高活性反硝化颗粒污泥的研究

以上流式厌氧污泥床（ＵＡＳＢ）反应器中的反硝化颗粒污泥为样品,研究了颗粒污泥的基本特性。测定出颗粒污泥中的优势无机元素为Ｃａ、Ｐ．颗粒表面以球菌和短杆菌为主。反硝化菌是颗拉中的优势菌群,数量可达６．５ｘ１０^８－１．５ｘ１０^１０个／ｍｌ颗粒污泥。初步鉴定了两株脱氮菌Ｍｉｃｒｏｃｏｃｃｕｓｓｐ．ｓｔｒａｉｎＮ_１和ＰｓｅｕｄｏｍｏｎａｓａｅｒｕｇｉｎｏｓａｓｔｒａｉｎＮ_２．     

乙肝病毒核衣壳启动子内三链DNA的形成

用电泳迁移分析方法研究了２１ｎｔ脱氧寡核苷酸Ｇ３ＴＧ２ＴＧＴ２Ｇ５ＴＧ２ＴＧＴ（ＣＰ１）与１２９ｂｐ的乙肝病毒（ＨＢＶ）核衣壳启动子（Ｃｐ）片段内一位点结合形成的三链ＤＮＡ的特异性及稳定性．在克隆有ＨＢＶ基因组的质粒ｐＣＰ１０的酶切产物中,ＣＰ１仅与含Ｃｐ的１２９ｂｐ片段结合．在２０ｍｍｏｌ／ＬＭｇ２＋溶液中其解离常数（Ｋｄ）为１．４×１０－７ｍｏｌ／Ｌ．不同离子稳定三链ＤＮＡ的效果依次为ｓｐ４＋（精胺）＞Ｍｇ２＋＞Ｚｎ２＋＞Ｎａ＋＞Ｋ＋,离子之间存在相互竞争作用．比ＣＰ１多一误配碱基的脱氧寡核苷酸Ｇ２ＴＧ２ＴＧＴＧ３ＴＧ２ＴＧ２ＴＧ２Ｔ（ＣＰ２）在２０ｍｍｏｌ／ＬＭｇ２＋溶液中与Ｃｐ结合的Ｋｄ值约为ＣＰ１的１／７,而在６０ｍｍｏｌ／ＬＫ＋或５ｍｍｏｌ／ＬＺｎ２＋溶液中检测不到它与Ｃｐ的结合,这进一步显示了三链ＤＮＡ形成的特异性．细胞的生理离子浓度被认为是：Ｓｐ４＋１ｍｍｏｌ／Ｌ,Ｍｇ２＋１０ｍｍｏｌ／Ｌ,Ｋ＋１４０ｍｍｏｌ／Ｌ,因此,ＣＰ１在细胞内将能特异地与Ｃｐ结合并具有较好的稳定性．     

紫色非硫细菌Rhodocista属一新分离株的鉴定及其系统学研究

从污水处理厂分离到一株紫色非硫细菌３ｐ ,该菌株具避光性,能形成孢囊,具片层状光合内膜,在波长７９８ ｎｍ 处的吸收峰很低,需硫胺素和维生素Ｂ１２ 作生长因子。该菌株可以琥珀酸为碳源,最佳生长温度为３４ ℃～４１ ℃,体内未发现Ｒ 体结构,醌类的主要成分是Ｑ９ 。对菌株３ｐ 的１６ＳｒＲＮＡ 基因的分子系统学分析结果表明,菌株３ｐ 与世纪红篓菌（ Ｒｈｏｄｏｃｉｓｔａ ｃｅｎｔｅｎａｒｉａ） 关系最近,相似性为９５ ％ ,二者可归为同一属;与Ｒｃ．Ｃｅｎｔｅｎａｒｉａ 的杂交结果表明,二者的ＤＮＡ 相关性为５６ ％ ,故可把３ｐ 定为一个新种：北京红篓菌（ Ｒｈｏｄｏｃｉｓｔａｐｅｋｉｎｅｎｓｅ ｓｐ ．Ｎｏｖ） ．     

高产稳产聚羟基烷酸的重组大肠杆菌的构建

重组大肠杆菌Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉＨＭＳ１７４（ｐＴＺ１８ＵＰＨＢ） 含有携带聚羟基烷酸（ＰＨＡ） 合成基因（ ｐｈａＣＡＢ）＊＊ 的质粒ｐＴＺ１８ＵＰＨＢ,是很有潜力的ＰＨＡ 生产菌,但存在着质粒不稳定和不能合成３羟基丁酸（３ＨＢ） 与３羟基戊酸（３ＨＶ） 共聚物［Ｐ（３ＨＢｃｏ３ＨＶ）］ 的缺陷。将ＲＫ２ 质粒上的ｐａｒ ＤＥ 基因引入ｐＴＺ１８ＵＰＨＢ 构成质粒ｐＪＭＣ２ ,该质粒可以在宿主Ｅ．ＣｏｌｉＨＭＳ１７４ 中稳定遗传。将培养基中的磷酸盐浓度降至１８ ｍ ｍｏｌ／Ｌ,发现Ｅ．Ｃｏｌｉ ＨＭＳ１７４（ｐＪＭＣ２） 能够以丙酸为前体合成Ｐ（３ＨＢｃｏ３ＨＶ） ,其中３ＨＶ 在共聚物中的含量为５ ％ ～８ ％ 。在５Ｌ自动发酵罐中分批补料培养Ｅ．Ｃｏｌｉ ＨＭＳ１７４（ｐＪＭＣ２） ,培养基初始磷酸盐浓度为１５ ｍ ｍｏｌ／Ｌ,３０ ｈ 后每升培养液中干菌体可达４２－５ ｇ,Ｐ（３ＨＢｃｏ３ＨＶ） 占干重的７０ ％ ,其中３ＨＶ 在共聚物中的含量为４－９ ％ 。     

产甘油假丝酵母胞浆3-磷酸甘油脱氢酶编码基因的克隆

当酵母细胞处于高渗压环境时,甘油被诱导合成以提高其胞内渗透压,这一过程受ＨＯＧ途径的调控。ＧＰＤ１基因为ＨＯＧ途径的重要靶基因,高效表达使胞内３磷酸甘油脱氢酶酶活水平提高可极大地提高甘油的产量。本研究将产甘油假丝酵母（Ｃａｎｄｉｄａｇｌｙｃｅｒｏｌｏｇｅｎｅｓｉｓ）染色体ＤＮＡ经Ｓａｕ３ＡＩ部分酶解后的５～１０ｋｂＤＮＡ片段与经ＢａｍＨＩ线性化及ＣＩＰ处理过的酵母大肠杆菌穿梭质粒ＹＥｐ５１连接,以大肠杆菌ＤＨ５α为受体,构建产甘油假丝酵母的染色体基因文库。通过遗传互补法,在含５０ｇ／Ｌ氯化钠的培养基上筛选出１５个转化子,对转化子０６０１进行了进一步鉴定,转化子０６０１所含质粒ＹＥｐ０６０１带有ＹＥｐ５１的标记并可以消除Ｓａｃｃｂａｒｏｍｙｃｅｓｃｅｒｅｖｉｓｉａｅ６４２菌株由于其ＧＰＤ１,ＧＰＤ２两基因的缺失突变而表现出的渗透压敏感性,表明已克隆到产甘油假丝酵母的编码胞浆３磷酸甘油脱氢酶的基因     

不产生胞外多糖的假单胞菌突变菌株E16

在适宜培养条件下,Ｐｓｅｕｄｏｍｏｎａｓｓｐ３１２６０能将木糖转化为酸性胞外多糖（ＥＰＳ）,用甲基磺酸乙醋（ＥＭＳ）诱变处理　Ｐｓｅｕｄｏｍｏｎａｓｓｐ３１２６０得到一株完全不产生胞外多糖的突变菌株Ｅ_１６。     

枯草芽孢杆菌B034拮抗蛋白的分离纯化及特性分析

枯草芽孢杆菌（Ｂａｃｉｌｕｓｓｕｂｔｉｌｉｓ）Ｂ０３４分离自水稻叶面,对水稻白叶枯病菌具有较强的拮抗能力。除去菌体培养液以７０％饱和度硫酸铵沉淀所得的拮抗物粗提液对热稳定,对胰蛋白酶不敏感,对蛋白酶Ｋ、链霉蛋白酶Ｅ部分敏感,对氯仿部分敏感,其作用的活性ｐＨ范围低至４,高至１２以上,比较耐碱性。粗提液经ＰｈｅｎｙｌＳｅｐｈａｒｏｓｅＣＬ４Ｂ柱层析、ＤＥＡＥＳｅｐｈａｃｅｌ柱层析和ＨＰＬＣ的Ｓｕｐｅｒｄｅｘ７５ＨＲ１０／３０柱层析,得到二个拮抗活性峰：Ｐ１和Ｐ２。Ｐ２经ＳＤＳＰＡＧＥ和ＰＡＧＥＩＥＦ电泳显示为单一蛋白带,分子量５０３ｋＤ,等电点６２５。自动Ｅｄｍａｎ降解法从Ｐ２的Ｎ端测出残基序列为ＩｌｅＳｅｒＡｓｎＰｒｏＸＩｌｅＡｓｐＶａｌ     

p53基因对人胃癌细胞系恶性生长的影响

以ｐ５３ｃＤＮＡ为探针,用Ｓｏｕｔｈｅｒｎ印迹法对人胃癌细胞系ＢＧＣ８２３进行了检测,发现该细胞中ｐ５３基因存在异常,将可在真核细胞表达的重组野生型Ｐ５３质粒ｐＣ５３－ＳＮ３和突变型ｐ５３质粒ｐＣ５３－ＳＣＸ３,用指质体介导法,分别导入ＢＣＧ８２３细胞,获得了较长时间受Ｇ４１８的多个阳性克隆。Ｓｏｕｔｈｅｒｎ抑迹法证实阳性克隆细胞中有外源性ｐ５３基因存在。     

蛋白激酶C对CNE-2Z细胞p53基因表达的影响

用抗人ｐ５３蛋白单抗,进行免疫细胞化学染色,研究了蛋白激酶Ｃ（ＰＫＣ）对ＣＮＥ－２Ｚ细胞ｐ５３基因表达的影响。结果发现：对照组Ｐ５３蛋白阳性细胞百分比为６７．６９±２．９７。ＰＫＣ催化区抑制剂Ｓｔａｕｒｏｓｐｏｒｉｎｅ（ＳＴ）和调节区抑制剂Ｓｐｈｉｎｇｏｓｉｎｅ（ＳＳ）终浓度分别为２×１０－６ｍｏｌ／Ｌ和４×１０－５ｍｏｌ／Ｌ诱导细胞２４ｈ后,Ｐ５３阳性细胞百分比分别为３０．４４±４．２５和２９．１９±２．３９,较对照组均明显降低,Ｐ＜０．０１。用终浓度为２×１０－６ｍｏｌ／Ｌ的ＴＰＡ和终浓度为４ｕｇ／ｍｌ的ＯＡＧ分别作用２４ｈ后,Ｐ５３阳性细胞百分比分别为３３．７５±４．３４和６８．１８±４．４２,前者较对照组明显降低,Ｐ＜０．０１,后者变化不明显。阳性细胞中对照组和ＯＡＧ组以胞核和胞浆均着色为主,而ＳＳ、ＳＴ和ＴＰＡ组以胞核着色为主。以上结果表明：突变型ｐ５３基因在ＣＮＥ－２Ｚ细胞中有较高表达;通过抑制细胞ＰＫＣ活性和耗竭ＰＫＣ含量后,均可降低ｐ５３基因的表达;ＰＫＣ激活剂ＯＡＧ对该细胞ｐ５３基因的表达无明显影响。     

嗜热脂肪芽孢杆菌质粒DNA的高压电穿孔转化研究*

采用高压电穿孔法将穿梭质粒导入了嗜热脂肪芽胞杆菌（Ｂａｃｉｌｌｕｓ　ｓｔｅａｒｏｔｈｅｒｍｏｐｈｉｌｕｓ）Ｋ１０４１和Ｔ５２１菌株。以对数生长后期的菌体制备Ｋ１０４１转化细胞,以ＬＢ平板上于５０℃培养的过夜菌制备Ｔ５２１转化细胞,细胞密度为５～７×１０^９细胞／ｍＬ。电击条件如下：电容２５μＦ,电场强度１０．０ＫＶ／ｃｍ,脉冲控制器设定２００。 Ｋ１０４１和 Ｔ５２１最高转化效率分别达２．０１×１０⁴和１．１９ ×１０²转化子／     

A highly efficient electroporation system for transformation of Bacillus licheniformis

Summary Transformation of intact cells of Bacillus licheniformis 5A24 with plasmids pLM6 (2.8 kb), pC194 (2.9 kb) and pCP49 (7.1 kb) by electroporation resulted in 1.5 × 10⁶, 1.2 × 10⁶ , and 5.2 × 10⁴ transformants per μg DNA, respectively. Transformation was possible with plasmid DNA, which was religated after restriction endonuclease digestions. In addition, evidence is presented that indicates that B. licheniformis possesses DNA restriction and modification systems.     

Mobilization and expression of bacteriocin plasmids from Carnobacterium piscicola isolated from meat

Mobilization and expression of bacteriocin plasmids from Carnobacterium piscicola isolated from meat. The nonconjugative plasmids pCP40 and pCP49 associated with bacteriocin production in Carnobacterium piscicola LV17, a lactic acid bacterium isolated from meat, were mobilized by the wide host range conjugative plasmid pAMβ1 by two stage conjugation. At the first stage, pAMβ1 was conjugally transferred into C. piscicola LV17 containing the two plasmids associated with bacteriocin production and a cryptic plasmid. Mobilization of the two bacteriocin plasmids by pAMβ1 was done by the second stage conjugation between the pAMβ1-containing C. piscicola LV17 and chloramphenicol (Cm)-resistant Bac^- mutant of C. piscicola LV17. The transconjugants had either partial bacteriocin activity associated with acquisition of pCP40 or pCP49, or complete bacteriocin activity associated with acquisition of all three of the resident plasmids from C. piscicola LV17 or an 89 MDa cointegrated plasmid derived from pCP40 and pCP49. Further manipulation of the transconjugants and a mutant strain of C. piscicola LV17 resulted in separate strains with only pCP40 or pCP49 which produce different bacteriocins. The bacteriocin gene from pCP49 was cloned into pCaT, a chloramphenicol resistance-encoding vector, and electrotransformed into another bacteriocin-producing strain of C. piscicola , enhancing the antagonistic spectrum of the recipient strain.     

Cloning of the determinants for microcin D93 production and analysis of three different D-type microcin plasmids

Three different microcin plasmids coding for D-type microcins were analyzed. Two of the plasmids (pMccD93 and pCP101) were small, multicopy plasmids and were closely related. The third plasmid (pCP106) was a conjugative, antibiotic multiresistance plasmid. Although plasmids pCP101 and pCP106 were previously classified as A-type microcin plasmids, we have determined that they are, in fact, D type. Furthermore, the determinants for microcin D93 production were cloned from plasmid pMccD93, and a DNA probe for the region implicated in the synthesis of microcin was obtained. This probe hybridized to plasmid C from Escherichia coli strain V517, indicating that this plasmid might be involved in the synthesis of a D-type microcin. The characteristics of replication of plasmid pCP106 were analyzed and appeared to be similar to those of ColEl plasmids, although pCP106 is a conjugative single-copy plasmid.     

Homology between clindamycin resistance plasmids in Bacteroides

Two different species of clindamycin-resistant Bacteroides were isolated from the same infection. One isolate contained a single 15-kb plasmid (pCP1) which encoded transferable clindamycin resistance. pCP1 appears similar to the Bacteroides clindamycin resistance plasmid pBFTM10 isolated independently by F.P. Tally, D.R. Snydman, M.J. Shimell, and M.H. Malamy (1982, J. Bacteriol. 151, 686-691). The second strain had a 10-kb plasmid (pCP2) but did not transfer resistance. DNA hybridization studies revealed that pCP1 shares a 5-kb region of homology with the B. fragilis R plasmid pBF4 studied by R.A. Welch and F.L. Macrina (1981, J. Bacteriol. 145, 867-872). This region in both plasmids was shown to be bounded by homologous direct repeats and contains the putative clindamycin resistance determinant. pCP1 and pCP2 were found to share extensive homology but sequences homologous to the clindamycin resistance region were missing from pCP2 and found instead in the whole cell DNA of the host strain. These results identify a transposon-like structure on Bacteroides clindamycin resistance plasmids.     

Transformation of Acidiphilium by electroporation and conjugation.

Two techniques, electroporation and conjugation, have been used to introduce the RK2-based broad-host-range plasmids pRK415 and pLAFR3 into strains of the bacterial genus Acidiphilium. Using electroporation, cells were also transformed with a series of chimeric plasmids constructed by cloning cryptic Acidiphilium plasmids into the Escherichia coli vector pBR328. Various parameters affecting electroporation were investigated. Transformation efficiency varied widely with different recipient strains. Growth at an elevated temperature (37 degrees C) prior to electroporation increased transformation efficiency 10-fold compared with growth at 32 degrees C. For three strains tested, optimum transformation efficiency was obtained with field strengths of 10-15 kV/cm. Transformation efficiency increased linearly with increasing DNA concentration up to 10 micrograms/mL. Transformation efficiencies in these experiments ranged up to 10(4) transformants/micrograms DNA. Mobilization of pRK415 and pLAFR3 from E. coli strain S17.1 into several Acidiphilium strains was achieved following incubation for 3 h on nutrient agar medium (pH 7.0). Conjugation frequencies in the range of 10(-5)-10(-9) per recipient cell were obtained. Conjugation frequency was also dependent on recipient strain.     

High-efficiency loading, transfection, and fusion of cells by electroporation in two-phase polymer systems.

A method to concentrate drugs, DNA, or other materials with target cells in two-phase polymer systems for high-efficiency electroloading is described. The two-phase polymer system is utilized for cell and loading material selection, as well as for cell aggregation before electrofusion. The phase mixing of several water-soluble polymers is characterized, and the polyethylene glycol-Dextran (PEG m.w. 8,000 + Dextran m.w. 71,000) mixture is selected to illustrate the advantage of the two-phase systems. Fluorescently labeled Dextran or DNA is loaded into Chinese hamster ovary (CHO) and JTL cells, using electroporation in either the two-phase polymer system or the conventional single-phase suspension. The loading efficiency is 4 to 30 times higher for the two-phase system, with the best advantage at lower applied field range. Transfections of CHO, COS, Melan C, and JTL lymphoid cells using pSV-beta-galactosidase (for CHO and COS), pBK-RSV-tyrosinase, and pCP4-fucosidase plasmids, respectively, by electroporation in the two-phase polymer system and the conventional single-phase electroporation method, are compared. The former method is far superior to the latter in terms of efficiency. The threshold and optimal field strengths for the former are significantly lower than those for the latter method, so the former method is more favorable in terms of equipment requirement and safety. Electrofusion efficiency in the two-phase system is comparable to that in polyethylene glycol suspension alone and is a significant improvement from the conventional electrofusion method with dielectrophoresis. The two-phase polymer method is, therefore, a valuable technique for gene delivery to a limited cell source, as in ex vivo gene therapy.     

针对乙型肝炎病毒（HBV）前S2基因三靶位串联核酶的实验研究

核酶（Ｒｉｂｏｚｙｍｅ）是一类具有核酸内切酶活性的ＲＮＡ分子,可特异地切割靶ＲＮＡ序列。核酶的催化中心有相对固定的序列,而切割特异性则由催化中心两侧序列与何种靶分子序列互补而决定。根据核酶这一特性,可以人工设计针对某一病毒ＲＮＡ的核酶分子,破坏病毒转...     

Development of genetic techniques for the psychrotrophic fish pathogen Flavobacterium psychrophilum

Flavobacterium psychrophilum, a member of the Cytophaga-Flavobacterium-Bacteroides group, is an important pathogen of salmonid fish. Previous attempts to develop genetic techniques for this fastidious, psychrotrophic bacterium have met with failure. Here we describe the development of techniques for the genetic manipulation of F. psychrophilum and the identification of plasmids, selectable markers, a reporter system, and a transposon that function in several isolates of this fish pathogen. The antibiotic resistance genes ermF, cfxA, and tetQ function in F. psychrophilum. Cloning vectors based on the F. psychrophilum cryptic plasmid pCP1 which carried these selectable markers were introduced by conjugation from E. coli, resulting in antibiotic-resistant colonies of F. psychrophilum. Conjugative transfer of DNA into F. psychrophilum was strain dependent. Efficient transfer was observed for two of the seven strains tested (THC02-90 and THC04-90). E. coli lacZY functioned in F. psychrophilum when expressed from a pCP1 promoter, allowing its development as a reporter for studies of gene expression. Plasmids isolated from F. psychrophilum were efficiently introduced into F. psychrophilum by electroporation, but plasmids isolated from E. coli were not suitable for transfer by this route, suggesting the presence of a restriction barrier. DNA isolated from F. psychrophilum was resistant to digestion by Sau3AI and BamHI, indicating that a Sau3AI-like restriction modification system may constitute part of this barrier. Tn4351 was introduced into F. psychrophilum from E. coli and transposed with apparent randomness, resulting in erythromycin-resistant colonies. The techniques developed in this study allow for genetic manipulation and analysis of this important fish pathogen.     

Improved method for electroporation of Staphylococcus aureus

We have developed a significantly improved method for the electroporation of plasmid DNA into Staphylococcus aureus. The highest transformation efficiency achieved with this procedure was 4.0 x 10(8) transformants per microgram of plasmid pSK265 DNA. This represents a 530-fold improvement over the previously reported optimum efficiency of 7.5 x 10(5) transformants per microgram of plasmid DNA after electroporation of S. aureus cells [9]. Identical results were obtained when electrocompetent cells, which had been stored frozen at -80 degrees C, were used. The improved efficiency is due primarily to the use of a modified medium (designated as B2 medium) and secondarily to the use of 0.1-cm cuvettes. Several other plasmids (pI258, pMH109, and pSK270) were also electrotransformed into competent cells using our procedure, and for each plasmid, the transformation efficiency was significantly reduced compared to that observed when pSK265 DNA was used. With respect to plasmid pI258, the transformation efficiency was 3500-fold higher than that reported previously for transformation of this plasmid into S. aureus RN4220 [9]. The optimized electroporation procedure was less successful in transforming other staphylococci. Electrocompetent cells of S. aureus ATCC 29213 and S. epidermidis ATCC 12228 produced 5.5 x 10(5) and 5 x 10(3) transformants per microgram of pSK265 DNA, respectively.     

Improved method for high-efficiency electrotransformation of Escherichia coli with the large BAC plasmids

High transformation competency of Escherichia coli is one of the critical factors in the bacterial artificial chromosome (BAC)-based DNA library construction. Many electroporation protocols have been published until now, but the majority of them was optimized for transformation of small plasmids. Large plasmids with a size above 50 kbp display reduced transformation efficiency and thereby require specific conditions in the preparation and electroporation of electrocompetent cells. In the present work, we have optimized the parameters critical to the application of BAC DNA electrotransformation into E. coli. Systematic evaluation of electroporation variables has revealed several key factors like temperature of growth, media supplements, washing buffer, and cell concentration. Improvements made in the transformation protocol have led to electrocompetent cells with transformation efficiency up to 7?×?10⁸ transformants per microgram of 120 kbp BAC plasmid DNA. We have successfully used in-house prepared competent cells, the quality of which is comparable with those produced by different companies, in the construction of metagenomic libraries from the soil. Our protocol can also be beneficial for other application with limited DNA source.