Functional analysis of PilT from the toxic cyanobacterium Microcystis aeruginosa PCC 7806

The evolution of the microcystin toxin gene cluster in phylogenetically distant cyanobacteria has been attributed to recombination, inactivation, and deletion events, although gene transfer may also be involved. Since the microcystin-producing Microcystis aeruginosa PCC 7806 is naturally transformable, we have initiated the characterization of its type IV pilus system, involved in DNA uptake in many bacteria, to provide a physiological focus for the influence of gene transfer in microcystin evolution. The type IV pilus genes pilA, pilB, pilC, and pilT were shown to be expressed in M. aeruginosa PCC 7806. The purified PilT protein yielded a maximal ATPase activity of 37.5 +/- 1.8 nmol P(i) min(-1) mg protein(-1), with a requirement for Mg(2+). Heterologous expression indicated that it could complement the pilT mutant of Pseudomonas aeruginosa, but not that of the cyanobacterium Synechocystis sp. strain PCC 6803, which was unexpected. Differences in two critical residues between the M. aeruginosa PCC 7806 PilT (7806 PilT) and the Synechocystis sp. strain PCC 6803 PilT proteins affected their theoretical structural models, which may explain the nonfunctionality of 7806 PilT in its cyanobacterial counterpart. Screening of the pilT gene in toxic and nontoxic strains of Microcystis was also performed.     

The rbcX gene product promotes the production and assembly of ribulose-1,5-bisphosphate carboxylase/oxygenase of Synechococcus sp. PCC7002 in Escherichia coli

The operon encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the cyanobacterium Synechococcus sp. PCC7002 contains three rbc genes, rbcL, rbcX and rbcS, in this order. Introduction of translational frameshift into the rbcX gene resulted in a significant decrease in the production of large (RbcL) and small (RbcS) subunits of the Rubisco protein in Synechococcus sp. PCC7002 and in Escherichia coli. To investigate the function of the rbcX gene product (RbcX), we constructed the expression plasmid for the rbcX gene and examined the effects of RbcX on the recombinant Rubisco production in Escherichia coli. The coexpression experiments revealed that RbcX had marked effects on the production of large and small subunits of Rubisco without any significant influence on the mRNA level of rbc genes and/or the post-translational assembly of the Rubisco protein. The present rbcX coexpression system provides a novel and useful method for investigating the Rubisco maturation pathway.     

An extracellular glycoprotein is implicated in cell-cell contacts in the toxic cyanobacterium Microcystis aeruginosa PCC 7806

Microcystins are the most common cyanobacterial toxins found in freshwater lakes and reservoirs throughout the world. They are frequently produced by the unicellular, colonial cyanobacterium Microcystis aeruginosa; however, the role of the peptide for the producing organism is poorly understood. Differences in the cellular aggregation of M. aeruginosa PCC 7806 and a microcystin-deficient Delta mcyB mutant guided the discovery of a surface-exposed protein that shows increased abundance in PCC 7806 mutants deficient in microcystin production compared to the abundance of this protein in the wild type. Mass spectrometric and immunoblot analyses revealed that the protein, designated microcystin-related protein C (MrpC), is posttranslationally glycosylated, suggesting that it may be a potential target of a putative O-glycosyltransferase of the SPINDLY family encoded downstream of the mrpC gene. Immunofluorescence microscopy detected MrpC at the cell surface, suggesting an involvement of the protein in cellular interactions in strain PCC 7806. Further analyses of field samples of Microcystis demonstrated a strain-specific occurrence of MrpC possibly associated with distinct Microcystis colony types. Our results support the implication of microcystin in the colony specificity of and colony formation by Microcystis.     

铜绿微囊藻对紫外辐射的生理代谢响应

采用紫外(UV)滤膜过滤日光UV以及紫外灯添加UV的方法,研究了UV辐射对铜绿微囊藻Microcystis aeruginosa单细胞藻株PCC7806和群体藻XW01生长及生理代谢的影响。结果显示,在室内条件下低剂量UV辐射可促进群体微囊藻XW01生长;室外条件下与滤除了UV的光照相比,含有UV的完全日光更有利于微囊藻生长;而相同的UV辐射强度均导致单细胞株死亡,群体株显示了较强的UV抗性;日光中的UV可促进XW01合成抗氧化相关的超氧化物歧化酶(SOD)和过氧化氢酶(CAT)、促进胞外多糖的产生并形成较大的群体、促进UV屏障物质类菌孢素氨基酸(MAAs)和伪枝藻素(Scy)积累。这些生理代谢的改变,消除了阳光辐射中UV对微囊藻的伤害。研究的结果提示,自然条件下阳光中的UV有助于群体微囊藻生长。     

Identification of pilus-like structures and genes in Microcystis aeruginosa PCC7806

Four putative type IV pilus genes from the toxic, naturally transformable Microcystis aeruginosa PCC7806 were identified. Three of these genes were clustered in an arrangement which is identical to that from other cyanobacterial genomes. Type IV pilus-like appendages were also observed by electron microscopy.     

甲磺隆对微囊藻生长和光合系统的影响

研究选取了水体常见蓝藻优势种类——铜绿微囊藻(Microcystis aeruginosa PCC7806)作为研究对象, 了解磺酰脲类除草剂甲磺隆(Metsulfuron-methyl)对铜绿微囊藻生长和光合系统的影响。研究表明, 当甲磺隆浓度大于80 mg/L时, 对铜绿微囊藻的生长具有显著抑制。通过回归分析和Probit分析, 甲磺隆对铜绿微囊藻生长的EC₅₀为81.998 mg/L。细胞色素研究结果显示, 实验第6天, 各浓度处理下单位细胞内Chl.a和类胡萝卜素含量均低于对照组, 且当甲磺隆浓度为80 mg/L时, 单位细胞内类胡萝卜素含量显著低于对照组。快速叶绿素荧光诱导动力学变化结果分析显示, 实验第6天甲磺隆胁迫下单位反应中心捕获的用于电子传递的能量(ET₀/RC)及单位反应中心用于电子传递的量子产额(φE₀)受到显著抑制, 综合细胞色素变化结果显示, 甲磺隆能显著抑制光合系统反应中心电子受体侧电子性能。综上所述, 甲磺隆可能作用于光合系统反应中心电子受体侧, 从而对铜绿微囊藻光合系统造成影响。     

A mannan binding lectin is involved in cell-cell attachment in a toxic strain of Microcystis aeruginosa

Microcystin, a hepatotoxin that represents a serious health risk for humans and livestock, is produced by the bloom-forming cyanobacterium Microcystis aeruginosa in freshwater bodies worldwide. Here we describe the discovery of a lectin, microvirin (MVN), in M. aeruginosa PCC7806 that shares 33% identity with the potent anti-HIV protein cyanovirin-N from Nostoc ellipsosporum. Carbohydrate microarrays were employed to demonstrate the high specificity of the protein for high-mannose structures containing alpha(1-->2) linked mannose residues. Lectin binding analyses and phenotypic characterizations of MVN-deficient mutants suggest that MVN is involved in cell-cell recognition and cell-cell attachment of Microcystis. A binding partner of MVN was identified in the lipopolysaccharide fraction of M. aeruginosa PCC7806. MVN is differentially expressed in mutants lacking the hepatotoxin microcystin. Additionally, MVN-deficient mutants contain much lower amounts of microcystin than the wild-type cells. We discuss a possible functional correlation between microcystin and the lectin and possible implications on Microcystis morphotype formation. This study provides the first experimental evidence that microcystins may have an impact on Microcystis colony formation that is highly important for the competitive advantage of Microcystis over other phytoplankton species.     

黑暗限气条件下铜绿微囊藻细胞死亡的形态结构和生理生化变化

【目的】研究铜绿微囊藻细胞死亡过程中形态和生理生化变化,探讨蓝藻细胞死亡机制。【方法】通过黑暗限气处理模拟水华爆发后期水体环境,在处理后不同时间取样,对藻液的OD值,溶氧含量和pH值进行监测,使用透射电镜对细胞形态结构变化进行观察,通过胱天蛋白酶(Cysteine-dependent aspartate specificprotease,Caspase)活性检测、活性氧含量测定、末端脱氧核糖核酸转移酶介导的dUTP缺口末端标记(Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling,TUNEL)染色和琼脂糖凝胶电泳对处理后藻细胞的死亡生理进行研究。【结果】黑暗限气处理后,藻培养液pH值和溶解氧含量下降,处理12 h后藻液开始变黄,48 h后藻细胞全部死亡。电镜观察结果表明,藻细胞在黑暗限气处理所导致的死亡过程中出现空泡和类囊体、核糖体等内部结构解体但细胞壁仍保持完整等现象。活性氧含量和caspase活性检测表明,在藻细胞死亡过程中活性氧含量和caspase活性上升。TUNEL染色和琼脂糖凝胶电泳分析发现,藻细胞在死亡过程中DNA发生断裂和降解。【结论】铜绿微囊藻细胞在黑暗和限气处理中表现出和真核生物细胞程序性死亡相类似的死亡特征,这说明细胞死亡机制是保守的,原核细胞和真核细胞一样具有程序性死亡机制。     

The diameter and critical collapse pressure of gas vesicles in Microcystis are correlated with GvpCs of different length

In cyanobacteria the protein on the outside of the gas vesicle, GvpC, is characterised by the presence of a 33 amino acid residue repeat (33RR), which in some genera is highly conserved. The number of 33RRs correlates with the diameter of the gas vesicle and inversely with its strength. Gas vesicles isolated from Microcystis aeruginosa strain PCC 7806 were found to be wider and have a lower critical collapse pressure than those from Microcystis sp. strain BC 8401. The entire gas-vesicle gene cluster of the latter strain was sequenced and compared with the published sequence of the former: the sequences of nine of the ten gvp genes differed by only 1-5% between the two strains; the only substantial difference was in gvpC which in strain BC 8401 lacked a 99-nucleotide section encoding a 33RR. This observation further narrows the correlation of gas vesicle width to the number of 33RRs and suggests how Microcystis strains might be used in experimental manipulation of gas vesicle width and strength.     

Characterization of the 2-hydroxy-acid dehydrogenase McyI, encoded within the microcystin biosynthesis gene cluster of Microcystis aeruginosa PCC7806

The cyanobacterium Microcystis aeruginosa is widely known for its production of the potent hepatotoxin microcystin. This cyclic heptapeptide is synthesized non-ribosomally by the thio-template function of a large modular enzyme complex encoded within the 55-kb microcystin synthetase gene (mcy) cluster. The mcy gene cluster also encodes several stand-alone enzymes, putatively involved in the tailoring and export of microcystin. This study describes the characterization of the 2-hydroxy-acid dehydrogenase McyI, putatively involved in the production of d-methyl aspartate at position 3 within the microcystin cyclic structure. A combination of bioinformatics, molecular, and biochemical techniques was used to elucidate the structure, function, regulation, and evolution of this unique enzyme. The recombinant McyI enzyme was overexpressed in Escherichia coli and enzymatically characterized. The hypothesized native activity of McyI, the interconversion of 3-methyl malate to 3-methyl oxalacetate, was demonstrated using an in vitro spectrophotometric assay. The enzyme was also able to reduce alpha-ketoglutarate to 2-hydroxyglutarate and to catalyze the interconversion of malate and oxalacetate. Although NADP(H) was the preferred cofactor of the McyI-catalyzed reactions, NAD(H) could also be utilized, although rates of catalysis were significantly lower. The combined results of this study suggest that hepatotoxic cyanobacteria such as M. aeruginosa PCC7806 are capable of producing methyl aspartate via a novel glutamate mutase-independent pathway, in which McyI plays a pivotal role.     

Immunogold localisation of microcystins in cryosectioned cells of Microcystis

Insights into the origins, function(s), and fates of cyanobacterial toxins may be obtained by an understanding of their location within cyanobacterial cells. Here, we have localised microcystins in laboratory cultures of Microcystis PCC 7806 and PCC 7820 by immunogold labelling. Cryosectioning was used for immunoelectron microscopy since microcystins were extracted during the ethanol-based dehydration steps routinely used for sample preparation. Microcystins were specifically localised in the nucleoplasm and were associated with all major inclusions of the microcystin-producing strains Microcystis PCC 7806 (MC(+)) and Microcystis PCC 7820, and labelling was preferentially associated with the thylakoids and around polyphosphate bodies. A mutant strain of Microcystis PCC 7806 (MC(-)) which does not produce microcystins was used as a control. Distribution of total gold label within each cell region or associated with inclusions indicated that most of the cells' microcystin pool was associated with the thylakoids (69%, PCC 7806 (MC(+)); 78%, PCC 7820), followed by the nucleoplasmic region (19%, PCC 7806 (MC(+)); 12%, PCC 7820). Cryosectioning is a useful technique since it reduces the extraction of microcystins during sample preparation for electron microscopy.     

The gas vesicle gene cluster from Microcystis aeruginosa and DNA rearrangements that lead to loss of cell buoyancy

Microcystis aeruginosa is a planktonic unicellular cyanobacterium often responsible for seasonal mass occurrences at the surface of freshwater environments. An abundant production of intracellular structures, the gas vesicles, provides cells with buoyancy. A 8.7-kb gene cluster that comprises twelve genes involved in gas vesicle synthesis was identified. Ten of these are organized in two operons, gvpA(I)A(II)A(III)CNJX and gvpKFG, and two, gvpV and gvpW, are individually expressed. In an attempt to elucidate the basis for the frequent occurrence of nonbuoyant mutants in laboratory cultures, four gas vesicle-deficient mutants from two strains of M. aeruginosa, PCC 7806 and PCC 9354, were isolated and characterized. Their molecular analysis unveiled DNA rearrangements due to four different insertion elements that interrupted gvpN, gvpV, or gvpW or led to the deletion of the gvpA(I)-A(III) region. While gvpA, encoding the major gas vesicle structural protein, was expressed in the gvpN, gvpV, and gvpW mutants, immunodetection revealed no corresponding GvpA protein. Moreover, the absence of a gas vesicle structure was confirmed by electron microscopy. This study brings out clues concerning the process driving loss of buoyancy in M. aeruginosa and reveals the requirement for gas vesicle synthesis of two newly described genes, gvpV and gvpW.     

Effects of light on the microcystin content of Microcystis strain PCC 7806

Many cyanobacteria produce microcystins, hepatotoxic cyclic heptapeptides that can affect animals and humans. The effects of photosynthetically active radiation (PAR) on microcystin production by Microcystis strain PCC 7806 were studied in continuous cultures. Microcystis strain PCC 7806 was grown under PAR intensities between 10 and 403 micro mol of photons m(-2) s(-1) on a light-dark rhythm of 12 h -12 h. The microcystin concentration per cell, per unit biovolume and protein, was estimated under steady-state and transient-state conditions and on a diurnal timescale. The cellular microcystin content varied between 34.5 and 81.4 fg cell(-1) and was significantly positively correlated with growth rate under PAR-limited growth but not under PAR-saturated growth. Microcystin production and PAR showed a significant positive correlation under PAR-limited growth and a significant negative correlation under PAR-saturated growth. The microcystin concentration, as a ratio with respect to biovolume and protein, correlated neither with growth rate nor with PAR. Adaptation of microcystin production to a higher irradiance during transient states lasted for 5 days. During the period of illumination at a PAR of 10 and 40 micro mol of photons m(-2) s(-1), the intracellular microcystin content increased to values 10 to 20% higher than those at the end of the dark period. Extracellular (dissolved) microcystin concentrations were 20 times higher at 40 micro mol of photons m(-2) s(-1) than at 10 micro mol of photons m(-2) s(-1) and did not change significantly during the light-dark cycles at both irradiances. In summary, our results showed a positive effect of PAR on microcystin production and content of Microcystis strain PCC 7806 up to the point where the maximum growth rate is reached, while at higher irradiances the microcystin production is inhibited.     

Cysteine and serine protease-mediated proteolysis in body homogenate of a zooplankter, Moina macrocopa, is inhibited by the toxic cyanobacterium, Microcystis aeruginosa PCC7806

The paper describes the characterization of proteases in the whole body homogenate of Moina macrocopa, which can possibly be inhibited by the extracts of Microcystis aeruginosa PCC7806. With the use of oligopeptide substrates and specific inhibitors, we detected the activities of trypsin, chymotrypsin, elastase and cysteine protease. Cysteine protease, the predominant enzyme behind proteolysis of a natural substrate, casein, was partially purified by gel filtration. The substrate SDS-polyacrylamide gel electrophoresis of body homogenate revealed the presence of nine bands of proteases (17-72 kDa). The apparent molecular mass of an exclusive cysteine protease was 60 kDa, whereas of trypsin, it was 17-24 kDa. An extract of M. aeruginosa PCC7806 significantly inhibited the activities of trypsin, chymotrypsin and cysteine protease in M. macrocopa body homogenate at estimated IC(50) of 6- to 79-microg dry mass mL(-1). Upon fractionation by C-18 solid-phase extraction, 60% methanolic elute contained all the protease inhibitors, and these metabolites could be further separated by reverse-phase liquid chromatography. The metabolites inhibitory to M. macrocopa proteases also inhibited the corresponding class of proteases of mammalian/plant origin. The study suggests that protease inhibition may contribute to chemical interaction of cyanobacteria and crustacean zooplankton.     

Protein: A proposal for a standard parameter to express cyanobacterial biomass in laboratory experiments

This study aims to check if the protein content of a cyanobacterial culture is a reliable biomass parameter for cyanobacteria in laboratory experiments, and therefore can be proposed as a standard biomass parameter in culture work to facilitate comparison of results from different studies. For this purpose, the cyanobacteria Microcystis aeruginosa PCC 7806 and Planktothrix agardhii PT2 were grown in 10-L batch cultures with O2 medium and under iron-, nitrate- or phosphate-limited conditions. A linear correlation was found between protein and biovolume in all cultures during exponential growth. We conclude that protein is a suitable biomass parameter for cyanobacteria in laboratory experiments during balanced growth.     

Iron uptake by toxic and nontoxic strains of Microcystis aeruginosa

Iron uptake by microcystin-producing and non-microcystin-producing strains of Microcystis aeruginosa was investigated through short-term uptake assays. Although strain-specific differences were observed, the siderophore-independent Fe uptake kinetics were essentially similar (e.g., maximum uptake rates of 2.0 to 3.3 amol·cell(-1)·h(-1)) for the wild-type toxic strain PCC7806 and a genetically engineered mutant unable to produce microcystin.     

Identification of Pilus-Like Structures and Genes in Microcystis aeruginosa PCC7806

Four putative type IV pilus genes from the toxic, naturally transformable Microcystis aeruginosa PCC7806 were identified. Three of these genes were clustered in an arrangement which is identical to that from other cyanobacterial genomes. Type IV pilus-like appendages were also observed by electron microscopy.