Characterization of Tn5-induced symbiotically defective mutants of cowpea rhizobia

Symbiotically defective mutants of cowpea rhizobia strain IRC256 were isolated by random Tn5 mutagenesis and characterized. One auxotroph (MS1) requiring adenine and thiamine was a non-nodulating mutant (Nod⁻) and three prototrophic mutants were Nod⁺ Fix⁻ which formed small and ineffective nodules on cowpeas (Vigna unguiculata). Acetylene reduction activity of the Nod⁺ Fix⁻ mutants was reduced to 80–94% of that of the wild-type strain. The non-nodulating mutant (MS1) induced root-hair curling but did not show any nodule initiation or nodule development. Ultrastructural examination of nodules formed by Fix⁻ mutants showed that these contained few bacteroids, indicating either early senescence or a reduction in bacterial release into the cytoplasm of the host cell. DNA hybridization of total DNAs from a representative number of Tn5 mutants showed that each of them had one copy of the transposon Tn5 which was randomly inserted into the genome of cowpea rhizobia.     

利用Tn5-1063转座诱变法分离苜蓿中华根瘤菌042BM noeB基因的研究

采用三亲本杂交方法将带有Tn5-1063(含luxAB)的质粒pRL1063a导入苜蓿中华根瘤菌(Sinorhizobium meldoti)042BM,进行转座子插入诱变,在含有氯霉素、卡那霉素的TY平板上筛选接合子。通过结瘤试验,从1000个突变株中,筛选到3个结瘤突变株042BMR5、042BMR11和042BRM29。它们都表现出发光酶活性,表明转座子正向插入到基因组中的某个启动子下游。Southern杂交结果证实,转座子均为单一位点插入。对042BMR5突变株基因组进行反向PCR,扩增位于Tn5-1063两端的侧翼序列。测序结果表明,转座子插入到苜蓿中华根瘤菌的共生质粒pSymA noeB基因内。根据基因组中noeB上游和下游序列扩增出042BM noeB,其与苜蓿中华根瘤菌1021 noeB的同源性为98％,而与NoeB蛋白的氨基酸序列相似性为95％。疏水性分析发现,NoeB是一个跨膜蛋白,在N末端有4个跨膜区,其中包含3个初级螺旋和1个次级螺旋。     

Molecular characterization of erm(B)- and mef(E)-mediated erythromycin-resistant Streptococcus pneumoniae in China and complete DNA sequence of Tn2010

Aims: To characterize the erm(B)‐ and mef(E)‐mediated erythromycin‐resistant Streptococcus pneumoniae clinical isolates obtained from ten hospitals located different cities in China. Methods and Results: Totally 83 S. pneumoniae were collected, and eighteen representative strains of 66 strains that exhibited erythromycin resistance were used for further characterization by antibiograms, serotyping, PFGE, MLST, DNA sequencing of the macrolide‐resistance elements and mapping of the elements on the chromosome. Twelve isolates showed a high‐level resistance to erythromycin, and six other isolates showed a low‐level resistance to erythromycin. Thirteen isolates harboured a Tn2010 transposon (26·4 kbp) encoding the erm(B), tet(M) and mef(E) genes and were classified into three types by Tn2010 structures. The remaining five isolates harboured a Tn6002 transposon (20·9 kbp) encoding the erm(B) and tet(M) genes and were classified into three types by Tn6002 locations on the chromosome. Three of the Tn6002 elements were located within the Tn5252‐like element, implying that these composed a large mobile element. The MLST analyses showed that several clones had been disseminated and that the CC271 strains carrying the Tn2010 element expressing the high‐level resistance to erythromycin were predominant in China. Four new MLST strains, which were designated as ST3262, ST3263, ST3397 and ST3398 were also identified. Conclusions: The erythromycin resistance determinant of S. pneumoniae that had been isolated in China was located in Tn2010 or the Tn6002 element and several clones had been disseminated, and the CC271 strains carrying the Tn2010 element expressing the high‐level resistance to erythromycin were predominant in China. Significance and Impact of the Study: This is the first molecular analysis of erythromycin‐resistant Streptococcus pneumoniae clinical isolates in China, and the first report of the complete nucleotide sequence of Tn2010 (26 390 bp).     

一株乳酸菌胞外多糖产生的影响因素及其提取

应用苯酚一硫酸法对乳酸菌胞外多糖产生的影响因素进行了研究,表明该菌株在培养温度为30℃,培养时间为4048h,pH值降到4时,胞外多糖的产量最大。葡萄糖是乳酸菌产生多糖的良好碳源。在对乳酸菌的培养物进行离心、透析、脱蛋白、脱色,最后用乙醇沉淀,得到粗品多糖,粗品多糖至少含有两种分子量和含量相差很大的多糖。经过SephadexG-200凝胶柱得到多糖精品EPS—Ⅱ,薄层层析结果显示其为一纯化的样品。     

Regiospecific hydroxylation of 3-(methylaminomethyl) pyridine to 5-(methylaminomethyl) -2 (1H) -pyridinone byArthrobacter ureafaciens

Microbial production of a 6-hydroxy-3-pyridylmethyl compound from 3-pyridylmethyl compound was investigated. The hydroxylation of 3-(methylaminomethyl)pyridine to 5-(methylaminomethyl)-2(1H)-pyridinone, tautomer of 2-hydroxy-5(methylaminomethyl)pyridine, by resting cells ofArthrobacter ureafaciens JCM3873 was found to proceed regio- and chemo-selectively with an almost quantitative yield. The addition of molybdate ion and nicotine as an inducer to the culture medium was required for the preparation of cells containing high hydroxylation activity. The optimal temperature and pH for the hydroxylation by using resting cells were 35°C and around 7, respectively. This hydroxylation enzyme does undergo inhibition by the substrate. The inhibitory effect could be eliminated by stepwise feeding of the substrate. Under adequate conditions, 23 mg/ml of 5-(methylaminomethyl)-2(1H)-pyridinone was produced with a molar yield of nearly 100% from 3-(methylaminomethyl)pyridine.     

Isolation and identification of urinary metabolites of AF-2 (3-(5-nitro-2-furyl)-2-(2-furyl)acrylamide) in rabbits

After oral administration of AF-2 (3-(5-nitro-2-furyl)-2-(2-furyl)acrylamide) to rabbits, the two unique metabolites, M-I and M-II, were isolated from the urine. M-I, yellow needles of mp 117°, was identified as a new type metabolite of nitrofuran derivative, 2-(β-carboxypropionyl)-3-(5-methylthio-2-furyl) acrylamide by its mass, ir and nmr spectrometries. M-II, yellow solid, appears to be cis-trans isomer of M-I considering from its uv and mass spectral data, and the behavior on tlc.     

Determination of (E)-5-(2-bromovinyl)-2′-deoxyuridine in plasma and urine by capillary electrophoresis

(E)-5-(2-Bromovinyl)-2′-deoxyuridine is an antiviral drug used for treatment of infections with Herpes simplex virus type 1 as well as Varicella zoster virus. Two fast methods for the determination of the drug and its metabolite in plasma and urine by capillary electrophoresis have been developed. The plasma method can be used for measurement of total as well as unbound drug and metabolite. Plasma and urine samples are prepared for measuring by liquid/liquid extraction resulting in a limit of quantification of 40 ng/ml for total and 10 ng/ml for free BVdU in plasma and 170 ng/ml in urine. Inter- as well as intra-day precision were found to be better than 10% and both methods have been used for drug monitoring of patients.     

5-(Hydroxymethyl)-2-furfural: a selective inhibitor of DNA polymerase lambda and terminal deoxynucleotidyltransferase

5-(Hydroxymethyl)-2-furfural (HMF), a pyrolysate of carbohydrate isolated from instant coffee (Coffea arabica L.), selectively inhibits the activities of mammalian DNA polymerase lambda (pol lambda) and terminal deoxynucleotidyltransferase (TdT) which are family X pols, in vitro. The compound influenced neither the activities of replicative DNA polymerases such as alpha, delta, and epsilon, nor even the activity of pol beta which is from the same family and thought to have a very similar three-dimensional structure to the pol beta-like region of pol lambda. Since parts of HMF such as furan, furfuryl alcohol, and 2-furaldehyde did not influence the activities of any enzymes tested, the substituted form of furan with a hyroxymethyl group and a formyl group might be important for the inhibition of pol lambda and TdT. The inhibitory effect of HMF on intact pol lambda (i.e., residues 1-575), a truncated pol lambda lacking the N-terminal BRCA1 C-terminus domain (133-575, del-1 pol lambda) and another truncated pol lambda lacking the N-terminal proline-rich region (245-575, del-2 pol lambda) was dose-dependent, and 50% inhibition was observed at a concentration of 26.1, 10.3, and 4.6 microM, respectively. The IC(50) value of HMF for TdT was the same as that for del-2 pol lambda (5.5 microM). The HMF-induced inhibition of both pol lambda and TdT activities was competitive with respect to both the DNA template-primer and the dNTP substrate. On the basis of these results, HMF was suggested to bind to the pol beta-like region of pol lambda and TdT.     

Action of 5-(2-thienyl) valeric acid as a biotin antagonist

5-(2-Thienyl)valeric acid (TVA), a biotin analogue which can be easily prepared through chemical process, inhibited the growth of a biotin synthesizing Rhodotorula glutinis. The growth inhibition was reversed by the addition of biotin. Among biotin intermediates, dethiobiotin and 7,8-diaminopelargonic acid reversed the inhibition by TVA, while 7-keto-8-amino-pelargonic acid and pimelic acid did not. From these results, it was concluded that TVA is a biotin antagonist which probably acts as an inhibitor of biotin biosynthesis.     

Structural analysis of the alpha-D-glucan (EPS35-5) produced by the Lactobacillus reuteri strain 35-5 glucansucrase GTFA enzyme

The neutral exopolysaccharide EPS35-5 (reuteran) produced from sucrose by the glucansucrase GTFA enzyme from Lactobacillus reuteri 35-5 was found to be a (1-->4,1-->6)-alpha-D-glucan, with no repeating units present. Based on linkage analysis and 1D/2D 1H and 13C NMR spectroscopy of intact EPS35-5, as well as MS and NMR analysis of oligosaccharides obtained by partial acid hydrolysis and enzymatic hydrolysis, using pullulanase M1 (Klebsiella planticola), of EPS35-5, a composite model, that includes all identified structural elements, was formulated as follows: [Formula: see text].