Distribution of plasmids in cyanobacteria of the LPP group

Abstract The distribution of plasmid DNA has been studied in 23 strains and variants of non-heterocystous filamentous cyanobacteria that are susceptible to infection by the LPP-1 cyanophage (archetype). 21 have identical plasmid profiles and contain 3 plasmids of M _rs 0.9, 10 and approx. 12 · 10⁶ respectively. In one strain, Plectonema FS180, the plasmids have been designated pMP1, pMP2, and pMP3, respectively. pMP1 shows sequence homology with pMP2 and pMP3 but not with DNA from an LPP-type cyanophage. Plectonema UTEX 598 lacks the small plasmid only, while Plectonema UTEX 1541 is distinct amongst all these strains with 3 plasmids of M _rs 3, 10, and > 30 · 10⁶, respectively. The findings support the view that the majority of these strains may be independent isolates of a single species.     

噬藻体研究进展

噬藻体（Cyanophage）是以蓝藻为寄主的浮游病毒类群,因其能特异性地感染蓝藻而具有重要的生态地位,是蓝藻＂水华＂潜在的控制因子。噬藻体的生物学、生态学特征是了解噬藻体的一扇窗,分子生物学研究仍将是主要的研究方向之一。同时,噬藻体研究手段也在不断更新。该文从这几个方面综述了噬藻体相关研究进展。     

Evidence for photosynthetic independence of viral multiplication in cyanophage LPP-1 infected cyanobacterium Phormidium uncinatum

Abstract Pigment decomposition, oxygen evolution and CO₂ fixation were measured in the cyanobacterium Phormidium uncinatum after infection with cyanophage LPP-1, under light and dark conditions. A gradual decrease in para benzoquinone supported O₂ evolution, chlorophyll a and phycocyanin level were noticed after 6 h of infection. These results demonstrated decreased photosynthetic activity of the host P. uncinatum prior to the start of LPP-1 multiplication. Metabolic inhibitor investigations confirmed that the cyanophage LPP-1 multiplication was independent of host photosynthesis.     

Characterisation of Host Growth after Infection with a Broad-Range Freshwater Cyanopodophage

Freshwater cyanophages are poorly characterised in comparison to their marine counterparts, however, the level of genetic diversity that exists in freshwater cyanophage communities is likely to exceed that found in marine environments, due to the habitat heterogeneity within freshwater systems. Many cyanophages are specialists, infecting a single host species or strain; however, some are less fastidious and infect a number of different host genotypes within the same species or even hosts from different genera. Few instances of host growth characterisation after infection by broad host-range phages have been described. Here we provide an initial characterisation of interactions between a cyanophage isolated from a freshwater fishing lake in the south of England and its hosts. Designated ΦMHI42, the phage is able to infect isolates from two genera of freshwater cyanobacteria, Planktothrix and Microcystis. Transmission Electron Microscopy and Atomic Force Microscopy indicate that ΦMHI42 is a member of the Podoviridae, albeit with a larger than expected capsid. The kinetics of host growth after infection with ΦMHI42 differed across host genera, species and strains in a way that was not related to the growth rate of the uninfected host. To our knowledge, this is the first characterisation of the growth of cyanobacteria in the presence of a broad host-range freshwater cyanophage.     

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。     

东北稻田水体噬藻体psbA基因多样性

【目的】揭示东北稻田噬藻体psbA基因多样性,分析其系统进化地位,为噬藻体生态学研究提供数据支持。【方法】采用滤膜分离并浓缩噬体、PCR-克隆测序技术对我国东北稻田水体中噬藻体psbA基因进行调查。【结果】在东北稻田水体中共得到17条来自于噬藻体的psbA基因,经系统进化分析表明,我国东北稻田具有新的噬藻体的类群,与日本稻田生态系统中psbA基因类群相比,两地间噬藻体类群存在显著的差异,稻田水体中噬藻体psbA基因类群有别于海洋、湖泊类群。【结论】采用PCR-克隆测序技术以psbA基因为分子标记首次对我国东北稻田水体噬藻体类群进行调查,发现有新的噬藻体类群。     

INTERACTION OF PLECTONEMA BORYANUM (CYANOPHYCEAE) AND THE LPP-CYANOPHAGES IN CONTINUOUS CULTURE1

Continuous culture techniques are used to study long-term population interactions between Plectonema boryanum Gomont, a filamentous bluegreen alga, and the LPP-viruses which infect it. After LPP-I (virulent cyanophage) infection of sensitive algae, 3 oscillations occur in cell density with concomitant oscillations in virus titer before final stabilization of both algal and viral concentrations. After LPP-ID and LPP-2 (temperate viruses) infection, oscillation in cell density occurred with burst of virus particles. Resistant algae always repopulated the chemostat; lysogeny was not established. The interaction between Plectonema that was resistant to virus infection and the 3 LPP-cyanophages resulted in rapid elimination of the viruses from the chemostat in the effluent. When lysogenic P. boryanum was tested, a law population of virus was present in the chemostat throughout the incubation period indicative of spontancous induction. Clones of lysogenic algae were isolated.     

新型聚球藻噬藻体Yong-L2-223的分离及基因组分析

【目的】噬藻体(cyanophages)是特异性侵染蓝藻(cyanobacteria)的病毒，广泛分布于各类水体中，在调节蓝藻种群动态和密度、推动生物地球水生生态系统循环中起着重要作用。本研究的目的在于分离、鉴定噬藻体。【方法】本研究以海洋聚球藻(Synechococcus sp.) PCC 7002为指示宿主，从淡水水样中分离培养一株新型噬藻体Yong-L2-223，对其进行了宿主范围实验、全基因组测序、基因功能注释和系统进化分析。【结果】针对31株供试蓝藻的宿主范围实验，结果除指示藻PCC 7002 [属于聚球藻目(Synechococcales)]外，Yong-L2-223能够感染2株淡水蓝藻，分别是来源于滇池的绿色微囊藻(Microcystis viridis) FACHB-1342 [属于色球藻目(Chroococcales)]和水华束丝藻(Aphanizomenon flos-aquae)FACHB-1209[属于念珠藻目(Nostocales)]。既可在高盐条件下感染海洋蓝藻，又可在低盐条件下感染淡水蓝藻，Yong-L2-223具有广盐性。透射电镜观察表明，Yong-L2...     

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.     

Evolution of an Eurasian Avian-like Influenza Virus in Na?ve and Vaccinated Pigs

Influenza viruses are characterized by an ability to cross species boundaries and evade host immunity, sometimes with devastating consequences. The 2009 pandemic of H1N1 influenza A virus highlights the importance of pigs in influenza emergence, particularly as intermediate hosts by which avian viruses adapt to mammals before emerging in humans. Although segment reassortment has commonly been associated with influenza emergence, an expanded host-range is also likely to be associated with the accumulation of specific beneficial point mutations. To better understand the mechanisms that shape the genetic diversity of avian-like viruses in pigs, we studied the evolutionary dynamics of an Eurasian Avian-like swine influenza virus (EA-SIV) in naïve and vaccinated pigs linked by natural transmission. We analyzed multiple clones of the hemagglutinin 1 (HA1) gene derived from consecutive daily viral populations. Strikingly, we observed both transient and fixed changes in the consensus sequence along the transmission chain. Hence, the mutational spectrum of intra-host EA-SIV populations is highly dynamic and allele fixation can occur with extreme rapidity. In addition, mutations that could potentially alter host-range and antigenicity were transmitted between animals and mixed infections were commonplace, even in vaccinated pigs. Finally, we repeatedly detected distinct stop codons in virus samples from co-housed pigs, suggesting that they persisted within hosts and were transmitted among them. This implies that mutations that reduce viral fitness in one host, but which could lead to fitness benefits in a novel host, can circulate at low frequencies.     

Growth and phage resistance of Anabaena sp. strain PCC 7120 in the presence of cyanophage AN-15

The cyanophage AN-15 was found to have a requirement for either 1 mM calcium or 1 mM magnesium ions to maintain viral stability, whereas 1 mM calcium ions alone were essential for the infection process to proceed in Anabaena sp. strain PCC 7120. Following prolonged incubation, phage-resistant cells were detected at a high frequency (approximately 10-⁵) in lysates, as either renewed growth in liquid cultures, or as colonies in confluently lysed lawns. Southern hybridisation failed to detect AN-15 DNA in any of the resistant strains, implying that resistance is unlikely to be due to the presence of temperate phages. A high rate of spontaneous mutation is therefore likely to be the cause of resistance. Two classes of resistant cells were identified; those in which AN-15 failed to attach to host cells, and those in which attachment occurred, but subsequent replication was defective. However, it was possible to overcome phage resistance by the isolation of spontaneous mutants of AN-15, capable of infecting phage-resistant cells. These observations imply that if cyanophages are to be assessed as a means of controlling cyanobacterial blooms in freshwater bodies, the ionic (notably calcium) concentration of the water must be considered, together with the possible need to employ alternative cyanophage strains if resistance to the original one arises. This revised version was published online in August 2006 with corrections to the Cover Date.     

新型淡水微囊藻噬藻体vB_MweS-yong2的分离与基因组分析

【目的】蓝藻(cyanobacteria)水华频繁暴发,引起水质恶化,使水生生物大量死亡,给水产养殖业造成巨大的经济损失;其代谢产物藻毒素具有肝毒性、神经毒性、生殖毒性、遗传毒性和肿瘤促进作用,并可在水生生物中富集,造成饮用水安全风险和水产品食用安全风险。噬藻体(cyanophages)是一类特异性侵染蓝藻的病毒,参与调控蓝藻的种群密度和丰度,被认为是极具潜力的蓝藻水华生物防控工具。以往的研究报道多集中于海水噬藻体,有关淡水噬藻体的报道寥寥无几,迄今尚无惠氏微囊藻(Microcystis wesenbergii)噬藻体的研究报道。本研究的目的在于分离、鉴定惠氏微囊藻噬藻体。【方法】以惠氏微囊藻FACHB-1112为指示宿主,采用双层平板法从淡水中分离出噬藻体vB_MweS-yong2,对其进行全基因组测序、基因功能注释和系统进化分析。【结果】vB_MweS-yong2的基因组长44 530 bp,G+C含量为71.6%,有61个开放阅读框(ORF)、1个tRNA基因。成对序列比较 (pairwise sequence comparison,PASC)表明,vB_MweS-yong2与所有已知噬菌体间的全基因组核苷酸序列相似度最高只有20.21%,小于<50%的属边界值。没有在vB_MweS-yong2基因组中发现抗生素耐药基因和毒力因子基因,显示该噬藻体在基因水平上的安全性。【结论】vB_MweS-yong2在有尾目的长尾科中代表一个新的属。本研究丰富了淡水噬藻体库、基因库,并为以后研发该噬藻体的功能基因、进一步研发用于治理以惠氏微囊藻为优势种引起的水华的产品与技术奠定了基础。     

Enhanced frequency of karyogamy in electrofusion of yeast protoplasts by means of preceding G1 arrest

Abstract The cellular glycogen pool and nitrate reductase activity were measured in the cyanobacterium Phormidium uncinatum after infection with cyanophage LPP-1, under both light and dark conditions. While dark incubation of the cyanobacterium reduced the glycogen level, the nitrate reductase (NR) activity remained almost unchanged. Furthermore, cyanophage multiplication enhances cellular glycogen level and NR activity in both illuminated and dark-incubated cyanobacterial cultures. Cyanophage-mediated increase in host nitrate assimilation appears to support the high protein demand for its reproductive cycle.     

云南不同高原湖泊噬藻体psbA基因多样性

【目的】噬藻体(cyanophage)广泛存在于自然水体生态系统中,通过侵染蓝藻进而调控蓝藻种群及群落结构,具有重要生态功能和生态地位,在控制蓝藻水华方面有巨大开发潜力。本研究旨在探究云南高原湖泊噬藻体psbA基因多样性,分析其系统进化地位,为深入了解高原湖泊生态功能、开发利用噬藻体资源奠定理论基础。【方法】以云南高原主要湖泊滇池、抚仙湖和星云湖等为研究对象,以psbA基因作为分子靶标,对湖泊水体中噬藻体遗传多样性进行研究。【结果】从不同湖泊中共获得100条环境噬藻体psbA基因序列,系统发育分析表明,湖泊的噬藻体psbA基因序列与中国东湖、中国东北稻田、日本稻田等淡水中的环境噬藻体psbA基因亲缘关系较近,与海洋环境噬藻体psbA基因亲缘关系较远;抚仙湖中的噬藻体psbA基因多样性高于滇池、星云湖和异龙湖中的噬藻体psbA基因多样性;云南高原湖泊中存在新的噬藻体类群;各湖泊秋冬季节噬藻体psbA基因遗传多样性差异不明显。【结论】云南主要高原湖泊噬藻体psbA基因遗传多样性高,与淡水环境噬藻体psbA基因亲缘关系较近,且存在独特的噬藻体类群。     

Photoreactivation of UV-irradiated blue-green algae and algal virus LPP-1

Ultraviolet (UV) sensitivity and photoreactivation of blue-green algae Cylindrospermum sp., Plectonema boryanum, spores of Fischerella muscicola and algal virus (cyanophage) LPP-1 were studied. The survival value after UV irradiation of filaments of Cylindrospermum sp. and Virus LPP-1 showed exponential trend and these were comparatively sensitive towards UV than F. muscicola and P. boryanum. Photoreactivation of UV-induced damage occurred in black, blue, green, yellow, red and white light in Cylindrospermum sp., however only black, blue and white light were capable of photorepair of UV-induced damage in P. boryanum, spores of F. muscicola and virus LPP-1 in infected host alga. Pre-exposure to yellow and black light did not show photoprotection. The non-heterocystous and nitrogen fixation-less mutants of Cylindrospermum sp. were not induced by UV and their spontaneous mutation frequency was not affected after photoreactivation. The short trichome mutants of P.boryanum were more resistant towards UV.The occurrence of photoreactivation of UV-induced killing in wide range of light in Cylindrospermum sp. is the first report in organisms.     

Identification of cyanophage Ma-LBP and infection of the cyanobacterium Microcystis aeruginosa from an Australian subtropical lake by the virus

Viruses can control the structure of bacterial communities in aquatic environments. The aim of this project was to determine if cyanophages (viruses specific to cyanobacteria) could exert a controlling influence on the abundance of the potentially toxic cyanobacterium Microcystis aeruginosa (host). M. aeruginosa was isolated, cultured, and characterized from a subtropical monomictic lake-Lake Baroon, Sunshine Coast, Queensland, Australia. The viral communities in the lake were separated from cyanobacterial grazers by filtration and chloroform washing. The natural lake viral cocktail was incubated with the M. aeruginosa host growing under optimal light and nutrient conditions. The specific growth rate of the host was 0.023 h(-1); generation time, 30.2 h. Within 6 days, the host abundance decreased by 95%. The density of the cyanophage was positively correlated with the rate of M. aeruginosa cell lysis (r(2) = 0.95). The cyanophage replication time was 11.2 h, with an average burst size of 28 viral particles per host cell. However, in 3 weeks, the cultured host community recovered, possibly because the host developed resistance (immunity) to the cyanophage. The multiplicity of infection was determined to be 2,890 virus-like particles/cultured host cell, using an undiluted lake viral population. Transmission electron microscopy showed that two types of virus were likely controlling the host cyanobacterial abundance. Both viruses displayed T7-like morphology and belonged to the Podoviridiae group (short tails) of viruses that we called cyanophage Ma-LBP. In Lake Baroon, the number of the cyanophage Ma-LBP was 5.6 x 10(4) cyanophage x ml(-1), representing 0.23% of the natural viral population of 2.46 x 10(7) x ml(-1). Our results showed that this cyanophage could be a major natural control mechanism of M. aeruginosa abundance in aquatic ecosystems like Lake Baroon. Future studies of potentially toxic cyanobacterial blooms need to consider factors that influence cyanophage attachment, infectivity, and lysis of their host alongside the physical and chemical parameters that drive cyanobacterial growth and production.     

Genetic diversity of marine Synechococcus and co-occurring cyanophage communities: evidence for viral control of phytoplankton

Unicellular cyanobacteria of the genus Synechococcus are a major component of the picophytoplankton and make a substantial contribution to primary productivity in the oceans. Here we provide evidence that supports the hypothesis that virus infection can play an important role in determining the success of different Synechococcus genotypes and hence of seasonal succession. In a study of the oligotrophic Gulf of Aqaba, Red Sea, we show a succession of Synechococcus genotypes over an annual cycle. There were large changes in the genetic diversity of Synechococcus, as determined by restriction fragment length polymorphism analysis of a 403- bp rpoC1 gene fragment, which was reduced to one dominant genotype in July. The abundance of co-occurring cyanophage capable of infecting marine Synechococcus was determined by plaque assays and their genetic diversity was determined by denaturing gradient gel electrophoresis analysis of a 118-bp g20 gene fragment. The results indicate that both abundance and genetic diversity of cyanophage covaried with that of Synechococcus. Multivariate statistical analyses show a significant relationship between cyanophage assemblage structure and that of Synechococcus. These observations are consistent with cyanophage infection being a major controlling factor in picophytoplankton succession.     

The virus's tooth: cyanophages affect an African flamingo population in a bottom-up cascade

Trophic cascade effects occur when a food web is disrupted by loss or significant reduction of one or more of its members. In East African Rift Valley lakes, the Lesser Flamingo is on top of a short food chain. At irregular intervals, the dominance of their most important food source, the cyanobacterium Arthrospira fusiformis, is interrupted. Bacteriophages are known as potentially controlling photoautotrophic bacterioplankton. In Lake Nakuru (Kenya), we found the highest abundance of suspended viruses ever recorded in a natural aquatic system. We document that cyanophage infection and the related breakdown of A. fusiformis biomass led to a dramatic reduction in flamingo abundance. This documents that virus infection at the very base of a food chain can affect, in a bottom-up cascade, the distribution of end consumers. We anticipate this as an important example for virus-mediated cascading effects, potentially occurring also in various other aquatic food webs.     

Effects of pesticides on cyanobacterium Plectonema boryanum and cyanophage LPP-1.

Cyanobacterium Plectonema boryanum IU 594 and cyanophage LPP-1 were used as indicator organisms in a bioassay of 16 pesticides. Experiments such as spot tests, disk assays, growth curves, and one-step growth experiments were used to examine the effects of pesticides on the host and virus. Also, experiments were done in which host or virus was incubated in pesticide solutions and then assayed for PFU. P. boryanum was inhibited by four herbicides: 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 1,1-dimethyl-3-(alpha, alpha,alpha-trifluoro-m-tolyl)urea ( Fluometeron ), 2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine (Atrazine), 2-(ethylamino)-4-(isopropylamino)-6-(methylthio)-s-triazine ( Ametryn ). One insecticide, 2-methyl-2-(methylthio)-propionaldehyde O-( methylcarbamoyl )oxime (Aldicarb), also inhibited the cyanobacterium. Two insecticides inactivated LPP-1, O,O-dimethyl phosphorodithioate of diethyl mercaptosuccinate (malathion) and Isotox . Isotox is a mixture of three pesticides: S-[2-( ethylsulfinyl )ethyl]O,O-dimethyl phosphorothioate ( Metasystox -R), 1-naphthyl methylcarbamate ( Sevin ) and 4,4'-dichloro-alpha- (trichloromethyl) benzhydrom ( Kelthane ). Two pesticide-resistant strains of P. boryanum were isolated against DCMU and Atrazine. These mutants showed resistance to all four herbicides, which indicates a relationship between these phototoxic chemicals. The results indicate that P. boryanum may be a useful indicator species for phototoxic agents in bioassay procedures.     

Effects of pesticides on cyanobacterium Plectonema boryanum and cyanophage LPP-1

Cyanobacterium Plectonema boryanum IU 594 and cyanophage LPP-1 were used as indicator organisms in a bioassay of 16 pesticides. Experiments such as spot tests, disk assays, growth curves, and one-step growth experiments were used to examine the effects of pesticides on the host and virus. Also, experiments were done in which host or virus was incubated in pesticide solutions and then assayed for PFU. P. boryanum was inhibited by four herbicides: 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 1,1-dimethyl-3-(alpha, alpha,alpha-trifluoro-m-tolyl)urea ( Fluometeron ), 2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine (Atrazine), 2-(ethylamino)-4-(isopropylamino)-6-(methylthio)-s-triazine ( Ametryn ). One insecticide, 2-methyl-2-(methylthio)-propionaldehyde O-( methylcarbamoyl )oxime (Aldicarb), also inhibited the cyanobacterium. Two insecticides inactivated LPP-1, O,O-dimethyl phosphorodithioate of diethyl mercaptosuccinate (malathion) and Isotox . Isotox is a mixture of three pesticides: S-[2-( ethylsulfinyl )ethyl]O,O-dimethyl phosphorothioate ( Metasystox -R), 1-naphthyl methylcarbamate ( Sevin ) and 4,4'-dichloro-alpha- (trichloromethyl) benzhydrom ( Kelthane ). Two pesticide-resistant strains of P. boryanum were isolated against DCMU and Atrazine. These mutants showed resistance to all four herbicides, which indicates a relationship between these phototoxic chemicals. The results indicate that P. boryanum may be a useful indicator species for phototoxic agents in bioassay procedures.