Valuable ingredients and feed toxicity evaluation of Microcystis aeruginosa acidolysis product in mice

This research studied the extraction from Microcystis aeruginosa using hydrochloric acid method as a potentially valuable protein resource from eutrophic lakes. Amino acid composition, residual algal toxins, and heavy metals of the acidolysis product were studied. After 18 h of hydrochloric acid treatment, the product of M. aeruginosa contained 17 amino acids, 51.34% of total amino acid requirements, and 30.25% of the livestock and poultry essential amino acid (Eaa). The residual microcystin-LR (MC-LR) was 0.94 µg kg⁻¹, which was less than WHO drinking water limit of microcystins. The removal ratio of microcystins was higher than 99.99% during the process of hydrolysis. The concentration of heavy metals of the product was in compliance with feed standards. Furthermore, using Horn’s method, Mouse Micronucleus Test and Sperm Shape Abnormality Test were conducted to study the forage safety of the product. Half lethal dose (LD₅₀) of acidolysis product in mice was >9.09 g kg⁻¹ body weight, actually belonging to non-toxic grade. Every dose treatment did not significantly increase activities of alkaline phosphatase (ALP), lactate dehydrogenase (LDH), and γ-glutamyltransferase (γ-GT). The results of both micronucleus test and sperm shape abnormality test were negative, which suggested the product with no mutagenicity and sperm malformation effects. This study indicated that the acidolysis product of M. aeruginosa was safe to be used as a feed ingredient.     

Bacteriocin-mediated competition in cystic fibrosis lung infections

Bacteriocins are toxins produced by bacteria to kill competitors of the same species. Theory and laboratory experiments suggest that bacteriocin production and immunity play a key role in the competitive dynamics of bacterial strains. The extent to which this is the case in natural populations, especially human pathogens, remains to be tested. We examined the role of bacteriocins in competition using Pseudomonas aeruginosa strains infecting lungs of humans with cystic fibrosis (CF). We assessed the ability of different strains to kill each other using phenotypic assays, and sequenced their genomes to determine what bacteriocins (pyocins) they carry. We found that (i) isolates from later infection stages inhibited earlier infecting strains less, but were more inhibited by pyocins produced by earlier infecting strains and carried fewer pyocin types; (ii) this difference between early and late infections appears to be caused by a difference in pyocin diversity between competing genotypes and not by loss of pyocin genes within a lineage over time; (iii) pyocin inhibition does not explain why certain strains outcompete others within lung infections; (iv) strains frequently carry the pyocin-killing gene, but not the immunity gene, suggesting resistance occurs via other unknown mechanisms. Our results show that, in contrast to patterns observed in experimental studies, pyocin production does not appear to have a major influence on strain competition during CF lung infections.     

白屈菜红碱对铜绿微囊藻生长和光合系统的影响

为了阐明白屈菜红碱(Chelerythrine)对铜绿微囊藻(Microcystis aeruginosa)生长及光合系统的影响, 研究了白屈菜红碱胁迫下铜绿微囊藻M. aeruginosa NIES-843生长、细胞色素含量及叶绿素光诱导荧光动力学变化. 结果显示, 当白屈菜红碱浓度10 g/L时, M. aeruginosa NIES-843的生长受到显著抑制. 通过线性回归分析, 白屈菜红碱对M. aeruginosa NIES-843生长50%抑制浓度(EC50)为(30.621.32) g/L. 当白屈菜红碱浓度为160 g/L时, M. aeruginosa NIES-843单位细胞内叶绿素a (Chl. a)和类胡萝卜素含量均显著低于对照. Chl. a光诱导荧光动力学分析结果显示, 白屈菜红碱胁迫下单位反应中心吸收的光能(ABS/RC)、单位反应中心捕获的用于还原QA的能量(TR0/RC)及单位反应中心捕获的用于电子传递的能量(ET0/RC)均受到显著抑制. 光合系统Ⅱ(PSⅡ)能量分配比率参数分析结果显示, 白屈菜红碱能显著抑制光合系统反应中心电子供体侧电子传递.     

Oxidative damage and antioxidant responses in Microcystis aeruginosa exposed to the allelochemical berberine isolated from golden thread

Berberine, extracted from golden thread (Coptis chinensis Franch), is an allelochemical exhibiting inhibitory effects on the growth of Microcystis aeruginosa. Berberine-induced oxidative damage and antioxidant responses in M. aeruginosa cells were investigated to elucidate the mechanisms involved in berberine inhibition on algal growth. Malondialdehyde content in M. aeruginosa cells exposed to berberine increased with increased exposure concentration and the prolongation of exposure time. The same changes were observed in O₂⁻ activity of M. aeruginosa cells exposed to berberine. Berberine upregulated superoxide dismutase (SOD) activity at low concentrations while downregulating it at high concentrations. SOD activity transitioned from an increase to a decrease from 0 to 72 h exposure to 0.10% berberine. We observed that berberine exposure increased glutathione content in M. aeruginosa cells. The results suggested that berberine-induced oxidative damage might be at least partially responsible for berberine inhibition on M. aeruginosa growth.     

从种群竞争的角度初步研究微囊藻的产毒机理

采用将微囊藻和栅藻混合培养的方法,从种群竞争的角度初步探讨了微囊藻毒素的产毒机理,结果显示：当起始接种浓度相同时,混合培养组比纯微囊藻培养组产生更多的毒素,由于其它培养条件完全一致,所以推论是由于栅藻的存在,增加了微囊藻的生存压力。当起始接种浓度微囊藻：栅藻为10：1时,此混合培养组比纯微囊藻产生的毒素少,并且毒素降解也更快,推论原因是微囊藻在种群数量上远远超过栅藻,竞争压力较小,同时由于栅藻的存在,增加了培养液中色素的含量,加快了光降解的速度。     

Early biochemical effects of Microcystis aeruginosa extracts on juvenile rainbow trout (Oncorhynchus mykiss)

Microcystins (MC) are usually the predominant cyanotoxins associated with cyanobacterial blooms in natural surface waters. These toxins are well-known hepatotoxic agents that proceed by inhibiting protein phosphatase in aquatic biota; recent studies have also reported oxidative stress and disruption of ion regulation in aquatic organisms. In the present study, young trout (Oncorhynchus mykiss) were exposed to crude extracts of Microsystis aeruginosa for four days at 15 °C. The level of microcystins was calculated to confirm the presence of toxins in these crude extracts: 0, 0.75, 1.8 and 5 μg/L. Protein phosphatase measured in the liver increased by at least 3-fold and is significantly as a result of exposure to these sublethal concentrations of crude extract, his indicates an early defense response against protein phosphatase inhibition from cyanotoxins. This was corroborated by the decreased phosphate content in proteins found in the liver and brain. No increase in glutathione-S transferase (GST) activity was observed and lipid peroxidation was unaffected in both liver and brain tissue exposed to the cyanobacterial extracts. The data revealed that the proportion of the reduced (metal-binding) form of metallothionein (MT) decreased by two-fold relative to the control group (with a concomitant increase in the proportion of the oxidized form). The level of phosphate associated with MT increased by 1.5-fold at the highest concentration of crude extract. Acetylcholinesterase (AChE) activity in brain tissue was decreased after exposure to the highest concentration of crude extract, suggesting a slowdown in neural activity. However, no biotransformation processes or detoxification of GST was triggered. Our findings show early sign of biochemical effects of MC-LR in young trout.     

Toxic blooms of cyanobacteria in the Patos Lagoon Estuary,southern Brazil

The Patos Lagoon is the largest lagoonal system in South America. Its waters are formed by a huge drainage basin (201,600 km²) situated in the most industrialized areas of the Southern state of Rio Grande do Sul. On its margins more than 3 million inhabitants live in several cities and towns. The lagoon waters are used for leisure, drinking, industry, fisheries, agriculture and navigation. A monitoring and sampling program was developed from February 1994 to January 1996 with emphasis on the estuarine area, aiming to evaluate the occurrence of algal blooms. In the last 15 years, several cyanobacterial (blue-green algal) blooms of theMicrocystis aeruginosa have been registered in the lagoon estuary. HighM. aeruginosa biomass (50 to 9,000 g chla l^–1) was observed in the whole region in late summer and autumn 1994, and early summer 1995. The LD50 of toxic bloom samples tested in mice varied from 22 to 250 mg dry weight kg body weight^–1 while levels of toxicity (LC50) in the brine shrimp varied from 0.47 to 2.44 mg ml^–1. Toxicity varied in different blooms, in the distances along the scum and with time, within the same bloom. The hepatotoxin microcystin-LR was identified in almost all samples.     

EVIDENCE THAT MICROCYSTIN IS A THIO-TEMPLATE PRODUCT1

The hepatotoxic cyclic heptapeptide toxins of cyanobacteria, collectively termed microcystins, are potent inhibitors of protein phosphatases PP1 and PP2A. The structure of microcystins resembles small, cyclic peptide secondary metabolites from fungi and eubacteria. Many of these metabolites are manufactured via a nonribosomal thio-template mechanism. We submit evidence that microcystin is synthesized by a similar mechanism. The organism used in this study was Microcystis aeruginosa PCC7820. Using the traditional ATP-³²PP_i exchange assay for thio-template activity, we found activity in the presence of the substrate d -amino acids occurring in microcystin. Thio-template mechanisms are known to be unaffected by protein synthesis inhibitors such as chloramphenicol. We subjected cultures in exponential and stationary growth to chloramphenicol and monitored culture health versus toxicity. Although the health of the treated cultures declined, the toxicity of the remaining cells increased. We developed an in vitro assay to measure microcystin synthesis in cell lysates in the presence of chloramphenicol. By supplementing the lysates with ATP and the substrate amino acids present in microcystin, we detected a fourfold increase in total microcystins over the course of 20 min.     

光、温限制后铜绿微囊藻和斜生栅藻的超补偿生长与竞争效应

研究了铜绿微囊藻(Microcystis aeruginosa)和斜生栅藻(Scenedesmus obliquus)低温和低光照限制后的超补偿效应,以及共培养条件下的竞争效应。结果表明,低温和低光照均显著抑制微藻的生长发育,但低温对铜绿微囊藻的抑制效应更强,而斜生栅藻则对低光胁迫更敏感。经过低光和低温培养后,铜绿微囊藻和斜生栅藻在恢复正常培养时藻细胞密度短期内都表现出超补偿增长效应,但不同藻类超补偿模式不同,斜生栅藻补偿生长时间不超过1周,而铜绿微囊藻的补偿效应可以持续10天;此外,统计结果表明铜绿微囊藻细胞密度对低温限制解除表现出更显著的补偿生长,斜生栅藻则在低光解除后表现出更强的超补偿效应。微藻叶绿素a指标在光恢复条件下都表现出显著的补偿效应,但温度恢复过程中叶绿素a含量与藻密度增长不同步,低温胁迫对恢复正常培养后微藻叶绿素a的形成产生了一定的负效应;铜绿微囊藻产毒株(912)在两种恢复模式下脱氢酶活性显著高于对照,产毒株(912)脱氢酶活性的补偿响应明显高于其它两种材料。共培养实验结果表明斜生栅藻同铜绿微囊藻产毒株(912)相比处于竞争劣势,而在同无毒株(469)的共培实验中,尽管连续正常培养情况下两者竞争能力差异不显著,但在恢复培养条件下斜生栅藻竞争能力显著高于后者。因此产毒型铜绿微囊藻低温和低光后的补偿生长效应以及对斜生栅藻的竞争优势可能是蓝藻爆发的内源性机制之一。     

An experimental study of strong reciprocity in bacteria

Strong reciprocity, whereby cooperators punish non-cooperators, may help to explain the evolutionary success of cooperative behaviours. However, theory suggests that selection for strong reciprocity can depend upon tight genetic linkage between cooperation and punishment, to avoid the strategy being outcompeted by non-punishing cooperators. We tested this hypothesis using experimental populations of the bacterium Pseudomonas aeruginosa, which cooperate by producing iron-scavenging siderophores and, in this context, punish non-cooperators with toxins. Consistent with theory, we show that cooperative punishers can indeed invade cheats, but only when the traits are tightly linked. These results emphasize that punishment is only likely to be favoured when the punishment itself leads to a direct or indirect fitness benefit to the actor.     

DEBtox theory and matrix population models as helpful tools in understanding the interaction between toxic cyanobacteria and zooplankton

Bioassays were performed to find out how field samples of the toxic cyanobacteria Microcystis aeruginosa affect Moina micrura, a cladoceran found in the tropical Jacarepagua Lagoon (Rio de Janeiro, Brazil). The DEBtox (Dynamic Energy Budget theory applied to toxicity data) approach has been proposed for use in analysing chronic toxicity tests as an alternative to calculating the usual safety parameters (NOEC, ECx). DEBtox theory deals with the energy balance between physiological processes (assimilation, maintenance, growth and reproduction), and it can be used to investigate and compare various hypotheses concerning the mechanism of action of a toxicant. Even though the DEBtox framework was designed for standard toxicity bioassays carried out with standard species (fish, daphnids), we applied the growth and reproduction models to M. micrura, by adapting the data available using a weight-length allometric relationship. Our modelling approach appeared to be very relevant at the individual level, and confirmed previous conclusions about the toxic mechanism. In our study we also wanted to assess the toxic effects at the population level, which is a more relevant endpoint in risk assessment. We therefore incorporated both lethal and sublethal toxic effects in a matrix population model used to calculate the finite rate of population change as a continuous function of the exposure concentration. Alongside this calculation, we constructed a confidence band to predict the critical exposure concentration for population health. Finally, we discuss our findings with regard to the prospects for further refining the analysis of ecotoxicological data.     

Large scale preparation of pure phycobiliproteins

This paper describes simple procedures for the purification of large amounts of phycocyanin and allophycocyanin from the cyanobacterium Microcystis aeruginosa. A homogeneous natural bloom of this organism provided hundreds of kilograms of cells. Large samples of cells were broken by freezing and thawing. Repeated extraction of the broken cells with distilled water released phycocyanin first, then allophycocyanin, and provides supporting evidence for the current models of phycobilisome structure. The very low ionic strength of the aqueous extracts allowed allophycocyanin release in a particulate form so that this protein could be easily concentrated by centrifugation. Other proteins in the extract were enriched and concentrated by large scale membrane filtration. The biliproteins were purified to homogeneity by chromatography on DEAE cellulose. Purity was established by HPLC and by N-terminal amino acid sequence analysis. The proteins were examined for stability at various pHs and exposures to visible light.Abbreviations A absorbance at wavelength in nanometers - DEAE cellulose diethylamino ethyl cellulose - HPLC high pressure liquid chromatography - UV ultraviolet     

The draft genome sequence of multidrug-resistant Pseudomonas aeruginosa strain CCBH4851, a nosocomial isolate belonging to clone SP (ST277) that is prevalent in Brazil

The high occurrence of nosocomial multidrug-resistant (MDR) microorganisms isconsidered a global health problem. Here, we report the draft genome sequence of aMDR Pseudomonas aeruginosa strain isolated in Brazil that belongsto the endemic clone ST277. The genome encodes important resistance determinantgenes and consists of 6.7 Mb with a G+C content of 66.86% and 6,347 predictedcoding regions including 60 RNAs.     

超声波对铜绿微囊藻超微结构和生理特性的影响

为了研究超声波对蓝藻细胞的影响,利用超声波（40W）处理200 mL铜绿微囊藻（Microcystis aeruginosa） 悬浮液20min,之后继续培养并于不同时间取样检测。检测悬浮藻细胞生物量发现其3d降低了97.84%;分别观察1、3、5d时沉降藻细胞超微结构变化,发现13d时细胞内脂质颗粒和藻青素颗粒增多、类囊体片层断裂、藻胆体脱落,5d时拟核区萎缩消失、细胞基础结构解体、胞质出现空洞、胞内结构颗粒降解;检测藻细胞光合放氧速率、叶绿素a （Chl.a）、超氧化物歧化酶（SOD）和过氧化氢酶（CAT）活性、膜透性以及跨膜ATP酶活性,发现光合放氧速率3d下降24.83%,Chl.a含量5d下降23.75%,超声组细胞SOD活性变化幅度比较大,但总体上活性降低,而CAT活性则表现为先增后减,活性始终大于对照组,同时胞内有机物渗出量增大,三种跨膜ATP酶活性（Na+/K+-ATPase、Mg2+-ATPase 和Ca2+-ATPase）均先升后降,并与膜透性变化相关。以上结果表明,超声波使铜绿微囊藻细胞沉降,并对其造成了胁迫,使部分藻细胞光合作用减弱,光合色素遭到损伤,细胞膜透性增大,甚至引起藻细胞程序性死亡。SOD活力的快速降低表明超声波使藻细胞内超氧离子（O2-）过量累积,从而对藻细胞造成氧化损伤,除此之外,超声波使藻细胞基础结构破坏、细胞内结构颗粒降解、细胞膜透性增大,这些都可能是致使部分铜绿微囊藻细胞死亡的重要原因。铜绿微囊藻细胞CAT以及跨膜ATP酶活性增大,表明藻细胞增强抗氧化酶活性以及离子调控和能量活动以抵御超声波的胁迫,而当胁迫随着时间减小后,细胞开始恢复生长和代谢,酶活力开始降低。     

Selective control of cyanobacteria by surfactin-containing culture broth of Bacillus subtilis C1

Of several types of chemical surfactants and biosurfactants, only the culture broth of Bacillus subtilis C1 containing surfactin at 10 mg l^–1 completely inhibited the growth of Microcystis aeruginosa, a bloom-forming cyanobacterium in highly eutrophic lakes. The broth with 10 mg surfactin l^–1 also removed 85% of the maximally grown M. aeruginosa (chlorophyll-a concentration, 1000 g l^–1) within 2 d, and the removal efficiency was enhanced by Ca²⁺ over 1 mM. The growth of Anabaena affinis, another bloom-forming cyanobacterium, was also inhibited about 70% with surfactin at 10 mg l^–1 broth. However, the effect of the broth was delayed over 3 d in the green algae, Chlorella vulgaris and Scenedesmus sp., and was negligible in a diatom, Navicula sp., indicating the potential for the selective control of cyanobacterial blooms.     

Viral clones from the GOS expedition with an unusual photosystem-I gene cassette organization

Cyanobacteria have a key role in marine photosynthesis, which contributes to the global carbon cycle and to the world oxygen supply. Genes encoding for photosystem-II (PSII) and photosystem-I (PSI) reaction centers are found in different cyanophage genomes, and it was suggested that the horizontal transfer of these genes might be involved in increasing phage fitness. We have further analyzed a rare viral Global Ocean Sampling (GOS) clone containing PSI genes. This clone contains the unusual PSI gene organization psaD->C->A, as opposed to the more frequently observed viral psaJF->C->A->B->K->E->D organization, and was detected only once in the GOS metagenome. Our analyses identified more occurrences with similar arrangement and indicate that this PSI viral gene organization (now psaD->C->A->B), although rare, is authentic and represents a new PSI gene arrangement.