噬藻体PP对野生宿主——丝状蓝藻的吸附率、裂解周期及释放量的影响

研究在日光光照条件下,以自然水体中存在的丝状蓝藻作为噬藻体PP的野生宿主,用离心法测定了噬藻体PP对野生宿主藻的吸附率,用一步生长曲线法获得了噬藻体PP对野生宿主藻的裂解周期及释放量。结果表明:噬藻体PP对野生宿主藻在60min时能够达到的吸附率为1.79‰,吸附系数为8.09%,噬藻体PP感染野生宿主藻的潜伏期介于45～75min之间,平均释放量为34.32PFU·Cell-1。上述结果一方面说明,噬藻体PP感染野生宿主藻的吸附系数、潜伏期及释放量均远小于以实验室培养的鲍氏织线藻作为宿主所得到的数据;另一方面也说明,噬藻体PP具有较强的吸附和裂解野生宿主的能力,这将有助于解释噬藻体PP在淡水富营养化水体中具有广泛分布的现象。     

CO_2浓度和温度升高对噬藻体PP增殖的联合作用

在4个条件下培养了鲍氏织线藻:(1)25℃+400μmol/mol(CK组),(2)29℃+400μmol/mol(温度升高组),(3)25℃+800μmol/mol(CO2升高组),(4)29℃+800μmol/mol(温室效应组),测定了藻的生物量及细胞大小,同时用离心法测定了噬藻体PP对相应条件下宿主藻的吸附率,用一步生长曲线法测定噬藻体PP的裂解周期和释放量。结果表明,不同培养条件对藻细胞的大小均没有影响;CO2升高提高了宿主藻的生物量;温度和CO2浓度的升高不仅使噬藻体PP的裂解周期提前,而且对吸附率和释放量存在交互作用,使其发生了明显改变:其中,温度和CO2升高对噬藻体PP吸附率的影响属于协同作用,而对其释放量的影响则能够互相抵消。上述结果说明温室效应将能够导致噬藻体PP增殖能力大幅度增加。     

感染丝状蓝藻的噬藻体的裂解周期和释放量的测定

近年来,随着浮游病毒的认识的深入,人们认识到浮游病毒对水体中初级生产力的影响是巨大的[1],其主要证据就是发现噬藻体在海洋蓝藻的种群控制上发挥着重要作用[2]. 噬藻体的释放量和裂解周期是衡量噬藻体感染力的重要指标,很多重要的生态指标如病毒在生态系统中对宿主的致死率、病毒种群得以维持的阈浓度等都需要使用病毒的释放量和裂解周期来加以推算[3,4], 因此准确地测定这两个基本参数是十分重要的.在自然界,很多丝状蓝藻,如颤藻、鱼腥藻、螺旋藻、席藻等是能够形成水华的,其中有些还具有产毒的功能[5].丝状蓝藻的形态特征有别于单细胞蓝藻, 在被噬藻体感染时,丝状蓝藻的感染周期和光合生理也与单细胞蓝藻有较大的差异[6],因此研究裂解丝状蓝藻的噬藻体的方法可能不同于感染单细胞的噬藻体.本次试验以一种感染丝状宿主的噬藻体为材料,探讨了确定其裂解周期和释放量的研究方法.     

感染丝状蓝藻的噬藻体的裂解周期和释放量的测定

近年来 ,随着浮游病毒的认识的深入 ,人们认识到浮游病毒对水体中初级生产力的影响是巨大的[1] ,其主要证据就是发现噬藻体在海洋蓝藻的种群控制上发挥着重要作用[2 ] 。噬藻体的释放量和裂解周期是衡量噬藻体感染力的重要指标 ,很多重要的生态指标如病毒在生态系统中对宿主的致死率、病毒种群得以维持的阈浓度等都需要使用病毒的释放量和裂解周期来加以推算[3,4 ] ,因此准确地测定这两个基本参数是十分重要的。在自然界 ,很多丝状蓝藻 ,如颤藻、鱼腥藻、螺旋藻、席藻等是能够形成水华的 ,其中有些还具有产毒的功能[5] 。丝状蓝藻的形态特征…     

不同光、温条件下野生宿主对噬藻体PP的光修复率

研究了1株野生宿主藻对UV-B损伤的噬藻体PP的光修复率,结果显示该野生宿主的光修复率显著高于实验室培养的坑形席藻(Phormidium foveolarumIU427)和鲍氏织线藻(Plectonema boryanum IU594)。不同理化条件(光质、光强、水温)下该野生宿主对经UV-B损伤的噬藻体PP光修复情况,结果表明:野生宿主的光修复率与UV-A强度、可见光强度、水温之间均呈显著正相关(P0.05);可见光所驱动的光修复能力明显高于UV-A所驱动的光修复,且修复率会在可见光强达到160μE.m-.2s-1时接近饱和。说明,自然条件下浅水湖泊中可见光介导的宿主光修复作用占主导地位,由于受水温和透明度的双重影响,野生宿主在秋季的修复能力最强,而在冬季的修复能力最弱。     

水华蓝藻噬藻体对不同条件培养的宿主细胞感染性分析

在从武汉东湖水样中培养分离水华蓝藻噬藻体(Planktothrix agardhii Virus from Lake Donghu,PaV-LD)的基础上,对在不同条件培养的宿主蓝藻细胞中,PaV-LD增殖效率及裂解作用进行了测定分析。分别将PaV-LD接种到生长期、半连续培养更新率或光照不同的宿主蓝藻液中,并采用稀释培养计数(Mostprobable number,MPN)方法与电镜观察,测定子代PaV-LD释放量及宿主细胞的裂解作用。结果显示:对数生长期宿主蓝藻单个细胞中子代PaV-LD的平均释放量为350感染单位(Infectious Units,IU/cell),显著高于稳定生长期的平均释放量110 IU/cell。在用新鲜培养基更新率为0%、35%、50%和65%的半连续培养宿主蓝藻中,接种PaV-LD 5d之后,噬藻体的释放量分别约为50 IU/cell、70 IU/cell、220 IU/cell或310 IU/cell,表明子代PaV-LD释放率随培养基更新率的增加而显著提高。在光照条件下感染3—4d后,宿主蓝藻细胞充分裂解,并释放大量子代PaV-LD,滴度可由初始7.00×103IU/mL快速增加到8.56×107IU/mL;但在遮光条件下,同样感染的蓝藻细胞未见裂解,也检测不到释放的子代噬藻体。电镜观察显示,在光照条件下感染的蓝藻细胞类囊体膜结构消失,而大量子代PaV-LD颗粒主要分布在原有类囊体的部位。显然,宿主蓝藻细胞的培养条件和状态可能对获得噬藻体纯培养有决定性影响。     

模拟生态系统中噬藻体裂解蓝藻宿主的生态学效应

实验采用模拟生态系统,研究了噬藻体裂解蓝藻宿主后营养物质循环变化的过程,以及细菌和漂浮植物对这个过程的影响。结果表明噬藻体裂解宿主所释放的营养元素在细菌的作用下迅速进入新的循环并形成新的水华,而加入浮萍则可以有效吸收水体营养,防止新的水华发生。因此推测单纯地利用包括噬藻体在内的微生物裂解藻类的方法,并非治理水华的有效方法,但如果能够结合漂浮植物等其它治理手段,则有可能同时实现水华的控制和水质的改善。     

营养盐限制条件下塔玛亚历山大藻对东海原甲藻的化感作用研究

为明确塔玛亚历山大藻(Alexandrium tamarense)对东海原甲藻(Prorocentrum donghaiense)生长的化感作用,研究了在N、P限制及正常营养盐条件下(又称富营养)塔玛亚历山大藻无细胞滤液对东海原甲藻生长的影响,并探讨了3种不同营养盐条件下两种藻共培养时的生长状况。结果表明,半连续培养时,营养盐限制下,塔玛亚历山大藻无细胞滤液对东海原甲藻的生长均有一定影响。N限制下,5 d后东海原甲藻藻密度显著低于未加滤液的对照组,藻密度为1.02×107 cells L-1,对照组为1.7×107 cells L-1;P限制下,东海原甲藻藻密度与对照组差异不显著,5 d后藻密度为1.44×107 cells L-1;富营养条件下,东海原甲藻藻密度与对照组无明显区别。共培养时,塔玛亚历山大藻对东海原甲藻生长的抑制作用更为显著,N、P限制下,4 d后东海原甲藻全部死亡,且聚集成团形成沉淀;富营养条件下,仍有少量东海原甲藻存活(藻密度3.3×104 cells L-1)。这表明,塔玛亚历山大藻对东海原甲藻的生长有一定的化感作用。营养盐限制可促进塔玛亚历山大藻化感物质的合成和释放,化感作用是塔玛亚历山大藻抑制东海原甲藻生长的原因之一。     

也西湖噬藻体的分离与鉴定

【背景】噬藻体是一类特异性侵染蓝藻的病毒,广泛存在于淡水和海水水体中,参与调控宿主蓝藻的丰度和种群密度,被认为是潜在的蓝藻水华生物防控工具。但目前的研究多集中于海洋噬藻体,对淡水噬藻体的生物学特性和结构生物学等研究较少。【目的】分离更多种类的淡水噬藻体,为研究淡水噬藻体的三维结构、侵染机制、与宿主的共进化关系,及其在蓝藻水华防治中的应用提供理论基础。【方法】采集中国科学技术大学西校区内景观湖也西湖水华暴发水域的水样,利用液体培养基和双层固体平板法对17种宿主蓝藻进行筛选,通过NaCl-PEG沉淀法和氯化铯密度梯度离心分离和纯化噬藻体,并利用负染电镜观察噬藻体的形态,同时采用梯度稀释法测定裂解液的效价。【结果】发现也西湖的水样可特异性侵染本实验室分离自巢湖的一株拟鱼腥藻Pan。侵染后的裂解液中存在4株形态各异的噬藻体,包括1株短尾噬藻体和3株长尾噬藻体,其中包括首次发现的1株含有非典型长轴状头部结构的淡水噬藻体。【结论】也西湖作为巢湖流域的一个小型水体,具有与巢湖类似的水华蓝藻及其噬藻体分布谱,因此可以用于模拟大型湖泊进行相关分子生态学和生物防控的研究。     

温度和营养盐限制对网状原角藻生长与产毒的影响

网状原角藻(Protoceratium reticulatum)是能够形成有毒赤潮的海洋甲藻之一,所产毒素为虾夷扇贝毒素(yessotoxins,YTXs),该藻在全球很多海域中普遍存在,其生长与产毒特征表现出较强的海域差异性。以分离自我国北黄海海域的网状原角藻为实验对象,研究了温度和营养盐(N、P)限制对该藻生长与产毒的影响。研究发现:温度和营养盐限制对藻细胞的生长和产毒均有影响,但营养盐限制影响更为显著。较低的温度更适宜P.reticulatum的生长,15℃时无营养盐限制的L1-Si培养基中藻细胞生长最好。营养盐限制尤其是P限制能够显著降低藻细胞的比生长速率和细胞密度(P0.01),缩短藻细胞指数生长期和稳定期的持续时间。所有温度下N、P限制均有利于藻细胞内毒素累积,15℃P限制培养基中单个藻细胞中YTX毒素最高,达到92.6 pg/细胞,分别是相同培养温度下N限制和L1-Si中藻细胞毒素含量的3.8倍和7.1倍。温度变化对N和P限制下藻细胞毒素含量影响不同:在5—15℃范围内,随温度升高,N限制培养基中藻细胞YTX含量增幅逐渐下降,而P限制条件下反之。在所有培养条件下,滤液中毒素含量在稳定期后开始增多,与L1-Si相比,N、P限制不利于毒素的释放。高温能促进L1-Si培养基和N限制培养基中毒素的释放,但对P限制影响不显著。     

THE EFFECT OF PHOSPHATE STATUS ON THE KINETICS OF CYANOPHAGE INFECTION IN THE OCEANIC CYANOBACTERIUM SYNECHOCOCCUS SP. WH78031

Phycoerythrin-containing Synechococcus species are considered to be major primary producers in nutrient-limited gyres of subtropical and tropical oceanic provinces, and the cyanophages that infect them are thought to influence marine biogeochemical cycles. This study begins an examination of the effects of nutrient limitation on the dynamics of cyanophage/Synechococcus interactions in oligotrophic environments by analyzing the infection kinetics of cyanophage strain S-PM2 (Cyanomyoviridae isolated from coastal water off Plymouth, UK) propagated on Synechococcus sp. WH7803 grown in either phosphate-deplete or phosphate-replete conditions. When the growth of Synechococcus sp. WH7803 in phosphate-deplete medium was followed after infection with cyanophage, an 18-h delay in cell lysis was observed when compared to a phosphate-replete control. Synechococcus sp. WH7803 cultures grown at two different rates (in the same nutritional conditions) both lysed 24 h postinfection, ruling out growth rate itself as a factor in the delay of cell lysis. One-step growth kinetics of S-PM2 propagated on host Synechococcus sp. WH7803, grown in phosphate-deplete and-replete media, revealed an apparent 80% decrease in burst size in phosphate-deplete growth conditions, but phage adsorption kinetics ofS-PM2 under these conditions showed no differences. These results suggested that the cyanophages established lysogeny in response to phosphate-deplete growth of host cells. This suggestion was supported by comparison of the proportion of infected cells that lysed under phosphate-replete and-deplete conditions, which revealed that only 9.3% of phosphate-deplete infected cells lysed in contrast to 100% of infected phosphate-replete cells. Further studies with two independent cyanophage strains also revealed that only approximately 10% of infected phosphate-deplete host cells released progeny cyanophages. These data strongly support the concept that the phosphate status of the Synechococcus cell will have a profound effect on the eventual outcome of phage-host interactions and will therefore exert a similarly extensive effect on the dynamics of carbon flow in the marine environment.     

The Ultrastructure of a Cyanophage Attack on Anabaena variabilis

Cyanophages multiplying on the nitrogen fixing blue-green alga Anabaena variabilis Kütz. were revealed by electron microscopy. Severe ultrastructural changes have been observed in the vegetative cells, whereas the heterocysts appeared resistant to the cyanophage. A lytic cycle was observed from adsorption to lysis.     

Development and evaluation of an organically certifiable growth medium for cultivation of cyanobacteria

On-farm cultivation of phototrophic, N-fixing cyanobacteria, grown in raceway ponds, could provide organic farmers an alternative N source to meet crop fertilizer needs. The application of cyanobacterial fertilizer in certified organic agriculture requires a growth medium of certified organic ingredients. This study compared growth and N-fixation of cyanobacteria cultured in two media, Allen and Arnon (AA) and a growth medium of organic-approved ingredients (RB) developed by the authors. A xenic culture of Anabaena sp. was grown for 2 weeks in the laboratory. The RB medium had significantly lower concentrations of P, Fe, B, Zn, and Cu than the AA medium. Cyanobacteria grown in RB had significantly greater exponential growth rate but significantly lower net total Kjeldahl nitrogen (TKN) than those grown in AA. In a follow-up replicated field study, the xenic culture of Anabaena sp. was inoculated into raceways aerated by paddle wheels. There was no significant difference in exponential or linear growth rate between the two treatments though the RB medium had lower concentrations of P, Co, Zn, and B than the AA medium. The lack of difference could be the result of an overarching limiting factor evident in both treatments such as light or C depletion or that the lower nutrient concentrations in RB were still sufficient for growth and N-fixation. There was no difference in net TKN between the two treatments, suggesting similar rates of N-fixation. Since bone meal contributed trace amounts of N to the RB media, it is possible that maximal N-fixation was not achieved. However, RB medium was able to support growth similar to that of the AA medium in raceway cultivation.     

紫外光和日光对噬藻体PP活性的影响

分别研究了实验室条件卜紫外灯照射及不同季节中目光照射对噬藻体PP活性的影响。结果表明紫外光(UV-A和UV-B)对噬藻体PP有较强的致失活作用,并且其致失活作用强弱与波长有关。日光的作用下,夏季时噬藻体PP的日失活率为88．60％,冬季时为58．86％。同时发现不同季节中,尽管在某些时段的目光强度相似,但噬藻体PP失活率的差别却较大,这一现象可能是由于光质不同引起的。上述结果有助于解释淡水环境中噬藻体种群大小的季节性波动。     

Effect of nutrient addition and environmental factors on prophage induction in natural populations of marine synechococcus species

A series of experiments were conducted with samples collected in both Tampa Bay and the Gulf of Mexico to assess the impact of nutrient addition on cyanophage induction in natural populations of Synechococcus sp. The samples were virus reduced to decrease the background level of cyanophage and then either left untreated or amended with nitrate, ammonium, urea, or phosphate. Replicate samples were treated with mitomycin C to stimulate cyanophage induction. In five of the nine total experiments performed, cyanophage induction was present in the non-nutrient-amended control samples. Stimulation of cyanophage induction in response to nutrient addition (phosphate) occurred in only one Tampa Bay sample. Nutrient additions caused a decrease in lytic (or control) phage production in three of three offshore stations, in one of three estuarine experiments, and in a lysogenic marine Synechococcus in culture. These results suggest that the process of cyanophage induction as an assay of Synechococcus lysogeny was not inorganically nutrient limited, at least in the samples examined. More importantly, it was observed that the level of cyanophage induction (cyanophage milliliter(-1)) was inversely correlated to Synechococcus and cyanophage abundance. Thus, the intensity of the prophage induction response is defined by ambient population size and cyanophage abundance. This corroborates prior observations that lysogeny in Synechococcus is favored during times of low host abundance.     

Effect of some environmental factors on cyanophage AS-1 development in Anacystis nidulans

The development cycle of the cyanophage AS-1 was studied in the host blue-green alga, Anacystis nidulans, under conditions that impair photosynthesis and under various light/dark regimes. Under standard conditions of incubation the 16-h development cycle consisted of a 5-h eclipse period and an 8-h latent period. Burst size was decreased by dark incubation to 2% of that observed in the light. An inhibitor of photosystem II, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU), reduced the burst size to 27% of that of the uninhibited control, whereas cyanophage production was completely abolished by carbonyl-cyanide m-chlorophenyl hydrazone (CCCP), an inhibitor of photosynthetic electron transport. Dark incubation of infected cells decreased the latent period by 1–2 h and the eclipse period by 1 h, once the cultures were illuminated. This suggests that adsorption took place in the dark. Intracellular growth curves indicated that light is necessary for viral development. Infected cells must be illuminated at least 13 h to produce a complete burst at the same rate as the continuously illuminated control. Low light intensities retarded the development cycle, and at lowest light intensities no phage yield was obtained. AS-1 is highly dependent on host cell photophosphorylation for its development.List of Abbreviations CCCP Carbonyl-cyanide m-chlorophenyl hydrazone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - m.o.i. multiplicity of infection - O.D. optical density - PFU plaque-forming unit Dedicated to Prof. Roger Y. Stanier on the occasion of his 60th birthday     

Effect of Nutrient Addition and Environmental Factors on Prophage Induction in Natural Populations of Marine Synechococcus Species

A series of experiments were conducted with samples collected in both Tampa Bay and the Gulf of Mexico to assess the impact of nutrient addition on cyanophage induction in natural populations of Synechococcus sp. The samples were virus reduced to decrease the background level of cyanophage and then either left untreated or amended with nitrate, ammonium, urea, or phosphate. Replicate samples were treated with mitomycin C to stimulate cyanophage induction. In five of the nine total experiments performed, cyanophage induction was present in the non-nutrient-amended control samples. Stimulation of cyanophage induction in response to nutrient addition (phosphate) occurred in only one Tampa Bay sample. Nutrient additions caused a decrease in lytic (or control) phage production in three of three offshore stations, in one of three estuarine experiments, and in a lysogenic marine Synechococcus in culture. These results suggest that the process of cyanophage induction as an assay of Synechococcus lysogeny was not inorganically nutrient limited, at least in the samples examined. More importantly, it was observed that the level of cyanophage induction (cyanophage milliliter⁻¹) was inversely correlated to Synechococcus and cyanophage abundance. Thus, the intensity of the prophage induction response is defined by ambient population size and cyanophage abundance. This corroborates prior observations that lysogeny in Synechococcus is favored during times of low host abundance.     

Induction of temperate cyanophage AS-1 by heavy metal - copper

Background  

It has been reported that some marine cyanophage are temperate and can be induced from a lysogenic phase to a lytic phase by different agents such as heavy metals. However, to date no significant reports have focused on the temperate nature of freshwater cyanophage/cyanobacteria. Previous experiments with cyanophage AS-1 and cyanobacteria Anacystis nidulans have provided some evidence that AS-1 may have a lysogenic life cycle in addition to the characterized lytic cycle.