链霉菌质粒pSET152电转化稀有放线菌小单孢菌的研究

利用链霉菌(Streptomyces)噬菌体ΦC31所构建的整合型载体pSET152作为供体质粒,分别以小单孢菌(Micromonospora)40027菌株的萌发孢子和新鲜菌丝体作为受体菌,在不同的电场强度下进行电转化实验,结果表明:以小单孢菌40027菌株萌发孢子为受体菌,未获得电转化子;以小单孢菌40027菌株新鲜菌丝体为受体菌,获得了电转化子。电场强度为13kV/cm时可获得最高转化效率。Southern杂交结果表明:质粒pSET152可通过菌丝体电转化法导入小单孢菌40027菌株,并整合到小单孢菌40027菌株的染色体上,暗示链霉菌噬菌体ΦC31的整合酶基因和整合位点在异源宿主小单孢菌40027菌株中仍具有相同的功能。质粒稳定性检测实验表明:质粒pSET152可稳定地存在于小单孢菌40027菌株中。     

放线菌噬菌体φHAU_3中噬菌斑形成必需功能区的克隆和定位

放线菌噬菌体φHAU3是以吸水链霉菌应城变种10-22为指示菌,筛选获得的一个广谱性噬菌体,其基因组大小约为53kb,具有粘性末端。已成功地将44.7kb的φHAU3 DNA克隆到pIJ255上,构建成噬粒(Phasmid)pIJ8300;46.8kb的φHAU3 DNA克隆到pIJ285上,构建成pIJ8301。采用部分酶解和Southern杂交的方法,制作了pIJ8300和pIJ8301被Asp718和Bgl Ⅱ酶切的限制酶图谱,又综合单,双和多酶切分析的结果,定位了单一的Pvu Ⅱ和NcoI切点。综合噬粒pIJ8300和pIJ8301的特性,已将φHAU3中噬菌体功能的必需区定位在41kb的范围内。这些结果为以φHAU3为母体发展新的放线菌噬菌体奠定了良好的基础。     

在变铅青链霉菌基因组中定域克隆外源DNA的置换性载体及通过反选择获得重组体的模式方法

ΦHAU3^R是变铅青链霉菌66中对噬菌体ΦHAU3显示抗性的基因,已从基因组中获得分离。将基因组中邻近于该基因两侧的一个3.5kb和另一个3.8kb的DNA片段分别以其在染色体上的天然取向插入到一个由pIJ101衍生的质粒pIJ653上,构建成pHZ806。然后在pHZ806上对应于pIJ101复制子的区域中插入一个spc/str抗性基因,同时在3.5kb和3.8kb片段之间插入一个潮霉素抗性基因(hyg),衍生出一个新质粒pHZ808。由于pHZ808中不具有完整的pIJ101复制功能区,所以它不能在链霉菌中复制。然而,在该质粒3.5kb和3.8kb片段之间插入的任何DNA片段,在导入到变铅青链霉菌中后都可借助于3.5kb和3.8kb两个片段与内源染色体的同源区域所发生的双交换而稳定地整入到内源染色体的特定区域(3.5kb和3.8kb片段之间),同时置换出染色体上的ΦHAU3-R基因。发生了这种基因置换的重组子菌株会对噬菌体ΦHAU3变得敏感,这种反选择方法可用来浓缩和初选携带定域插入片段的重组子。已利用潮霉素抗性基因(hyg)作为一个模式基因片段阐明了这种载体和这种在染色体上定域克隆外源基因片段的方法学和适用性。同时,用pHZ808作载体克隆另外的基因片段时还有另一个优越性：hyg可作为报告基因一同参与外源基因片段的定域整合,携带插入片段的重组子除了对噬菌体ΦHAU3显示敏感性以外,还对潮霉素显示抗性。     

一株感染铜绿假单胞菌的双链RNA噬菌体PaP6的分离和鉴定

【目的】鉴定一株新分离的铜绿假单胞菌噬菌体PaP6的生物学特性。【方法】利用铜绿假单胞菌临床分离株PA038为宿主,从西南医院污水中分离得到一株裂解性噬菌体PaP6,观察其噬斑特点;氯化铯密度梯度离心纯化噬菌体颗粒后,用透射电子显微镜观察噬菌体形态;提取PaP6基因组,通过DNA酶和RNA酶酶切,做基因组酶切图谱分析;按照感染复数(MOI)分别为10、1、0.1、0.01、0.001和0.000 1加入噬菌体和宿主菌,裂解细菌后,测定噬菌体滴度;以MOI=10的比例加入噬菌体和宿主菌,绘制一步生长曲线;用112株铜绿假单胞菌临床分离株检测PaP6宿主谱。【结果】PaP6的噬斑直径约2 mm-4 mm,圆形透明,边缘清晰;PaP6噬菌体呈多面体立体对称的头部,直径约45 nm;酶切图谱表明PaP6基因组对DNase不敏感,对RNase敏感,未酶切基因组具有3节段双链RNA(dsRNA),长度分别约为9.0、4.5、3.5 kb,共约17 kb;当MOI为0.1时PaP6感染其宿主菌产生的子代噬菌体滴度最高,达到3.4×109 PFU/m L;用一步生长曲线描绘了其生长特性;PaP6可以感染40.1%的临床分离株,是一株比较广谱的噬菌体。【结论】首次报道了一株铜绿假单胞菌的ds RNA分节段噬菌体,分类学上属于囊病毒科,该噬菌体具有较广的宿主谱,在噬菌体治疗领域具有应用前景。     

一株裂解性低温假单胞菌噬菌体的分离及特性研究

从明永冰川低温土壤中分离、纯化获得一株低温假单胞菌噬菌体PFV1,并对其生物学特性(噬菌体基因组限制性酶切片段长度多态性、衣壳蛋白组分分析及生理特征)进行了初步研究。基于16S rRNA基因测序分析结果,将宿主菌初步鉴定为荧光假单胞菌菌株。噬菌体PFV1为球形,直径约50 nm。PFV1在4℃时具有侵染活性,4~25℃范围内均可形成噬菌。对氯仿具有一定的敏感性,具有热不稳定性,基因组为双链DNA,大小约38 kb。     

8株动物源编码Stx2短尾噬菌体的生物学特性

[目的]对8株源自大肠杆菌O157编码Stx2毒素的噬菌体生物学特性进行研究.[方法]丝裂霉素C诱导8株大肠杆菌O157菌株释放噬菌体,采用PCR作初步鉴定,分离、纯化噬菌体基因组,随机引物法地高辛(DIG)标记stx2基因片段作为探针,对纯化的噬菌体采用Southernblot进行Stx2噬菌体再次鉴定,透射电子显微镜观察纯化的8株Stx2噬菌体的形态特征,通过限制性内切酶图谱分析,确定噬菌体的核酸类型和基因组大小、以及限制性内切酶酶切片段多态性,并分析噬菌体的蛋白质组成特征.[结果]Southern blot证实分离的8株噬菌体为Stx2噬菌体,电镜下观察的各株Stx2噬菌体形态一致,头部均为正六边形,尾部很短,属于短尾噬菌体科,各株噬菌体之间存在相同的蛋白结构模式,基因组为双链DNA,限制性内切酶片段长度表现出一定的多态性,噬菌体的基因组大小从48.0-65.3 kb不等.[结论]来源不同菌株的8株编码Stx2噬菌体均为短尾噬菌体,其蛋白结构模式一致,但基因组具有不同组成.     

噬菌体ΦEc-SL25对大肠埃希菌临床分离株裂解作用的研究

目的分离鉴定大肠埃希菌噬菌体并分析其裂解特性,为噬菌体疗法应用于大肠埃希菌感染提供实验依据。方法采用双层琼脂噬斑法从污水中分离噬菌体,通过透射电镜观察噬菌体的形态学特征,利用限制性酶切图谱初步分析噬菌体的基因组,测定噬菌体对宿主菌的最佳感染复数和一步生长曲线,分析噬菌体对宿主菌的裂解谱,观察噬菌体在不同的pH及温度下对宿主菌的裂解特性,SDS-PAGE分析噬菌体的主要和次要蛋白。结果通过噬斑法从污水中分离出1株能裂解大肠埃希菌的噬菌体,命名为ΦEc-SL25;电镜显示,噬菌体ΦEc-SL25的形态特征符合有尾病毒目、管尾病毒科噬菌体;ΦEc-SL25的最佳感染复数为0.01;一步生长曲线表明,噬菌体ΦEc-SL25的潜伏期为5 min,爆发期为10 min;ΦEc-SL25对26株大肠埃希菌的裂解率可达30.8%;在温度70℃20min时以及在pH 4~10的范围内,噬菌体ΦEc-SL25仍保持其裂解活性;蛋白电泳可观察到2条主要蛋白带和至少3条次要蛋白。结论噬菌体ΦEc-SL25是一种潜伏期短、裂解较性强的毒性噬菌体,可用于开发针对大肠埃希菌感染的生物制剂。     

鲍曼不动杆菌噬菌体生物学特性的研究

从污水中分离鉴定鲍曼不动杆菌噬菌体,并对其生物学特性进行分析,为开发针对细菌感染的噬茵体生物制剂提供前期工作.采用双层琼脂法分离可裂解鲍曼不动杆菌的噬菌体,通过负染法电镜观察噬茵体的大小和形态,将噬菌体和宿主菌以不同比例混合,测定噬菌体的最佳感染复数并观察一步生长曲线,提取噬菌体核酸进行酶切电泳分析,通过SDS-PAGE分析噬菌体的结构蛋白和非结构蛋白.成功分离3株可裂解鲍曼不动杆菌的噬菌体(分别命名为ΦAb-1、ΦAb-2和ΦAb-3),电镜显示噬菌体的头部呈二十面体,直径约50nm,有一短尾.噬菌体ΦAb-1的最佳感染复数为10-2,一步生长曲线表明噬菌体在裂解宿主菌时,潜伏期为20 min,爆发期为30 min,裂解量为190 PFU/cell.限制性酶切电泳显示噬菌体ΦAb-1的基因组大小约40 kb左右,为双股环状DNA.SDS-PAGE的噬菌体蛋白电泳包括7种蛋白,分子量在29 ～ 116 ku.噬菌体ΦAb-1、ΦAb-2和ΦAb-3对鲍曼不动杆菌具有很强的裂解毒性.根据其形态、结构和噬菌体分类法,鲍曼不动杆菌噬菌体属于有尾病毒目,足尾病毒科.     

金黄色葡萄球菌中PVL基因与噬菌体DNA的同源性

为了探讨金黄色葡萄球菌PVL基因与噬菌体的相关性,从4株含有PVL基因的菌株中分离出DNA,用HindⅢ或EcoRI酶切后,分别与PVL和Lukm-lukF-PV探针进行Southern印迹杂交,以及对含有PVL基因及其下游区域的片段克隆、测序和同源性分析。结果表明3株菌的PVL基因及其下游区域的序列与V8菌株噬菌体ΦPVL的PVL基因及其下游噬菌体stt site的序列一致。另一菌株的LukM-lukF-PV基因与ΦPVL,的PVL基因有78％的同源性,并且在LukM-lukF-PV基因的下游区域发现与金黄色葡萄球菌噬菌体Φ11整合位点有98％同源性的序列,根据PVL基因存在于噬菌体的基因组上,推测可通过噬菌体转导在金黄色葡萄球菌中传播。     

明永冰川融水中一株裂解性低温噬菌体的分离及特征

【目的】高山冰川是一类独特的生态系统,本研究探索从明永冰川地区分离和培养低温菌噬菌体,并对其特征进行研究。【方法】利用已分离的低温菌为宿主,采用"双层平板法"从明永冰川融水中分离纯化低温菌噬菌体;对噬菌体及其宿主进行电镜形态观察,并进行噬菌体基因组限制性酶切片段长度多态性分析、衣壳蛋白组成分析及噬菌体生理特征研究。【结果】从明永冰川融水中分离获得一株裂解性低温噬菌体,命名为MYSP03(Mingyong Flavobacterium Siphoviridae Bacteriophage),其宿主菌MYB03鉴定为Flavobacterium菌株。噬菌体MYSP03为长尾型,无囊膜,头部具典型的正多面体立体对称结构,直径约72 nm;尾管长约240 nm,直径约10 nm;4℃时具侵染活性,在4℃-20℃范围内均可产生边缘清晰、透明的噬菌斑,最适感染温度约10℃,pH耐受范围较广,最适感染pH约9.4,对氯仿不敏感,基因组为双链DNA,大小约66 kb。     

Isolation and characterization of Micromonospora phage PhiHAU8 and development into a phasmid

PhiHAU8, a temperate Micromonospora phage, which is capable of infecting Micromonospora sp. strains 40027 and A-M-01, was isolated. The PhiHAU8 virion has a polyhedral head and a flexible tail and has a small genome (ca. 42.5 kb) with double-stranded DNA and cohesive ends. PhiHAU8 was most stable at 4 degrees C in Difco nutrient broth within a pH range of 6 to 12. PhiHAU8 plaque formation on Micromonospora sp. strain 40027 was optimal with 32 mM Ca(2+) and 30 mM Mg(2+). A lysogen, LXH8, was isolated from turbid plaques, and a phasmid derivative that functions as a lambda cosmid vector in Escherichia coli and as a phage in Micromonospora sp. strain 40027 was constructed. Pulsed-field gel electrophoresis of AseI-digested total DNA showed that PhiHAU8 DNA integrates into the 500-kb AseI fragment of Micromonospora sp. strain 40027.     

Isolation and Characterization of Micromonospora Phage ΦHAU8 and Development into a Phasmid

ΦHAU8, a temperate Micromonospora phage, which is capable of infecting Micromonospora sp. strains 40027 and A-M-01, was isolated. The ΦHAU8 virion has a polyhedral head and a flexible tail and has a small genome (ca. 42.5 kb) with double-stranded DNA and cohesive ends. ΦHAU8 was most stable at 4°C in Difco nutrient broth within a pH range of 6 to 12. ΦHAU8 plaque formation on Micromonospora sp. strain 40027 was optimal with 32 mM Ca²⁺ and 30 mM Mg²⁺. A lysogen, LXH8, was isolated from turbid plaques, and a phasmid derivative that functions as a λ cosmid vector in Escherichia coli and as a phage in Micromonospora sp. strain 40027 was constructed. Pulsed-field gel electrophoresis of AseI-digested total DNA showed that ΦHAU8 DNA integrates into the 500-kb AseI fragment of Micromonospora sp. strain 40027.     

Vector systems allowing efficient autonomous or integrative gene cloning in Micromonospora sp. strain 40027

Vector systems allowing autonomous or site-specific integrative gene cloning were developed for Micromonospora sp. strain 40027, a producer of the antibiotic fortimicin A. The autonomous system depends on the discovery of a low-copy-number, self-transmissible covalently closed circular plasmid, pJTU112 (ca. 14.1 kb), which was shown to be present in the progenitor strain in both integrated and autonomous states. The copy numbers of both wild-type pJTU112 and three derivatives of it can be amplified at least sixfold by addition of streptomycin to the culture medium. The integrative system was developed by the use of a pBR322-derived Escherichia coli plasmid vector, pSET152, mediated by the attP site of the Streptomyces phage PhiC31. Both vectors can be transferred by conjugation from E. coli into Micromonospora sp. strain 40027. The heterologous cloning and expression of the dnd gene cluster originating from Streptomyces lividans 1326 into Micromonospora sp. strain 40027 demonstrated the use of the two systems.     

Characteristics of bacteriophages for Micromonospora purpurea.

Chemical and physical stabilities of bacteriophages ?UW 21 and ?UW 51 infecting Micromonospora purpurea ATCC 15835 were examined. Both phages were stable over the pH range of 5 to 8 and to heating at temperatures up to 50 degrees C and especially stable in buffer containing magnesium ion. Exposure to 1 M Ca(NO3)2 inactivated both phages, and phage ?UW 51 was also susceptible to 1 M CaCl2, 0.1 M tris(hydroxymethyl)aminomethane, and 0.3% H2O2. Phage plating efficiency was highest on the cultures at logarithmic phase and sometimes much influenced by host growth. Phage ?UW 51 has a latent period of 2 h at 34 degrees C and a burst size between 35 and 40. The latent period for phage ?UW 21 is about 12 h, and the burst size is smaller than 30.     

Extracellular proteases from eight psychrotolerant Antarctic strains

Extracellular proteases from 8 Antarctic psychrotolerant Pseudomonas sp. strains were purified and characterised. All of them are neutral metalloproteases, have an apparent molecular mass of 45kDa, optimal activity at 40 degrees C and pH 7-9, retaining significant activity at pH 5-11. With the exception of P96-18, which is less stable, all retain more than 50% activity after 3 h of incubation at pH 5-9 and show low thermal stability (their half-life times range from 20 to 60 min at 40 degrees C and less than 5 min at 50 degrees C). These proteases can be used in commercial processes carried out at neutral pH and moderate temperatures, and are of special interest for their application in mixtures of enzymes where final thermal selective inactivation is needed. Results also highlight the relevance of Antarctic biotopes for the isolation of protease-producing enzymes active at low temperatures.     

Biochemical analysis of a native and proteolytic fragment of a high-molecular-weight thermostable lipase from a mesophilic Bacillus sp.

An extracellular lipase was isolated from the cell-free broth of Bacillus sp. GK 8. The enzyme was purified to 53-fold with a specific activity of 75.7 U mg(-1) of protein and a yield of 31% activity. The apparent molecular mass of the monomeric protein was 108 kDa as estimated by molecular sieving and 112 kDa by SDS-PAGE. The proteolysis of the native molecule yields a low molecular weight component of 11.5 kDa that still retains the active site. It was stable at the pH range of 7.0-10.0 with optimum pH 8.0. The enzyme was stable at 50 degrees C for 1 h with a half life of 2 h, 40 min, and 18 min at 60, 65, and 70 degrees C, respectively. With p-nitrophenyl laurate as substrate the enzyme exhibited a K(m) and V(max) of 3.63 mM and 0.26 microM/min/ml, respectively. Activity was stimulated by Mg(2+) (10 mM), Ba(2+) (10 mM), and SDS (0.1 mM), but inhibited by EDTA (10 mM), phenylmethane sulfonyl fluoride (100 mM), diethylphenylcarbonate (10 mM), and eserine (10 mM). It hydrolyzes triolein at all positions. The fatty acid specificity of lipase is broad with little preference for C(4) and C(18:1). Thermostability of the proteolytic fragment at 60 degrees C was observed to be 37% of the native protein. The native enzyme was completely stable in ethylene glycol and glycerol (30% v/v each) for 60 min at 65 degrees C.     

Characterization of a novel fungal chitosanase Csn2 from Gongronella sp. JG

A 28kDa chitosanase designated as Csn2 was purified from the culture broth of the fungus Gongronella sp. JG through three chromatography steps: CM-Sepharose FF, Superdex 200 and SP-Sepharose FF. Its optimal reaction pH and temperature were pH 5.6 and between 55 degrees C and 60 degrees C. The half-lives of Csn2 at 50 degrees C and 55 degrees C were estimated to be 30min and 11min, respectively. The K(m) value of Csn2 in sodium acetate buffer (pH 5.6) at 55 degrees C was 8.86mg/mL. Mn(2+), Ca(2+) and Sr(2+) were activators of Csn2; ETDA was an inhibitor. Cu(2+) stimulated Csn2 at 1mM, but inhibited Csn2 activity at 10mM. Csn2 displayed strong activity on colloidal chitosan, but did not hydrolyze colloidal chitin and carboxylmethyl cellulose. Thin layer chromatography analysis showed the end products of colloidal chitosan hydrolyzed by Csn2 were chitobiose, chitotriose and chitotetraose with chitotriose as the major product. The N terminus of Csn2 was determined to be YQLPANLKKIYDSHKSGTC. Part of the genomic DNA sequence corresponding to Csn2 was cloned. Sequence alignment showed DNA sequence of Csn2 was partly identical to chitosanase genes from Metarhizium anisopliae var. acridum, Hypocrea lixii and Aspergillus fumigatus. Based on sequence similarity, Csn2 was classified as a GH-75 chitosanase.     

Characteristics of bacteriophages for Micromonospora purpurea.

Chemical and physical stabilities of bacteriophages øUW 21 and øUW 51 infecting Micromonospora purpurea ATCC 15835 were examined. Both phages were stable over the pH range of 5 to 8 and to heating at temperatures up to 50 degrees C and especially stable in buffer containing magnesium ion. Exposure to 1 M Ca(NO3)2 inactivated both phages, and phage øUW 51 was also susceptible to 1 M CaCl2, 0.1 M tris(hydroxymethyl)aminomethane, and 0.3% H2O2. Phage plating efficiency was highest on the cultures at logarithmic phase and sometimes much influenced by host growth. Phage øUW 51 has a latent period of 2 h at 34 degrees C and a burst size between 35 and 40. The latent period for phage øUW 21 is about 12 h, and the burst size is smaller than 30.     

嗜热硫杆菌启动子的克隆及定位

用DNA体外重组技术,将嗜热硫扦菌(Thiobacillus sp．)染色体DNA的Hind III片段插人启动子探测质粒pSDSI(Apr、Tc2)的Hind III位点,在四环素平板上获得一批转化子。四环素抗性能力测定表明,有12个转化子可以在含240／μg／ml四环素的平板上生长,有2个可以在360μg／ml的四环素平板上生长,对这二个抗性表达能力较高、分子量较小的质粒pSDH7和pSDH11作了限制性酶切图谱分析,并利用PstI位点,通过亚克隆将pSDH11插人片段上的启动子定位在0．25kb片段内。分子杂交实验证明克隆的启动子活性片段确实来源于嗜热硫杆菌染色体DNA。