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1.
[目的]鉴定一株新分离的铜绿假单胞菌噬菌体PaP4的生物学特性.[方法]双层琼脂培养法制备PaP4的单个噬斑,观察噬斑特点;用聚乙二醇8000浓缩PaP4颗粒后,再用氯化铯密度梯度离心纯化;用透射电子显微镜观察磷钨酸负染色的PaP4颗粒;提取PaP4基因组核酸,通过限制性内切酶图谱分析其核酸类型;按照感染复数(MOI)分别为0.000 1、0.001、0.01、0.1、1和10加入噬菌体纯培养液和宿主菌,充分裂解细菌后,测定噬菌体滴度;以MOI=10的比例加入噬菌体及宿主菌,进行一步生长实验,绘制一步生长曲线.[结果]PaP4的噬斑直径约3 mm-5 mm,圆形透明边缘清晰;PaP4噬菌体呈多面体立体对称的头部,直径约50 nm,有一个约30 nm的短尾;限制性酶切实验表明PaP4基因组为双链DNA;当MOI为0.001时PaP4感染其宿主菌产生的子代噬菌体滴度最高;用一步生长曲线描绘了其生长特性.[结论]PaP4属dsDNA短尾科裂解性噬菌体;最佳感染复数是0.001;由一步生长曲线得出感染宿主菌的潜伏期是25 min,裂解期是20 min,平均裂解量是150.  相似文献   

2.
When Escherichia coli B, labeled by prior growth in 14C-glucose, are infected with T4 phage there is a rapid release of 14C-nondialyzable material into the medium. About half of this material is derived from the cell envelope as evidenced by its content of phospholipid and lipopolysaccharide and its buoyant density upon isopycnic ultracentrifugation of 1.19 g/cm3. It is similar in its gross chemical and physical properties to envelope material released at a lower rate from growing uninfected cells or from cells whose protein synthesis is inhibited by chloramphenicol (22). The rate of release of this envelope material at a multiplicity of infection (MOI) of 10 is greatest in the first minute after infection, and release is completed by 4 min. The rate of its release, as a function of MOI at 2 min after infection, is greatest at low MOI (e.g., MOI 2 and 4); in addition, the release does not continue above MOI 30. The main conclusion derived from the data is that phage, as part of the process of adsorption and injection of DNA, cause an increased release of envelope substance from the cells. With the assumption that all of the envelope material released is derived from the outer envelope, it is estimated that uninfected cells release 20 to 30% of their outer envelope per hour, whereas infected cells release 30% in 2 min at MOI 30. Further, because release does not continue at high MOI, this phenomenon is not considered to be a direct cause of lysis from without. Data are also presented on the amounts of other non-dialyzable 14C-components released and on the differences in the kinetics of release from chloramphenicol-treated cells compared to phage-infected cells. To avoid the possibility that the release is due to phage lysozyme which is an adventitious “contaminant” of wild-type phage, a phage mutant (T4BeG59s) devoid of this enzyme was used in these experiments.  相似文献   

3.
一株副溶血弧菌噬菌体的分离鉴定及生理特性   总被引:1,自引:0,他引:1  
为了探寻有效的控制副溶血弧菌的方法, 从水产品市场的污水中分离出来一株烈性噬菌体qdvp001。采用双层平板法分离纯化噬菌体qdvp001, 电镜观察其形态特征, 并利用双层平板法测定其生理特性, 包括热稳定性试验、最适pH、最佳感染复数及一步生长曲线, 然后提取基因组进行酶切和序列分析。结果显示该烈性噬菌体qdvp001头部直径大约为79 nm, 尾长大约118 nm, 属于肌尾噬菌体科。它对60 °C以下的温度耐受力较强, 最适pH为7.0?8.0左右, 最佳感染复数是0.000 1, 感染宿主菌的潜伏期约20 min, 裂解期约70 min, 并获得部分DNA片段的序列。将获得的DNA序列在NCBI上进行比对, 结果显示, qdvp001与其他噬菌体的同源性较低。该噬菌体很可能是一种新发现的噬菌体。  相似文献   

4.
Summary Escherichia coli cells lysogenic for temperate phage HK239 exclude phages , HK022, P1 vir, P2, and rII mutants of phage T4. After mutagenic treatment, four isolates were obtained for their inability to exclude T4rII. It is shown that this mutation, designated exc, is located in the prophage HK239, and that, it also abolishes the exclusion of phages , HK022, P1 vir, and P2.  相似文献   

5.
Summary Phage cl + gives clear plaques whereas phage cIind - gives turbid plaques on a lawn of a mutant strain of E. coli K12. This strain, called STS, carries mutation spr in a tif sfi genetic background. I hypothesize that upon temperate phage infection, STS bacteria spontaneously inactivate phage repressor by the same mechanism involved in normal lysogenic induction which results in obligatory lytic growth of +. The use of the STS mutant facilitates the isolation and genetic analysis of phage mutants with an abnormal response to lysogenic induction.  相似文献   

6.
一株粘质沙雷氏菌烈性噬菌体污水分离及特性   总被引:1,自引:0,他引:1  
[目的]以粘质沙雷氏菌(8039)为宿主菌从医院污水中分离噬菌体并对其基本生物学特点进行研究.[方法]四步法污水分离噬菌体;单、双层平板噬菌斑实验筛选烈性噬菌体并观察噬菌斑形态;纯化后2%磷钨酸染色电镜观察;手工法提取噬菌体核酸酶切后琼脂糖凝胶电泳分析;利用双层平板噬菌斑实验测定最佳感染复数和完成一步生长实验.[结果]从医院污水中成功分离出粘质沙雷氏菌烈性噬菌体一株(SM701),该噬菌体有一个正多面体立体对称的头部,头径约64nm,无囊膜,有一长尾,无收缩尾鞘,尾长约143nm;基因组核酸能被双链DNA内切酶BamH Ⅰ及Hind Ⅲ切开,大小约57kb;噬菌斑圆形透明,直径1mm左右(培养12h,),边界清楚;当感染复数(multiplicity of infection,MOI)为10时,子代噬菌体滴度较高;按照一步生长实验结果绘制出一步生长曲线,可知感染宿主菌的潜伏期是约为30min,爆发期约100min,平均爆发量约为630[结论]按照国际病毒分类委员会分类标准,该噬菌体属于长尾噬菌体科(siphoviridae)烈性噬菌体,按照Bradley和Ackermann形态分类法属于B1亚群;噬菌斑与周围红色细菌生长区,颜色差异明显,非常便于观察和计数;噬菌体头部大小和形态与呼吸道病毒中的呼肠病毒和腺病毒最为接近;国内尚未见粘质沙雷氏菌噬菌体相关报道.  相似文献   

7.
[目的]分离喜马拉雅旱獭肠内容物样本中的噬菌体,并研究其生物学特性和基因组特征。[方法]以大肠杆菌为宿主菌,利用双层琼脂平板法从喜马拉雅旱獭肠内容物样本中分离噬菌体;用透射电镜观察形态特征;测定其最佳感染复数、一步生长曲线、酸碱耐受度及宿主裂解谱等生物学特性,并进行全基因组测序。[结果]从喜马拉雅旱獭肠内容物样本中分离得到一株裂解性大肠杆菌噬菌体,命名为vB_EcoM_TH18,其噬菌斑呈无晕环的透亮圆形,透射电镜观察发现该噬菌体头部直径为(90±5) nm,尾部长度为(115±5) nm;最佳感染复数为1;一步生长曲线显示其潜伏期为10 min,110 min后进入平台期,平均裂解量为15 PFU/mL;在pH 4.5-9.5的范围内具有稳定活性;可裂解多种致病型和血清型大肠杆菌和宋内志贺氏菌,无法裂解沙门氏菌、屎肠球菌、金黄色葡萄球菌、肺炎克雷伯杆菌及鲍曼不动杆菌;基因组测序结果表明,其基因组长度为133 882 bp,GC含量为39.95%。基因组共注释到210个编码序列(CDS)和13个tRNAs,不含毒力基因及耐药基因。BLASTn比对结果表明该基因组与Avunavirus属噬菌体Av-05同源性为95.17%。基于噬菌体全基因组、主要衣壳蛋白和终止酶大亚基分别构建系统进化树,结果表明vB_EcoM_TH18是一株肌尾噬菌体科(Myoviridae) Avunavirus属的新型噬菌体。[结论]从喜马拉雅旱獭肠内容物中成功分离并鉴定了一株新型宽谱大肠杆菌噬菌体vB_EcoM_TH18,可裂解多种致病型和血清型的大肠杆菌及宋内志贺菌。  相似文献   

8.
【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。  相似文献   

9.
1. An anti-Escherichia coli phage has been isolated and its behavior studied. 2. A plaque counting method for this phage is described, and shown to give a number of plaques which is proportional to the phage concentration. The number of plaques is shown to be independent of agar concentration, temperature of plate incubation, and concentration of the suspension of plating bacteria. 3. The efficiency of plating, i.e. the probability of plaque formation by a phage particle, depends somewhat on the culture of bacteria used for plating, and averages around 0.4. 4. Methods are described to avoid the inactivation of phage by substances in the fresh lysates. 5. The growth of phage can be divided into three periods: adsorption of the phage on the bacterium, growth upon or within the bacterium (latent period), and the release of the phage (burst). 6. The rate of adsorption of phage was found to be proportional to the concentration of phage and to the concentration of bacteria. The rate constant ka is 1.2 x 10–9 cm.8/min. at 15°C. and 1.9 x 10–9 cm.8/min. at 25°. 7. The average latent period varies with the temperature in the same way as the division period of the bacteria. 8. The latent period before a burst of individual infected bacteria varies under constant conditions between a minimal value and about twice this value. 9. The average latent period and the average burst size are neither increased nor decreased by a fourfold infection of the bacteria with phage. 10. The average burst size is independent of the temperature, and is about 60 phage particles per bacterium. 11. The individual bursts vary in size from a few particles to about 200. The same variability is found when the early bursts are measured separately, and when all the bursts are measured at a late time.  相似文献   

10.
Summary The UV-sensitivity of phage and its infectious DNA have been compared in experiments involving infection of normal cells by phage and transfection of lysozyme-EDTA spheroplasts or Ca++-treated cells by phage DNA. It is shown that UV-irradiated DNA undergoes extensive HCR. Since intact phage and free phage DNA have the same survival after UV-irradiation in Hcr- spheroplasts and cells, resp., and since survival is also identical in Ca++-treated Hcr+ cells it is concluded that DNA in solution or packaged in the phage head provides the same target for the induction of lethal UV lesions. This conclusion is supported by the observation that cysteamine provides a similar radioprotection to the intact phage and its free DNA. Spheroplasts of Hcr+ cells, however, have an HCR capacity reduced by about 20% when compared with normal or Ca++-treated cells. Moreover, UV-reactivation of irradiated DNA, which is absent in spheroplasts, occurs efficiently in Ca++-treated cells. Possible reasons for the physiological difference between spheroplasts and normal cells are discussed. c-mutations, which are readily induced by UV in phage assayed with E. coli mul -, could not be induced in DNA when assayed with spheroplasts or Ca++-treated cells of this strain. No mutants were also found with DNA extracted from UV-irradiated phage. The significance of the mode of entry of UV-irradiated DNA into a cell for the production of mutations is discussed.  相似文献   

11.
Summary A nonlysogenic cell has twenty fold higher (26% versus 1.3%) probability to survive phage infection than entry of the same genome via conjugation (prophage infection). When the entering genome bears a cIII- mutation, this difference increases to one hundred fold (6% versus 0.06%). A lysogenic im- cell harbouring a defective prophage able to synthetize anti-immunity (product of gene tof) has ten fold higher probability to survive prophage infection than phage infection (20% versus 2%). Here, cIII- mutation does not affect the survival. When the cell is simultaneously infected with the phage and prophage, the decision of the phage whether to enter the lytic cycle (in im- cells) or not (in nonlysogens) is always epistatic to that of the prophage.  相似文献   

12.
Summary Genetic recombination induced by structural damage in DNA molecules was investigated in E. coli K12 () lysogens infected with genetically marked phage . Photoproducts were induced in the phage DNA before infection by exposing them either to 313 nm light in the presence of acetophenone or to 254 nm light. To test the role of the replication of the damaged phage DNA on the frequency of the induced recombination, both heteroimmune and homoimmune crosses were performed.First, samples of a heteroimmune phage imm434 P80 exposed to these treatments were allowed to infect cells lysogenic for prophage cI857 P3. Phage DNA replication and maturation took place, and the resulting progeny phages were assayed for the frequency of P + recombinants. Recombination was less frequent in infected cells exposed to visible light and in wild type cells able to perform excision repair than in excision-defective lysogens. Therefore, much of the induced recombination can be atributed to the pyrimidine dimers in the phage DNA, the only photoproducts known to be dissociated by photoreactivating enzyme.Second, in homoimmune crosses, samples of similarly treated homoimmune P3 phages were allowed to infect lysogens carrying cI857 P80. Replication of the phage DNA containing ultraviolet photoproducts was repressed by immunity, and was futher blocked by the lack of the P gene product needed for replication. The lysogens were purified and scored for both colony forming ability and for P + recombinant prophages. The 254 nm photoproducts increased the frequency of recombination in these homimmune crosses, even though phage DNA replication was blocked. Irradiation with 313 nm light and acetophenone M, which produces dimers and unknown photoproducts, was not as effective per dimer as the 254 nm light.It is concluded from these results that certain unidentified 254 nm photoproducts can cause recombination even in the absence of DNA replication. They are not pyrimidine dimers, as they are not susceptible to excision repair or photoreactivation. In contrast, pyrimidine dimers appear to cause recombination only when the DNA containing them undergoes replication.  相似文献   

13.
A DNA-containing bacteriophage, designated RS1, infecting Rhodopseudomonas spheroides 2.4.1, has been isolated from sewage. The buoyant density of RS1 in CsCl equilibrium centrifugation is 1.50 g/cm(3), and the buoyant density of RS1 DNA is 1.706. The phage possesses a polyhedral head, approximately 65 nm in diameter, and a tail 60 nm long. When grown on aerobic cells, RS1 has a latent period of 120 min and an average burst size of 20. When grown on anaerobic cells, RS1 has a latent period of 150 min, and a burst size similar to that observed during aerobic infection. The adsorption rate constant of RS1 to aerobic cells is 1.2 x 10(-9) ml/min, and 0.58 x 10(-9) ml/min to anaerobic cells. Adsorption of RS1 to R. spheroides requires the presence of divalent cations.  相似文献   

14.
Aims: To identify and characterize bacteriophages specific for Edwardsiella ictaluri, the causative agent for enteric septicemia of catfish (ESC). Methods and Results: Two bacteriophages were isolated that infect Edw. ictaluri. They both produce clear plaques, have icosahedral heads with a non‐rigid tail, and are tentatively classified as Siphoviridae. Phages ΦeiDWF and ΦeiAU are dsDNA viruses with approximate genome sizes of 40 and 45 kb, respectively. The addition of 500 μmol l?1 CaCl2 enhanced phage titres. Both phages have a latent period of 40 min and an estimated burst size of 270. Every Edw. ictaluri strain tested was susceptible to phage infection with variable plaquing efficiencies and with no evidence of lysogeny, with no plaques detected on other bacterial species. Conclusions: Two unique bacteriophages were isolated that show host‐specificity for Edw. ictaluri, have temperature and metal cation‐dependent infectivity, and are tentatively placed within the family Siphoviridae. Significance and Impact of the Study: This is the first report of bacteriophages specific to Edw. ictaluri, an important fish pathogen affecting farm‐raised channel catfish. Initial characterization of these bacteriophages has demonstrated their potential use as biotherapeutic and diagnostic agents associated with ESC.  相似文献   

15.
Summary In UV-irradiated cells of Escherichia coli K-12 a partial release of the restriction of non-modified phage is observed when the cells are recA + lexA +. We show here that the induction of this restriction allevation (RA) also depends on the recBC enzyme and that the expression of RA requires protein synthesis. Maximum expression was reached within 60 to 90 min after irradiation. Experiments are presented which show that upon UV-irradiation a signal is created which triggers the development of RA when protein synthesis is allowed. This signal decayed with a half-life of only a few minutes in cells treated with chloramphenicol. The decay kinetics were similar in uvr + and uvrA mutants. RA appeared to be specific for EcoK insofar as no allevation of restriction by EcoRI, EcoRII and EcoP1 occurred. During maximum expression of RA no gross reduction of the activities of the recBC enzyme (exonuclease V) and the restriction endonuclease EcoK was observed and no new DNA modifying activity appeared in the cells. Since, in fully expressed cells, up to 75% of the infecting DNA was converted to acid-soluble material within 20 min after infection we suggest that only a small specific fraction of infections may undergo RA.  相似文献   

16.
17.
Traditional technology of cell disruption has become one of the bottlenecks restricting the industrialization of genetic engineering products due to its high cost and low efficiency. In this study, a novel bioprocess of phage lysis coupled with salting-out extraction (SOE) was evaluated. The lysis effect of T7 phage on genetically engineered Escherichia coli expressing κ-carrageenase was investigated at different multiplicity of infection (MOI), meanwhile the phage and enzyme released into the lysate were separated by SOE. It was found that T7 phage could lyse 99.9% of host cells at MOI = 1 and release more than 90.0% of enzyme within 90 min. After phage lysis, 87.1% of T7 phage and 71.2% of κ-carrageenase could be distributed at the middle phase and the bottom phase, respectively, in the SOE system composed of 16% ammonium sulfate and 20% ethyl acetate (w/w). Furthermore, κ-carrageenase in the bottom phase could be salted out by ammonium sulfate with a yield of 40.1%. Phage lysis exhibits some advantages, such as mild operation conditions and low cost. While SOE can efficiently separate phage and intracellular products. Therefore, phage lysis coupled with SOE is expected to become a viable alternative to the classical cell disruption and intracellular product recovery.  相似文献   

18.
【目的】铅黄肠球菌是医源感染的机会致病菌,可引起危及生命的败血症、脑膜炎等,但针对其噬菌体的研究尚属空白。噬菌体作为细菌病毒,具有宿主特异性。本研究首次分离到可培养的铅黄肠球菌烈性噬菌体,对其基因组序列的分析和其他特征研究为进一步探讨噬菌体与宿主的作用机制及治疗应用提供参考。【方法】噬菌体Ecf_virus_SZ01以健康人粪便中分离的铅黄肠球菌(DO55)作为宿主菌,分离自深圳市南山区未经处理的生活污水样本,利用透射电镜观察噬菌体形态并对其生物学特征和基因组特点进行研究。【结果】透射电镜显示,噬菌体Ecf_virus_SZ01头部直径约为106 nm,尾部直径约为150 nm,尾长且无伸缩性尾鞘,属长尾噬菌体科;该噬菌体的最佳感染复数为0.01;一步生长曲线显示,潜伏期约为30 min,每个受感染细胞产生子代的平均数量为50 PFU/cell;抑菌曲线显示MOI=0.01时对宿主菌具有很好的抑制效果;宿主特异性强,不能实现跨属侵染;测序结果显示其基因组为dsDNA,长度为59 409 bp,GC含量为43.2%;该噬菌体共有102个开放阅读框,BLASTn比对显示该噬菌体与NCBI数据库中其他噬菌体相似性极低。【结论】首次分离到宿主为铅黄肠球菌的噬菌体,具有潜伏期短、裂解能力强、宿主专一的特征,基因组与数据库中现有噬菌体相比十分新颖,并对其生物学特性和基因组进行了分析。  相似文献   

19.
一株肺炎克雷伯菌噬菌体的生物学特性及全基因组分析   总被引:1,自引:0,他引:1  
【背景】随着抗生素的广泛使用甚至滥用,细菌耐药性问题日益显著,利用噬菌体治疗耐药致病菌的方法重新开始被人们关注。【目的】对一株烈性肺炎克雷伯菌噬菌体vB_KpnP_IME279进行生物学特性研究及生物信息学分析。【方法】以一株多重耐药的肺炎克雷伯菌为宿主菌,从医院污水中分离噬菌体,应用双层平板法检测噬菌体效价、最佳感染复数(Optimal MOI)、一步生长曲线以及裂解谱,纯化后通过透射电镜观察噬菌体形态;应用蛋白酶K/SDS法提取噬菌体全基因组,使用Illumina MiSeq测序平台进行噬菌体全基因组测序,测序后对噬菌体全基因组序列进行组装、注释、进化和比较基因组学分析。【结果】分离到一株新的肺炎克雷伯菌噬菌体,命名为vB_KpnP_IME279;其最佳感染复数为0.1,一步生长曲线显示潜伏期为20 min,平均裂解量140 PFU/cell,电镜观察显示该噬菌体属于短尾噬菌体科(Podoviridae)。基因组测序表明,噬菌体基因组全长为42 518 bp,(G+C)mol%含量为59.3%。BLASTn比对结果表明,该噬菌体与目前已知噬菌体的相似性较低,基因组仅70%区域与已知噬菌体有同源性。构建噬菌体主要衣壳蛋白的基因进化树,分析了噬菌体IME279与其他短尾科噬菌体的进化关系,结果表明该噬菌体是短尾科噬菌体的一名新成员。【结论】分离鉴定了一株新的肺炎克雷伯菌噬菌体,进行了生物学特性、全基因组测序和生物信息学分析,为研究肺炎克雷伯菌噬菌体与宿主之间的相互作用关系以及治疗多重耐药细菌感染奠定了基础。  相似文献   

20.
The increasing emergence of antibiotic-resistant bacteria has produced a growing interest among scientists in bacteriophages as alternative antimicrobial agents. This article reports a lytic phage against an antibiotic-resistant strain of Pseudomonas aeruginosa. Phage BVPaP-3 is a member of the Podoviridae family and morphologically similar to the T7-like phage gh-1. The phage has a hexagonal head of 58–59 nm in diameter and a short tail of 10 × 8 nm. It is stable at a wide range of pH (6–10) and temperatures (4–40°C). Its optimal growth temperature is 37°C and the adsorption rate constant is 1.19 × 10−9. Latent and eclipse periods are 20 and 15 min, respectively, and the burst size is 44 after 35 min at 37°C. The phage has a DNA size of 41.31 kb and a proteome of 11 proteins. The major protein is 33 kDa in size.  相似文献   

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