变铅青链霉菌与放线菌噬菌体φHAU3相互关系的研究

变铅青链霉菌ＺＸ１,是由变铅青链霉菌ＪＴ４６经ＮＴＧ诱变后产生的一株修饰基因突变株,与其亲本ＪＴ４６相比,ＺＸ１除了与ＤＮＡ降解有关的基因发生了突变（Ｄｎｄ－,即该突变株的ＤＮＡ在含有Ｆｅ２＋的缓冲液中电泳不受到降解,而野生型变铅青链霉菌在同样条件下则遭到降解）以外,对噬菌体　ＨＡＵ３的抗性也随之消失,这项特征主要表现在噬菌斑大小和成斑单位（效价）上的显著变化。研究结果表明,噬菌体　ＨＡＵ３以同等的频率吸附野生型变铅青链霉菌及其突变菌株ＺＸ１,从　ＨＡＵ３基因组中没有克隆到被变铅青链霉菌识别的特异性靶位点;噬菌体ＨＡＵ３的ＤＮＡ也可以转染野生型变铅青链霉菌原生质,但其释放的噬菌体粒子只能感染突变菌株ＺＸ１,而不能感染野生型变铅青链霉菌。噬菌体ＨＡＵ３在突变株ＺＸ１中的繁殖遵循一步生长曲线,单菌释放量大约为１００,而　ＨＡＵ３感染野生型变铅青链霉菌后,则检测不到噬菌体的释放。     

限制修饰系统cglI赋予钝齿棒杆菌抗噬菌体功能活性

为解决氨基酸发酵工业中的噬菌体污染问题, 对cglI基因复合体在钝齿棒杆菌中的功能活性表达进行研究。通过PCR从谷氨酸棒杆菌基因组扩增cglI基因复合体, 构建重组质粒pJL23-cglI, 转化钝齿棒杆菌T6-13后得到重组菌株。定性和定量检测重组菌株的噬菌体抗性。实验结果表明, 携带cglI基因复合体的重组钝齿棒杆菌显示了明显的抗噬菌体功能活性和较广的抗噬菌体谱, 进而证实了cglI基因复合体用于构建钝齿棒杆菌抗噬菌体菌株的可行性, 为解决氨基酸发酵生产中的噬菌体污染问题提供了一种有效方法。     

限制修饰系统cglI赋予钝齿棒杆菌抗噬菌体功能活性

为解决氨基酸发酵工业中的噬菌体污染问题, 对cglI基因复合体在钝齿棒杆菌中的功能活性表达进行研究。通过PCR从谷氨酸棒杆菌基因组扩增cglI基因复合体, 构建重组质粒pJL23-cglI, 转化钝齿棒杆菌T6-13后得到重组菌株。定性和定量检测重组菌株的噬菌体抗性。实验结果表明, 携带cglI基因复合体的重组钝齿棒杆菌显示了明显的抗噬菌体功能活性和较广的抗噬菌体谱, 进而证实了cglI基因复合体用于构建钝齿棒杆菌抗噬菌体菌株的可行性, 为解决氨基酸发酵生产中的噬菌体污染问题提供了一种有效方法。     

一株苏云金杆菌噬菌体的形态结构及其蛋白质性质

从武汉微生物药厂苏云金杆菌发酵裂解液中分离到1株具有独特形态结构的苏云金杆菌噬菌体GP-1。电镜观察发现,这株噬菌体的头部呈长六棱柱状,具一短直尾和一“衣领”状结构,并首次发现了“衣领”状结构是由8～10个颗粒亚单位组成。该株噬菌体所具有的这8～10个颗粒亚单位对于噬菌体牢固地吸附于宿主表面应具有很强的促进作用,对于进一步研究噬菌体与宿主之间的关系提供一个结构上的证据。该株噬菌体的蛋白经SDS-聚丙烯酰胺凝胶电泳法测定,呈现一条主带,分子量为58892 D,一条次主带和七条次带,表明该株噬菌体的蛋白是由9种蛋白质构成。     

噬菌体吸附及受体结合蛋白(RBP)的研究进展与应用

噬菌体通过受体结合蛋白(Receptor binding protein,RBP)结合到细菌表面,其过程需要复杂的原子结构的参与和构象改变。针对噬菌体侵染,细菌发展了多种抗性机制,同时,噬菌体也进化出多种逃逸宿主抗性的机制。对噬菌体与细菌间"吸附-抗吸附-逃逸过程"的探索有助于我们理解噬菌体与细菌共进化的过程,对科学发展噬菌体治疗技术以及噬菌体的生物应用技术具有重要意义。本文概述了噬菌体吸附相关蛋白及吸附发生过程、基于RBP改变的噬菌体逃逸机制和RBP相关的生物技术研究进展。     

枯草芽孢杆菌噬菌体载体的构建

以φ0105DI：It为原始株构建的重组噬菌体φ105S35和φ10 5S36具有自主侵染能力和溶源化特征。其基因组内插入的lkb片段上的cat,基因赋予二者所在宿主以氯霉素抗性,在两株噬菌体中插入位点相同,即原φ105DI ：It的smal酶切片段D、E之间,但插入片段在二者中的定向相反。与cat基因同时引入的单一BamHI和Xbal位点提供了外源DNA的插入位置。重组噬菌体DNA可高效转染枯草芽孢杆菌原生质体。因此φ105S35和币φ105S36可作为枯草芽孢杆随系统的载体而被利用。     

快速从医院废水中大规模分离铜绿假单胞杆菌噬菌体

目的:从医院废水中快速分离多株不同的铜绿假单胞杆菌噬菌体,研究其生物学特性,为建立铜绿假单胞杆菌噬菌体库做准备。方法:利用噬菌斑法从未经处理的医院污水中分离和鉴定铜绿假单胞杆菌噬菌体,根据感染谱的不同确定它们为不同的铜绿假单胞杆菌噬菌体;重点研究其中一株宿主谱较广的噬菌体的生物学特性,采用负染法电镜观察噬菌体的形态和大小,提取该噬菌体的基因组并进行酶切电泳分析,测定噬菌体感染复数并观察其一步生长曲线。结果:通过噬菌斑法分离出90株铜绿假单胞杆菌噬菌体。电镜观察显示,噬菌体Pa27P1头部呈立体对称,有一长尾;酶切结果显示,噬菌体Pa27P1的基因组为双链DNA;生长曲线表明噬菌体Pa27P1感染宿主菌的潜伏期为25 min,爆发时间为25 min,裂解量为514。结论:90株铜绿假单胞杆菌噬菌体中有5株具有较广的噬菌谱,其组合能裂解所有18株铜绿假单胞杆菌,为深入研究铜绿假单胞杆菌噬菌体的生物学特性及其功能提供了依据。     

卡那霉素链霉菌噬菌体sKJl——温和性噬菌体

从土壤中分离出一株卡那霉素链霉菌噬菌体SKJl,该噬菌体在卡那霉素链霉菌及林肯链霉菌菌苔上产生混浊的噬菌斑。从噬斑中长出的菌落后代对SKJl噬菌体的感染产生了抗性。单株传代未见自发释放游离噬菌体。紫外线照射亦未发现诱导现象。抗性菌株经10ug／ml丝裂霉素C处理后,其中一株能释放出约5．9×103pfu／ml游离噬菌体颗粒,推测这些抗性菌株可能是溶源性菌株。菌落原位杂交实验证实抗性菌株的DNA与SKJl噬菌体DNA有同源性,说明这些抗性菌株已被SKjL噬菌体溶源化,从而确证SKJl噬菌体为一温和性噬菌体。     

卡那霉素链霉菌噬菌体SKJ1—温和性噬菌体

从土壤中分离出一株卡那霉素链霉菌噬菌体ＳＫＪ１,该噬菌体在卡那霉素链霉菌及林肯链霉菌菌苔上产生混浊的噬菌斑。从噬斑中长出的菌落后代对ＳＫＪ１噬菌体的感染产生了抗性。单株传代未风自发释放游离噬菌体。紫外线照射亦未发现诱导现象。抗性菌株经１０μｇ／ｍｌ丝裂霉素Ｃ处理后,其中一株能释放出约５．９×１０＾３ｐｆｕ／ｍｌ游离噬菌体颗粒,推测这些抗性菌株可能是溶源性菌株。菌落原位杂交实验证实抗性菌株的ＤＮＡ与     

明永冰川三株黄杆菌低温噬菌体的生物学特性研究

对分离自明永冰川地区的3株黄杆菌低温噬菌体(MYSP03、MYSP08和MYTP08)的生物学特征开展了研究,采用氯化铯密度梯度离心法对3株低温噬菌体进行了纯化并系统比较了3株低温噬菌体的形态、宿主专一性、吸附速率、最佳感染复数及一步生长曲线等生物学特性。虽然3株低温噬菌体的宿主均为黄杆菌,但它们在形态及生物学特性上均表现不同。低温噬菌体MYSP03和08为头尾型噬菌体,呈复合结构,二者头部直径与尾管长度明显不同;噬菌体MYTP08与MYSP08形态差异较大,但它们都能感染同一宿主菌,MYTP08为复层噬菌体,衣壳直径为58 nm。噬菌体MYSP03、MYSP08和MYTP08分别在MOI为0.1、10和1时所产生的子代噬菌体数量最多,3株噬菌体的裂解量显著不同,其中MYTP08最大为137。     

Park-Williams Number 8 Strain of Corynebacterium diphtheriae

Five clones of the Park-Williams number 8 strain of Corynebacterium diphtheriae, previously maintained in separate laboratories, were examined for their colonial and biochemical properties, for the restriction and modification system which operates to obscure their lysogeny, and for their capacity to produce large amounts of toxin under ordinary laboratory conditions. The phenotypes of their phage, P, produced in strain 603 and C7 (P.603 and P.C7) differ both as to stability to storage in the cold and to inactivation by antiphage serum. Evidence for a high degree of stability in the integration of P prophage in the PW8 genome is presented.     

The Clostridium difficile cell wall protein CwpV confers phase‐variable phage resistance

Bacteriophages are present in virtually all ecosystems, and bacteria have developed multiple antiphage strategies to counter their attacks. Clostridium difficile is an important pathogen causing severe intestinal infections in humans and animals. Here we show that the conserved cell‐surface protein CwpV provides antiphage protection in C. difficile. This protein, for which the expression is phase‐variable, is classified into five types, each differing in their repeat‐containing C‐terminal domain. When expressed constitutively from a plasmid or the chromosome of locked ‘ON’ cells of C. difficile R20291, CwpV conferred antiphage protection. Differences in the level of phage protection were observed depending on the phage morphological group, siphophages being the most sensitive with efficiency of plaquing (EOP) values of < 5 × 10^?7 for phages ?CD38‐2, ?CD111 and ?CD146. Protection against the myophages ?MMP01 and ?CD52 was weaker, with EOP values between 9.0 × 10^?3 and 1.1 × 10^?1. The C‐terminal domain of CwpV carries the antiphage activity and its deletion, or part of it, significantly reduced the antiphage protection. CwpV does not affect phage adsorption, but phage DNA replication is prevented, suggesting a mechanism reminiscent of superinfection exclusion systems normally encoded on prophages. CwpV thus represents a novel ubiquitous host‐encoded and phase‐variable antiphage system in C. difficile.     

Identification of structural genes for Clostridium botulinum type C neurotoxin-converting phage particles

The structural genes for strain C-Stockholm (c-st) phage particles, a representative type C toxin-converting phage of Clostridium botulinum, have been determined. First, by determining the N-terminal amino acid sequences of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) bands of c-st phage particles, it became clear that four proteins, 14, 25, 32 and 42 kDa, are the products of the ORFs, cst166, cst165, cst160 and cst164, respectively, of the c-st phage genome. The Western blot analyses reacting these phage bands with an antiphage serum prepared previously indicated that the products of cst165 and cst160 are the main proteins of the phage particles. Then, six candidates for the phage structural proteins, including cst165 and cst160 gene products, were prepared as recombinant proteins. Also, the protein corresponding to the cst164 gene product was excised from SDS-PAGE gels. The antibodies against these seven proteins were prepared in rabbits, and finally, the reaction of these antibodies to the c-st phage particles was analyzed by electron microscopy. It was concluded that a sheath protein and a head protein of the c-st phage are the products of genes cst160 and cst165, respectively, and that these two proteins are conserved in the other three converting phages, but not in the nonconverting phage.     

Screening,Purification and Properties of a Specific Antiphage Substance,AFS, against Filamentous Phage of Escherichia coli

We have made screening for antiphage antibiotics using an assay system of a male-specific filamentous phage, fl, and Escherichia coli as its host, and found a proteinous active substance named AFS (anti-filamentous phage substance) which was produced by an Actinomyces belonging to the group of Streptomyces lavendulae. It was purified from the filtered broth of the strain by ammonium sulfate precipitation and chromatographies of SP-Sephadex, CM-Sephadex and Sephadex G–50. The purified preparation was judged to be homogeneous by several column chromatographies and electrophoresis. Its molcular weight was about 5100 and isoelectric point was pH 9.9. Fourty two residues of amino acids, rich in the hydrophobic ones, were contained in one mole of AFS, but no carbohydrate was detected.     

Phage A25-mediated transfer induction of a prophage in Streptococcus pyogenes

Summary The group A streptococcal strain 56188 used as standard donor in transduction with the virulent phage A25 is lysogenic for a phage called P56188. By using specific antiphage sera it is shown that A25 lysates obtained from 56188 contain a fraction of about 10^-4 phenotypically A25 but genotypically P56188 particles. A25-mediated transduction of prophage P56188 is measured by scoring plaques produced by transfer induction on 5004, a lysogenic strain unable to support the growth of A25. Data are obtained suggesting that A25 can also transduce a prophage carried by strain T25₃.Prophage P5004 present in 5004 is found to interfere with the propagation of A25 but does not seem to exert its action by directing extensive degradation of A25 DNA. Lysogenization of SM27 with P5004 leads to dramatically decreased burst sizes of A25, associated with the loss of its ability to plaque on this strain. Furthermore, P5004 lysogens of SM27 yield fewer streptomycin resistant transductants than their parent but gain the ability to serve as donors in A25-mediated transduction. A comparison of the burst size and the yield of transducing particles of A25 on various lysogenic and nonlysogenic hosts suggests that interfering with A25 growth is a widespread property of streptococcal prophages, which might favour processes leading to the formation of transducing A25 particles.     

Characterization of the two-component abortive phage infection mechanism AbiT from Lactococcus lactis

During the production of fermented dairy products, virulent bacteriophages infecting Lactococcus lactis can delay or stop the milk acidification process. A solution to this biological problem consists of introducing natural phage barriers into the strains used by the dairy industry. One such hurdle is called abortive infection (Abi) and causes premature cell death with no or little phage progeny. Here, we describe the isolation and characterization of a novel Abi mechanism encoded by plasmid pED1 from L. lactis. The system is composed of two constitutively cotranscribed genes encoding putative proteins of 127 and 213 amino acids, named AbiTi and AbiTii, respectively. Site-directed mutagenesis indicated that a hydrophobic region at the C-terminal extremity of AbiTi is essential to the antiphage phenotype. The AbiT system is effective against phages of the 936 and P335 species (efficiency of plaquing between 10(-5) and 10(-7)) and causes a 20-fold reduction in the efficiency to form centers of infection as well as a 10- to 12-fold reduction in the burst size. Its efficacy could be improved by raising the plasmid copy number, but changing the intrinsic ratio of AbiTi and AbiTii did not greatly affect the antiphage activity. The monitoring of the intracellular phage infection process by DNA replication, gene expression, and electron microscopy as well as the study of phage mutants by genome mapping indicated that AbiT is likely to act at a later stage of the phage lytic cycle.     

Mutational Analysis of the Antitoxin in the Lactococcal Type III Toxin-Antitoxin System AbiQ

The lactococcal abortive phage infection mechanism AbiQ recently was classified as a type III toxin-antitoxin system in which the toxic protein (ABIQ) is regulated following cleavage of its repeated noncoding RNA antitoxin (antiQ). In this study, we investigated the role of the antitoxin in antiphage activity. The cleavage of antiQ by ABIQ was characterized using 5′ rapid amplification of cDNA ends PCR and was located in an adenine-rich region of antiQ. We next generated a series of derivatives with point mutations within antiQ or with various numbers of antiQ repetitions. These modifications were analyzed for their effect on the antiphage activity (efficiency of plaquing) and on the endoribonuclease activity (Northern hybridization). We observed that increasing or reducing the number of antiQ repeats significantly decreased the antiphage activity of the system. Several point mutations had a similar effect on the antiphage activity and were associated with changes in the digestion profile of antiQ. Interestingly, a point mutation in the putative pseudoknot structure of antiQ mutants led to an increased AbiQ antiphage activity, thereby offering a novel way to increase the activity of an abortive infection mechanism.     

Structural characteristics of the Corynebacterium lilium bacteriophage CL31

Bacteriophage CL31 was isolated on a Corynebacterium lilium strain. Out of 30 strains tested, only CL31 was able to form plaques on Corynebacterium glutamicum ATCC 13287, Brevibacterium lactofermentum ATCC 21086, and Arthrobacter sp. strain SI55, but at a very low frequency. This phage belongs to group B of Bradley's classification (D. E. Bradley, Bacteriol. Rev. 31:230-314; 1967). Its head is 53 nm in diameter, and its tail is 396 nm in length. The phage capsid contains three major proteins, of 12.5, 29.0, and 37.0 kilodaltons, and five minor ones (23.9, 26.0, 27.0, 40.0, and 55.4 kilodaltons). CL31 DNA is a linear molecule of 48 kilobases with cohesive ends. Restriction mapping was performed for endonucleases BglII, EcoRI, SalI, and KpnI. The expression of CL31 genes in Escherichia coli was studied by the maxicell technique; 12 different proteins were detected.     

Studies on transducing phage M-1 for Rhizobium japonicum D211

Phage M-1 produced clear plaques with a halo in the lawn of Rhizobium japonicum D211. A one step growth curve of phage M-1 showed a latent period of 3 h, burst size of 55 and rise period of 2 h. The inactivation of phage M-1 was found to be dependent upon the concentraion of d-glucosomanine. The neutralization kinetics of phage M-1 by antiphage serum gave a K value (velocity constant) of 83.1 min^–1. Transduction of str and kan was studied in the presence of antiphage serum and d-glucosamine. Cotransduction of different antibiotic resistance markers suggested that the system can be further explored for high resolution mapping in R. japonicum.Abbreviations YM yeast mannitol medium - PFU plaque forming unit - moi multiplicity of infection - EOP efficiency of plating