红霉素链霉菌2-62溶源性菌株的确证与特性研究

我们从红霉紊产生菌Streptomyces erythreus的27株系谱菌株中分离到一株带确噬菌体的2—62菌株,并对2—62菌株作了多方面的考察。根据单菌落传代、孢子加热,柠檬酸钠洗涤消除噬菌体后仍继续释放噬菌体;2—62菌株释放的噬菌体经纯化后制备抗血清,用此抗血清处理2—62菌株的斜面孢子,以中和游离的噬菌体,这种孢子在培养过程中仍继续释放噬菌体;2—62菌株对其自身释放的噬菌体具有免疫力等特性,确证2—62菌株为溶源性菌株。用紫外线或丝裂霉素C进行诱导试验,噬菌体释放量增加不多。将2—62菌株释放的温和性噬菌体感染P32一102敏感菌,得到的溶源化菌林亦释放噬菌体,并且溶源化菌林的性状保持稳定。     

检测噬菌体DNA法鉴别细菌的溶原性

根据前噬菌体的可诱导性,将细菌培养物经丝裂霉素C诱导,诱导液滤过除菌,经核酸酶处理和聚乙二醇(PEG 6000)浓缩,再用苯酚进行抽提。通过检测抽提物中有无DNA,以确定菌株的溶原性。实验证明从溶原菌诱导液中可提取DNA,同时表明该DNA确为溶原菌诱导出的噬菌体DNA,而非溶原性菌以同样方法不能取得DNAo用此方法,可以作为鉴别细菌溶原性的一个手段。     

N_3质粒对λ噬菌体发育途径的影响

诱导试验表明N_3质粒抑制λ溶源菌诱导处理时的裂解性发育途径,使λ噬菌体产率大大降低,仅为对照的10~(-5)—10~(-6)。感染实验表明N_3质粒主要抑制λ噬菌体的溶源性发育途径,其溶源菌形成率仅为对照的1.3—1.9％。从N_3质粒对λ噬菌体感染时与λ溶源菌诱导时的发育途径的不同作用的事实推测N_3质粒的某种产物(蛋白质)与λ噬菌体cI蛋白相作用,以障碍cI蛋白正常解离或聚合的方式而干扰其发育途径。     

诱导溶源性嗜盐古生菌产生噬菌体的方法

目的:用简单易行的诱导手段,从溶源性的嗜盐古生菌中诱导产生新的噬菌体,为分离嗜盐古生菌噬菌体提供一种新的途径.方法:分别用紫外线与丝裂霉素C对10株对数期的嗜盐古生菌菌株进行诱导,上清液采用双层平板法进行噬菌斑鉴定,并用脉冲场凝胶电泳对噬菌体基因组进行分析.结果:经1 μg/mL丝裂霉素C诱导的嗜盐古生菌融合子F5产生了一株新的嗜盐古生菌噬菌体SNJ1,该噬菌体能感染Natrinema属的菌株J7.结论:丝裂霉素C能诱导原噬菌体从宿主中分离,为嗜盐古生菌噬菌体分离提供了一条新的途径.     

双启动子对重组溶源性枯草杆菌中外源蛋白表达的增强作用

探讨了双启动子对基于溶源性噬菌体构建的重组枯草杆菌中外源蛋白表达的影响。分别将不含或含有本身启动子的α-淀粉酶基因(来源于Bacillus amyloliquefaciens)和青霉素酰化酶基因(来源于Bacillus megaterium)克隆到溶源性枯草杆菌中,得到重组菌B.subtilisAMY1,B.subtilisAMY2,B.subtilisPA1以及B.subtilisPA2。由于同源重组,所克隆的片段整合到溶源性枯草杆菌中的噬菌体基因组上,并处于噬菌体强启动子的下游。在重组菌AMY1和PA1中,在热诱导的情况下外源基因的转录只受到噬菌体启动子的作用,而在重组菌AMY2和PA2中,在热诱导下外源基因的转录同时受到噬菌体启动子和基因本身所带启动子的作用。双启动子的应用使重组α-淀粉酶的表达量提高了133%,使重组青霉素酰化酶的表达量提高了113%。     

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。     

枯草芽孢杆菌携带PBSX类缺陷性原噬菌体的普遍性调查

【目的】对来自中国典型培养物保藏中心(CCTCC)的39株枯草芽孢杆菌中的PBSX类缺陷性原噬菌体进行普遍性调查。【方法】对实验菌株进行丝裂霉素C(MMC)诱导,得到诱导后的裂解液上清。通过裂解液上清中13 kb DNA片段的存在情况,及其对PBSX敏感菌Bacillus subtilis W23的攻击作用,判断该菌株是否携带PBSX类缺陷性原噬菌体。同时利用透射电镜检测裂解液上清中噬菌体状颗粒。【结果】39株检测菌株中,24株菌裂解液上清含有13 kb DNA片段,对W23也具有较强的攻击能力,为PBSX溶源菌;1株菌裂解液上清中含有13 kb DNA片段,但不能攻击W23;5株菌裂解液上清中不含13 kb DNA片段,但依然对W23具有一定的攻击能力;另外9株裂解液上清中不含13 kb DNA片段,对W23也不具备攻击能力,其中3株菌株裂解液上清中能检测到大小不同于PBSX的噬菌体状颗粒。【结论】39株检测菌株中,携带有PBSX的菌株占61.5%的比重,具有一定普遍性;而工业菌株以及分离自神农架土壤中的野生菌株中含有不同于PBSX的多种噬菌体状颗粒。本文结果为进一步揭示PBSX对于宿主菌的作用提供了更多的理论依据。     

分枝杆菌噬菌体整合及裂解的分子机理

结核病仍旧威胁着全球人类健康,中国是结核病高发国家之一,寻求新的药物和疫苗势在必行。随着对噬菌体研究的深入,分枝杆菌噬菌体成为结核病新型药物发现和药敏实验的研究热点之一。噬菌体进入宿主菌体内,以裂解和溶源两种途径进入循环。以分枝杆菌的溶源性噬菌体为例,综述了分枝杆菌噬菌体整合和裂解分子机理。分枝杆菌溶源性噬菌体的整合需噬菌体基因组的附着位点attachment site(attP),宿主菌分枝杆菌基因组的附着位点attachment site(attB),整合酶integrase(Int)和整合宿主因子integration host factor(mIHF)。部分溶源性噬菌体如Ms6进入裂解循环,复制转录组装成新的子代噬菌体,在裂解素(Lysin)和穿孔素(Holin)的协同作用下裂解宿主菌,释放子代噬菌体。目前国内未见对分枝杆菌噬菌体的研究报道。研究分枝杆菌噬菌体整合及裂解机理对结核病治疗新药开发有一定的启示。     

测定λ原噬菌体诱导频率的新方法

本文报道两种测定λ原噬菌体紫外诱导频率的新方法—菌落计数法和平板诱导法。将溶源菌液经紫外诱导暗培养稀释后直接涂布在平板上培养 ,根据平板上菌落形成单位数计算λ噬菌体紫外诱导频率。另一种是将溶源菌与指示菌混合制备的平板用紫外线诱导 ,根据平板上噬菌体形成单位确定λ噬菌体紫外诱导频率。这两种方法不仅能准确测定噬菌体紫外诱导频率 ,而且操作简便 ,节省时间和用具 ,重复性好。本研究还将冬虫夏草浸出汁与溶源菌混合后进行紫外辐射 ,通过几种方法进行比较 ,结果证明建立的新方法确实可行 ,易操作 ;同时也表明冬虫夏草具有较强的抗紫外辐射作用     

测定λ原噬菌体诱导频率的新方法

本文报道两种测定λ原噬菌体紫外诱导频率的新方法 - 菌落计数法和平板诱导法.将溶源菌液经紫外诱导暗培养稀释后直接涂布在平板上培养,根据平板上菌落形成单位数计算λ噬菌体紫外诱导频率.另一种是将溶源菌与指示菌混合制备的平板用紫外线诱导,根据平板上噬菌体形成单位确定λ噬菌体紫外诱导频率.这两种方法不仅能准确测定噬菌体紫外诱导频率,而且操作简便,节省时间和用具,重复性好.本研究还将冬虫夏草浸出汁与溶源菌混合后进行紫外辐射,通过几种方法进行比较,结果证明建立的新方法确实可行,易操作;同时也表明冬虫夏草具有较强的抗紫外辐射作用.     

Effects of Mitomycin C and Thymidine Deprivation on Lysogenic and Sensitive Strains of Bacillus megaterium

A triple auxotroph of Bacillus megaterium strain KM was lysogenized with a phage suspension from B. megaterium 899a. The lysogenic and phage-sensitive derivatives of KM were found to die at the same exponential rate during thymineless incubation, despite the fact that the lysogenic strain became induced. The lysogenic strain was also induced by mitomycin C, and died at an exponential rate which was approximately twice that of the sensitive strain. With both strains, the lethality of mitomycin C was the same in the presence and absence of thymidine; thymidine was required for maximal phage production. Mitomycin C preferentially inhibited deoxyribonucleic acid (DNA) synthesis of both strains for the first 60 min. The (DNA) synthetic ability of the lysogenic strain was subsequently restored, due to phage production. Since there was no evidence that sensitive strains of KM contained other inducible elements (prophage or probacteriocins), it is concluded that both thymineless death and mitomycin C death can occur via mechanisms not involving induction.     

Comparative Study of 35 Bacteriophages of Lactobacillus helveticus: Morphology and Host Range

This survey included 23 phages isolated from cheese whey and 12 temperate phages induced with mitomycin from their lysogenic host strains. All of the phages had an isometric head and a tail with a contractile sheath. In addition, short-tailed (160-nm-long) and long-tailed (260-nm-long) phages were distinguished. Short-tailed phages were by far the most widespread in French cheese factories (32 of the 35 phages studied). The study of phage relationships enabled two large groups of strains to be distinguished: those not or slightly sensitive to phages and those very sensitive to phages. There was an obvious relationship in the first group between phage sensitivity (or resistance) and the geographic origin of the strains. The second group contained primarily strains from large international collections and those isolated from commercial starters. The relationships among short-tailed phages, either temperate or isolated as lytic, suggest that lysogenic strains could be the major source of phages in French cheese factories.     

Potential Significance of Lysogeny to Bacteriophage Production and Bacterial Mortality in Coastal Waters of the Gulf of Mexico

The potential effect that induction of lysogenic bacteria has on bacteriophage production and bacterial mortality in coastal waters was investigated, and we present estimates for the percentage of lysogenic cells in a natural aquatic bacterial community. Various concentrations of mitomycin C and exposure times to UV C radiation (UV-C) (wavelength of 254 nm) were used to induce the lytic cycle in lysogenic cells of natural communities of marine bacteria. UV-C treatment occasionally resulted in phage production, but phage production induced by UV-C was always less than that caused by the addition of mitomycin C. There was no evidence that high growth rates of bacteria resulted in lysogenic phage production. The burst size of cells induced by mitomycin C was determined by transmission electron microscopy and ranged from 11 to 45. Dividing the induced phage production by the burst size provided an estimate of the number of lysogenic bacterial cells, which ranged from 0.07 to 4.4% (average, 1.5%) of the total bacterial population. The percentages of lysogenic bacteria that were induced by mitomycin C were similar for samples collected nearshore from the pier of the Marine Science Institute (chlorophyll a, 1.6 to 2.9 (mu)g liter(sup-1)) and in relatively oligotrophic water (chlorophyll a, 0.2 to 0.9 (mu)g liter(sup-1)) collected 25 to 100 km offshore. By using a steady-state model, if all lysogenic bacteria were induced simultaneously, 0.14 to 8.8% (average, 3.0%) of the total bacterial mortality would result from induction of lysogenic cells. If mitomycin C induces all or the majority of lysogenized cells, our results imply that lysogenic phage production is generally not an important source of phage production or bacterial mortality in the coastal waters of the western Gulf of Mexico.     

Transformation and transfection in lysogenic strains of Bacillus subtilis: evidence for selective induction of prophage in competent cells.

Lysogenic strains of Bacillus subtilis 168 were reduced in their level of transformation as compared to non-lysogenic strains. The level of transformation decreased even further if the competent lysogenic cells were allowed to incubate in growth media prior to selection on minimal agar. This reduction in the frequency of transformation was attributable to the selective elimination of transformed lysogenic cells from the competent population. Concurrent with the decrease in the number of transformants from a lysogenic competent population was the release of bacteriophage by these cells. The lysogenic bacteria demonstrated this dramatic release of bacteriophage only if the cells were grown to competence. Both the selective elimination of transformed lysogens and the induction of prophage was prevented by the inhibition of protein synthesis. Additionally, competent lysogenic cells released significantly higher amounts of exogenous donor transforming deoxyribonucleic acid than did competent non-lysogenic cells or competent lysogenic cells incubated with erythromycin. These data establish that the induction of the prophage from the competent lysogenic cells was responsible for the selective elmination of the lysogenic transformants. A model is presented that accounts for the induction of the prophage from competent lysogenic bacteria via the induction of a repair system. It is postulated that a repair system is induced or derepressed by the accumulation of gaps in the chromosomes of competent bacteria. This hypothetical enzyme(s) is ultimately responsible for the induction of the prophage and the selective elimination of transformants.     

Conditions for induction of bacteriophage from lysogenic Bacillus megaterium with aflatoxin B1.

The present study was conducted to determine whether or not aflatoxin B1 was an effective inducing agent for lysogenic bacteria and to characterize some of the parameters involved in induction. A lysogenic strain of Bacillus megaterium (NRRL-B-3695) and an indicator strain of this species (NRRL-B-3694) were used. Cultures of the lysogenic strain were incubated for various periods of time in the presence of aflatoxin B1. Plaque-forming units as well as colony-forming units were then determined. Results of the present study indicated that bacteriophage lysogenizing B. megaterium could be induced with aflatoxin B1. The optimum concentration for induction was 25 micrograms of toxin per ml of early-log-phase culture. Evidence suggested that: (i) higher concentrations of aflatoxin B1 formed hydrophobic complexes which would not efficiently induce B. megaterium; (ii) the toxic effect of aflatoxin B1 severely limited the number of cells which could be induced prior to killing action of the toxin; and (iii) concentrations less than 25 micrograms of aflatoxin B1 per ml were not efficient inducers of bacteriophage production nor did they demonstrate the toxic effect observed at higher concentrations.     

Induction of Phage Production in the Lysogenic Escherichia coli by Hydroxyurea

1) Hydroxyurea, a reversible DNA synthesis inhibitor, was used to study the mechanism of prophage λ induction in Escherichia coli K12. Induction of prophage was judged on two criteria: increase of phage-producing cells and loss of colony-forming ability of the cells. 2) Hydroxyurea induced an increase of phage-producing cells only in lysogenic strains known to be inducible with ultraviolet irradiation for prophage development and not in strains such as E. coli K12 (λind^–) or E. coli K12 recA (λ⁺). 3) When protein synthesis was inhibited, hydroxyurea did not increase phage-producing cells of lysogenic strains; it showed a bacteriocidal effect on lysogenic recA⁺ strains, but not on nonlysogenic strains. 4) The sensitivity of E. coli K12 recA to hydroxyurea was independent of whether or not the cells were lysogenic. 5) From the results it is suggested that certain steps leading to loss of colony-forming ability (i.e. prophage induction) do not require de novo protein synthesis but require the presence of the host recA⁺ gene.     

Simultaneous detection of submicrogram quantities of hyaluronic acid and dermatan sulfate on agarose-gel by sequential staining with toluidine blue and Stains-All

A new discontinuous agarose-gel electrophoresis in 0.05 M HCl/0.04 M barium acetate combined with the highly sensitive visualization technique using toluidine blue/Stains-All has been developed for the simultaneous assaying of hyaluronic acid (HA) and dermatan sulfate (DS) with a detection limit at submicrogram level greater than other conventional procedures. Furthermore, this procedure also separates and reveals chondroitin sulfate (CS). The densitometric analysis of bands resulted in a linear response between 0.01 and 0.5 microg of glycosaminoglycans (GAGs) with correlation coefficients greater than approximately 0.94. Hyaluronic acid and dermatan sulfate extracted and purified from the abdominal skin of six rats were separated and quantified in comparison with the evaluation made by treatment of chondroitin ABC lyase and separation of Delta-disaccharides from hyaluronic acid (DeltadiHA) and dermatan sulfate/chondroitin sulfate (Deltadi4s and Deltadi6s) by HPLC. The total amount of rat skin polysaccharides (hyaluronic acid and dermatan sulfate) was 1.24+/-0.26 microg/mg of tissue by discontinuous agarose-gel electrophoresis and 1.20+/-0.33 microg/mg by HPLC with hyaluronic acid and dermatan sulfate percentages of 50.32+/-2.38 and 49.66+/-2.53, respectively. The analyses also confirmed that hyaluronic acid and dermatan sulfate are the main rat abdominal skin polysaccharides with chondroitin sulfate present in trace amounts. This new agarose-gel electrophoresis could be particularly useful in the study of the distribution of glycosaminoglycans in the skin from different body sites of animals and normal human subjects and may be of importance in understanding the changes that occur in the skin, especially the metabolism of extracellular matrix constituents, in connective tissue disorders.     

Disaccharide analysis and molecular mass determination to microgram level of single sulfated glycosaminoglycan species in mixtures following agarose-gel electrophoresis.

The separation of sulfated glycosaminoglycans in mixtures by agarose-gel electrophoresis and the recovery of single polysaccharide bands has been applied to the characterization of polysaccharides extracted from tissues without previous purification of single species. Sulfated glycosaminoglycans, heparin with its two components, slow-moving and fast-moving, heparan sulfate, dermatan sulfate, and chondroitin sulfate, were separated to microgram level by conventional agarose-gel electrophoresis. After their separation, they were fixed in the agarose-gel matrix by precipitation in a cetyltrimethylammonium bromide solution, making them visible on a dark background. After recovery of gel containing the fixed bands, high temperatures (90 degrees C for 15 min) were necessary to dissolve the gel matrix, and a solution of NaCl (3 M) was used to release sulfated polysaccharides from the complex with cetyltrimethylammonium. After precipitation of glycosaminoglycans in the presence of ethanol, the recovery of slow-moving heparin, fast-moving heparin, heparan sulfate, dermatan sulfate, and chondroitin sulfate was from 1 to 10 microg, with a percentage greater than 45% and a purity above 90%. Sulfated glycosaminoglycans in mixtures recovered from gel matrix as single species were evaluated for purity and characterized for unsaturated disaccharides after treatment with bacterial lyases (heparinases for heparin and heparan sulfate samples, and chondroitinases for dermatan sulfate and chondroitin sulfate) and molecular mass. Bovine lung and heart Glycosaminoglycans were extracted and separated into single species by agarose-gel electrophoresis and recovered from gel matrix after treatment in cetyltrimethylammonium solution. Unsaturated disaccharides pattern, the sulfate to carboxyl ratio, and the molecular mass of each single polysaccharide species were determined.     

Conditions for induction of bacteriophage from lysogenic Bacillus megaterium with aflatoxin B1.

The present study was conducted to determine whether or not aflatoxin B1 was an effective inducing agent for lysogenic bacteria and to characterize some of the parameters involved in induction. A lysogenic strain of Bacillus megaterium (NRRL-B-3695) and an indicator strain of this species (NRRL-B-3694) were used. Cultures of the lysogenic strain were incubated for various periods of time in the presence of aflatoxin B1. Plaque-forming units as well as colony-forming units were then determined. Results of the present study indicated that bacteriophage lysogenizing B. megaterium could be induced with aflatoxin B1. The optimum concentration for induction was 25 micrograms of toxin per ml of early-log-phase culture. Evidence suggested that: (i) higher concentrations of aflatoxin B1 formed hydrophobic complexes which would not efficiently induce B. megaterium; (ii) the toxic effect of aflatoxin B1 severely limited the number of cells which could be induced prior to killing action of the toxin; and (iii) concentrations less than 25 micrograms of aflatoxin B1 per ml were not efficient inducers of bacteriophage production nor did they demonstrate the toxic effect observed at higher concentrations.     

Ultraviolet light and mitomycin C induced sister-chromatid exchanges in fibroblasts from patients with retinoblastoma

Ultraviolet light and mitomycin C (MMC) induced sister-chromatid exchanges (SCEs) were investigated in 6 diploid fibroblast strains derived from 3 patients with deletion 13 and retinoblastoma, one patient with a hereditary form of retinoblastoma, one patient with trisomy 13, and one normal control. Two fibroblast strains with del(13)(q14q22) showed a significant increase in SCEs compared to the control after UV and MMC treatments. In contrast, cell strains with del(13)(q12q14) and trisomy 13 did not show increased SCEs. The frequency of SCEs in fibroblasts from a patient with autosomal dominant retinoblastomas (no deletions) was significantly increased by UV, but not by MMC. The results suggest that cell strains with different deletions of chromosome 13 have different SCE responses to UV and MMC inductions. The cells with del(13)(q14q22) may have a DNA-repair defect.