Lake Erie Microcystis: Relationship between microcystin production, dynamics of genotypes and environmental parameters in a large lake

Cyanobacteria of genus Microcystis sp. have been commonly found in Lake Erie waters during recent summer seasons. In an effort to elucidate relationships between microcystin production, genotypic composition of Microcystis community and environmental parameters in a large lake ecosystem, we collected DNA samples and environmental data during a three-year (2003–2005) survey within Lake Erie and used the data to perform a series of correlation analyses. Cyanobacteria and Microcystis genotypes were quantified using quantitative real-time PCR (qPCR). Our data show that Microcystis in Lake Erie forms up to 42% of all cyanobacteria, and that Microcystis exists as a mixed population of potentially toxic and (primarily) non-toxic genotypes. In the entire lake, the total abundance of Microcystis as well as the abundance of microcystin-producing Microcystis is strongly correlated with the abundance of cyanobacteria suggesting that Microcystis is a significant component of the cyanobacterial community in Lake Erie during summer seasons. The proportion of total Microcystis of all cyanobacteria was strongly linked to the microcystin concentrations, while the percentage of microcystin-producing genotypes within Microcystis population showed no correlation with microcystin concentrations. Correlation analysis indicated that increasing total phosphorus concentrations correlate strongly with increasing microcystin concentrations as well as with the total abundance of Microcystis and microcystin-producing Microcystis.     

Genetic analysis of the peptide synthetase genes for a cyclic heptapeptide microcystin in Microcystis spp.

Peptide-synthetase-encoding DNA fragments were isolated by a PCR-based approach from the chromosome of Microcystis aeruginosa K-139, which produces cyclic heptapeptides, 7-desmethylmicrocystin-LR and 3,7-didesmethylmicrocystin-LR. Three open reading frames (mcyA, mcyB, mcyC) encoding microcystin synthetases were identified in the gene cluster. Sequence analysis indicated that McyA (315 kDa) consists of two modules with an N-methylation domain attached to the first and an epimerization domain attached to the second; McyB (242 kDa) has two modules, and McyC (147 kDa) contains one module with a putative C-terminal thioesterase domain. Conserved amino acid sequence motifs for ATP binding, ATP hydrolysis, adenylate formation, and 4'-phosphopantetheine attachment were identified by sequence comparison with authentic peptide synthetase. Insertion mutations in mcyA, generated by homologous recombination, abolished the production of both microcystins in M. aeruginosa K-139. Primer extension analysis demonstrated light-dependent mcy expression. Southern hybridization and partial DNA sequencing analyses of six microcystin-producing and two non-producing Microcystis strains suggested that the microcystin-producing strains contain the mcy gene and the non-producing strains can be divided into two groups, those possessing no mcy genes and those with mcy genes.     

Genetic variation of the bloom-forming Cyanobacterium Microcystis aeruginosa within and among lakes: implications for harmful algal blooms

To measure genetic variation within and among populations of the bloom-forming cyanobacterium Microcystis aeruginosa, we surveyed a suite of lakes in the southern peninsula of Michigan that vary in productivity (total phosphorus concentrations of approximately 10 to 100 microg liter(-1)). Survival of M. aeruginosa isolates from lakes was relatively low (i.e., mean of 7% and maximum of 30%) and positively related to lake total phosphorus concentration (P = 0.014, r2 = 0.407, n = 14). In another study (D. F. Raikow, O. Sarnelle, A. E. Wilson, and S. K. Hamilton, Limnol. Oceanogr. 49:482-487, 2004), survival rates of M. aeruginosa isolates collected from an oligotrophic lake (total phosphorus of approximately 10 mug liter(-1) and dissolved inorganic nitrogen:total phosphorus ratio of 12.75) differed among five different medium types (G test, P of <0.001), with higher survival (P = 0.003) in low-nutrient media (28 to 37% survival) than in high-nutrient media. Even with the relatively low isolate survivorship that could select against detecting the full range of genetic variation, populations of M. aeruginosa were genetically diverse within and among lakes (by analysis of molecular variance, Phi(sc) = 0.412 [Phi(sc) is an F-statistic derivative which evaluates the correlation of haplotypic diversity within populations relative to the haplotypic diversity among all sampled populations], P = 0.001), with most clones being distantly related to clones collected from lakes directly attached to Lake Michigan (a Laurentian Great Lake) and culture collection strains collected from Canada, Scotland, and South Africa. Ninety-one percent of the 53 genetically unique M. aeruginosa clones contained the microcystin toxin gene (mcyA). Genotypes with the toxin gene were found in all lakes, while four lakes harbored both genotypes possessing and genotypes lacking the toxin gene.     

Diversity of Microcystin Genes within a Population of the Toxic Cyanobacterium Microcystis spp. in Lake Wannsee (Berlin, Germany)

In order to find out how many genotypes determine microcystin production of Microcystis spp. in field populations, single colonies (clones) were sampled from Lake Wannsee (Berlin, Germany), characterized morphologically, and subsequently analyzed by PCR for a region within the mcyB gene encoding the activation of one amino acid during microcystin biosynthesis. The different morphospecies varied considerably in the proportion of microcystin-producing genotypes. Most colonies (73%) of M. aeruginosa contained this gene whereas only 16% of the colonies assigned to M. ichthyoblabe and no colonies of M. wesenbergii gave a PCR product of the mcyB gene. Restriction fragment length polymorphism revealed seven restriction profiles showing low variability in nucleotide sequence within each restriction type (0.4-4%) and a low to high variability (1.6-38%) between restriction types. In addition, the sequences of amino acids within the mcyB gene were analyzed to compare the specificity of the amino acid activation during microcystin biosynthesis between restriction types and with the occurrence of amino acids in microcystin variants as detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Most of the microcystin-producing colonies showed high similarity in the sequence of amino acids and contained microcystin-LR (LR refers to leucine and arginine in the variable positions of the heptapeptide), microcystin-RR, and microcystin-YR, as well as other variants in minor concentrations. It is concluded that the gene product found for most of the microcystin-producing colonies in the lake is rather unspecific and the diversity of microcystin variants in the lake results from activation of various amino acids during microcystin biosynthesis in the same genotypes.     

Abundance of active and inactive microcystin genotypes in populations of the toxic cyanobacterium Planktothrix spp

To investigate the abundance of active and inactive microcystin genotypes in populations of the filamentous cyanobacterium Planktothrix spp., individual filaments were grown as clonal strains in the laboratory and analysed for microcystin synthetase (mcy) genes and microcystin. Twenty-three green-pigmented strains of P. agardhii originating mostly from shallow water bodies fell into two groups, those possessing mcyA and those lacking mcyA. In contrast, all of the 49 strains that were assigned to the red-pigmented P. rubescens contained mcyA. One strain of P. agardhii and eight strains of P. rubescens contained the total microcystin synthetase gene cluster but were found inactive in microcystin synthesis. To investigate the natural abundance of inactive mcy genotypes in P. rubescens individual filaments sampled from Lake Irrsee and Lake Mondsee (Austria) were analysed directly for the presence of mcyA and microcystin by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. All filaments assigned to P. rubescens contained mcyA. The proportion of inactive microcystin genotypes in populations with a low (Irrsee) or high density (Mondsee) of P. rubescens was 5% and 21%, each. The results of this study demonstrate that P. rubescens typically contain mcy genes whereas P. agardhii have a patchy distribution of mcy genes. In both species microcystin producers co-occur with non-microcystin producers due to the absence/inactivation of mcy genes.     

Polyketide synthase gene coupled to the peptide synthetase module involved in the biosynthesis of the cyclic heptapeptide microcystin

The peptide synthetase gene operon, which consists of mcyA, mcyB, and mcyC, for the activation and incorporation of the five amino acid constituents of microcystin has been identified [T. Nishizawa et al. (1999) J. Biochem. 126, 520-529]. By sequencing an additional 34 kb of DNA from microcystin-producing Microcystis aeruginosa K-139, we identified the residual microcystin synthetase gene operon, which consists of mcyD, mcyE, mcyF, and mcyG, in the opposite orientation to the mcyABC operon. McyD consisted of two polyketide synthase modules, and McyE contained a polyketide synthase module at the N-terminus and a peptide synthetase module at the C-terminus. McyF was found to exhibit similarity to amino acid racemase. McyG consisted of a peptide synthetase module at the N-terminus and a polyketide synthase at the C-terminus. The microcystin synthetase gene cluster was conserved in another microcystin-producing strain, Microcystis sp. S-70, which produces Microcystin-LR, -RR, and -YR. Insertional mutagenesis of mcyA, mcyD, or mcyE in Microcystis sp. S-70 abolished microcystin production. In conclusion, the mcyDEFG operon is presumed to be responsible for 3-amino-9-methoxy-2,6, 8-trimethyl-10-phenyldeca-4,6-dienoic acid (Adda) biosynthesis, and the incorporation of Adda and glutamic acid into the microcystin molecule.     

Evidence of the cost of the production of microcystins by Microcystis aeruginosa under differing light and nitrate environmental conditions

The cyanobacterium Microcystis aeruginosa is known to proliferate in freshwater ecosystems and to produce microcystins. It is now well established that much of the variability of bloom toxicity is due to differences in the relative proportions of microcystin-producing and non-microcystin-producing cells in cyanobacterial populations. In an attempt to elucidate changes in their relative proportions during cyanobacterial blooms, we compared the fitness of the microcystin-producing M. aeruginosa PCC 7806 strain (WT) to that of its non-microcystin-producing mutant (MT). We investigated the effects of two light intensities and of limiting and non-limiting nitrate concentrations on the growth of these strains in monoculture and co-culture experiments. We also monitored various physiological parameters, and microcystin production by the WT strain. In monoculture experiments, no significant difference was found between the growth rates or physiological characteristics of the two strains during the exponential growth phase. In contrast, the MT strain was found to dominate the WT strain in co-culture experiments under favorable growth conditions. Moreover, we also found an increase in the growth rate of the MT strain and in the cellular MC content of the WT strain. Our findings suggest that differences in the fitness of these two strains under optimum growth conditions were attributable to the cost to microcystin-producing cells of producing microcystins, and to the putative existence of cooperation processes involving direct interactions between these strains.     

温度和铜绿微囊藻毒性对萼花臂尾轮虫生活史参数的影响

文章开展了25℃下两个品系铜绿微囊藻(有毒与无毒)不同浓度对萼花臂尾轮虫生活史的影响研究, 及在5个温度下不同浓度有毒铜绿微囊藻对萼花臂尾轮虫生活史影响的研究。结果表明铜绿微囊藻毒性、浓度及二者交互作用对轮虫生活史参数净生殖率(R₀; F=31.83, P<0.01; F=30.36, P<0.01; F=13.51, P<0.01)、内禀增长率(r_m; F=34.67, P<0.01; F=18.73, P<0.01; F=12.99, P<0.01)均有显著影响; 温度、铜绿微囊藻浓度及二者交互作用对轮虫生活史参数净生殖率、内禀增长率也均有显著影响。无毒铜绿微藻在低浓度(1×104 cells/mL)下对轮虫种群有促进作用, 可作为轮虫食物来源, 但缺乏脂肪酸等营养物质, 食物质量比蛋白核小球藻低; 在高浓度(1×105和5×105 cells/mL)下轮虫摄食无毒铜绿微囊藻机率变大, 整体食物品质下降, 对轮虫有抑制作用。有毒铜绿微囊藻对轮虫种群的抑制作用更加明显, 微囊藻浓度升高, 净生殖率和内禀增长率显著下降。研究结果还表明30℃和35℃高温下轮虫生长繁殖变快, 世代时间缩短。在高温(30℃和35℃)环境下, 铜绿微囊藻浓度升高对轮虫抑制作用更加明显。     

Distribution of microcystin-producing and non-microcystin-producing Microcystis sp. in European freshwater bodies: detection of microcystins and microcystin genes in individual colonies

Microcystis is a well-known cyanobacterial genus frequently producing hepatotoxins named microcystins. Toxin production is encoded by microcystin genes (mcy). This study aims (i) to relate the mcy occurrence in individual colonies to the presence of microcystin, (ii) to assess whether morphological characteristics (morphospecies) are related to the occurrence of mcy genes, and (iii) to test whether there are geographical variations in morphospecies specificity and abundance of mcy genes. Individual colonies of nine different European countries were analysed by (1) morphological characteristics, (2) PCR to amplify a gene region within mcyA and mcyB indicative for microcystin biosynthesis, (3) matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) to detect microcystins. Almost one hundred percent of the colonies predicted to produce microcystins by PCR analysis were found to contain microcystins. A high similarity in microcystin variants in the different colonies selected from lakes across Europe was demonstrated. The different morphospecies varied in the frequency with which they contained mcy genes. Most colonies (>75%) of M. aeruginosa and M. botrys contained the mcy genes, whereas < or = 20% of the colonies identified as M. ichthyoblabe and M. viridis gave a PCR product of the mcy genes. No colonies of M. wesenbergii gave a PCR product of either mcy gene. In addition, a positive relationship was found between the size of the colony and the frequency of those containing the mcy genes. It is concluded that the analysis of morphospecies is indicative for microcystin production, although the quantitative analysis of microcystin concentrations in water remains indispensable for hazard control.     

The abundance of microcystin-producing genotypes correlates positively with colony size in Microcystis sp. and determines its microcystin net production in Lake Wannsee

The working hypotheses tested on a natural population of Microcystis sp. in Lake Wannsee (Berlin, Germany) were that (i) the varying abundance of microcystin-producing genotypes versus non-microcystin-producing genotypes is a key factor for microcystin net production and (ii) the occurrence of a gene for microcystin net production is related to colony morphology, particularly colony size. To test these hypotheses, samples were fractionated by colony size with a sieving procedure during the summer of 2000. Each colony size class was analyzed for cell numbers, the proportion of microcystin-producing genotypes, and microcystin concentrations. The smallest size class of Microcystis colonies (<50 microm) showed the lowest proportion of microcystin-producing genotypes, the highest proportion of non-microcystin-producing cells, and the lowest microcystin cell quotas (sum of microcystins RR, YR, LR, and WR). In contrast, the larger size classes of Microcystis colonies (>100 microm) showed the highest proportion of microcystin-producing genotypes, the lowest proportion of non-microcystin-producing cells, and the highest microcystin cell quotas. The microcystin net production rate was nearly one to one positively related to the population growth rate for the larger colony size classes (>100 microm); however, no relationship could be found for the smaller size classes. It was concluded that the variations found in microcystin net production between colony size classes are chiefly due to differences in genotype composition and that the microcystin net production in the lake is mainly influenced by the abundance of the larger (>100- microm) microcystin-producing colonies.     

Variation of microcystin content of microcystis aeruginosa relative to medium N:P ratio and growth stage

Changes in the microcystin content of Microcystis aeruginosa UTEX 2388 were investigated at several N:P ratios of the medium and various growth stages. Under the P-fixed condition, the microcystin content of the cells changed with different medium N:P ratios, with the highest at 2748 microg g-1 at a N:P ratio of 16 after incubation for 7 d. The microcystin content of M. aeruginosa exhibited a high correlation with the total N content regardless of an N-fixed or P-fixed culture. When the N:P ratio of the medium was fixed to 16 : 1, the microcystin content of M. aeruginosa at various growth stages was highest at 2191 microg g-1 after an incubation of 4 d and the chlorophyll-a content showed a similar tendency. There was a highly significant relationship between the microcystin content of M. aeruginosa and the chlorophyll-a concentration in the culture during the incubation. Accordingly, the microcystin content of M. aeruginosa during incubation can be easily estimated and monitored by measuring the in vivo fluorescence changes in the culture.     

Quantitative real-time PCR for determination of microcystin synthetase e copy numbers for microcystis and anabaena in lakes

Cyanobacterial mass occurrences in freshwater lakes are generally formed by Anabaena, Microcystis, and Planktothrix, which may produce cyclic heptapeptide hepatotoxins, microcystins. Thus far, identification of the most potent microcystin producer in a lake has not been possible due to a lack of quantitative methods. The aim of this study was to identify the microcystin-producing genera and to determine the copy numbers of microcystin synthetase gene E (mcyE) in Lake Tuusulanj?rvi and Lake Hiidenvesi in Finland by quantitative real-time PCR. The microcystin concentrations and cyanobacterial cell densities of these lakes were also determined. The microcystin concentrations correlated positively with the sum of Microcystis and Anabaena mcyE copy numbers from both Lake Tuusulanj?rvi and Lake Hiidenvesi, indicating that mcyE gene copy numbers can be used as surrogates for hepatotoxic Microcystis and ANABAENA: The main microcystin producer in Lake Tuusulanj?rvi was Microcystis spp., since average Microcystis mcyE copy numbers were >30 times more abundant than those of ANABAENA: Lake Hiidenvesi seemed to contain both nontoxic and toxic Anabaena as well as toxic Microcystis strains. Identifying the most potent microcystin producer in a lake could be valuable for designing lake restoration strategies, among other uses.     

Diversity of microcystin-producing cyanobacteria in spatially isolated regions of Lake Erie

The diversity of microcystin-producing cyanobacteria in the western basin of Lake Erie was studied using sequence analysis of mcyA gene fragments. Distinct populations of potentially toxic Microcystis and Planktothrix were found in spatially isolated locations. This study highlights previously undocumented diversity of potentially toxic cyanobacteria.     

Variation of Microcystin Content of Cyanobacterial Blooms and Isolated Strains in Lake Grand-Lieu (France)

Abstract Cyanobacterial blooms were sampled at five locations in Lake Grand-Lieu on seven different occasions during May–October 1994. Strains of Microcystis aeruginosa and Anabaena circinalis were isolated from the samples. Microcystins were detected in freeze-dried field samples and the isolated strains by HPLC. The toxins were present in the blooms sampled between June and October. The microcystin content in the blooms varied with site and time, from undetectable concentrations to 0.23 mg g⁻¹. The highest concentrations of microcystin were found in blooms sampled in September. Microcystin-LR and microcystins with retention times close to the retention time of [Dha⁷]microcystin-RR (probably varieties of microcystin-RR) were found in the field samples. Sixteen of the 98 isolated M. aeruginosa strains and 2 of the 24 A. circinalis strains produced microcystins. The total amount of microcystins varied from undetectable concentrations to 5.06 mg g⁻¹ in the M. aeruginosa isolates, and from undetectable concentrations to 1.86 mg g⁻¹ in the A. circinalis strains. Microcystin-LR was the main toxin found in strains of M. aeruginosa, but was not present in strains of A. circinalis. Both microcystin-producing strains and strains that did not produce microcystin coexisted in the bloom samples. Received: 23 January 1997; Accepted: 25 March 1997     

铜绿微囊藻对轮虫生命表参数和表型特征的影响

为评价铜绿微囊藻的有毒(Microcystin-producing Microcystis aeruginosa)、无毒(Microcystin-free M. aeruginosa)品系对轮虫种群增长和表型特征的影响, 研究探讨了萼花臂尾轮虫(Brachionus calyciflorus)在不同微囊藻溶液中的生活史参数及形态变化。实验中各处理组单位体积总含碳量为(20.61±0.15) g C/mL, 以使轮虫获得等碳量的食物供应。实验组轮虫分别用蛋白核小球藻(Chlorella pyrenoidosa)、斜生栅藻(Scenedesmus obliquus)、有毒和无毒微囊藻溶液单独投喂, 并用有毒、无毒蓝藻菌分别与不同绿藻的混合液投喂。生命表实验结果表明, 不同微囊藻混合液投喂的轮虫净生殖率R₀ (F=102.71, df=32, P<0.001)、世代时间T (F=17.05, df=32, P<0.001)和内禀增长率r_m(F=18.89, df=32, P<0.001)与对照组相比降低1.36%—210.34%。侧棘刺长(F=28.18, df=65, P<0.001)和游泳速度(F=181.69, df=65, P<0.001)下降2.63%—39.07%, 轮虫体长(F=690.04, df=65, P<0.001)变化显著。与绿藻投喂的轮虫参数值相比, 轮虫的生命表参数和表型特征变化规律随微囊藻溶液浓度改变。萼花臂尾轮虫受到铜绿微囊藻胁迫时, 生长繁殖受到抑制并通过改变自身形态以抵御不利的生存环境。     

太湖水华期间有毒和无毒微囊藻种群丰度的动态变化

采用荧光定量PCR技术分析太湖3个湖区(梅梁湾、贡湖湾和湖心)水体中有毒和无毒微囊藻基因型丰度及有毒微囊藻比例的季节变化(2010年4-9月),并与环境因子进行统计分析。结果表明,有毒微囊藻基因型丰度及所占比例存在季节和空间差异:从4-8月,有毒微囊藻基因型丰度及其比例呈逐渐增加趋势,到9月开始下降;梅梁湾水体中有毒微囊藻基因型丰度及其比例高于贡湖湾和湖心。梅梁湾、贡湖湾和湖心有毒微囊藻在微囊藻种群中的比例变化范围分别为(26.2±0.8)%-(64.3±2.2)%、(4.4±0.2)%-(22.1±1.8)%和(10.4±0.4)%-(20.6±1.5)%。相关分析结果表明,有毒微囊藻丰度、总微囊藻丰度和叶绿素a浓度呈极显著正相关(P<0.01),均与温度呈显著正相关(P<0.05);有毒微囊藻比例与磷浓度呈显著正相关(P<0.05),与温度呈极显著正相关(P<0.01)。研究结果表明,温度和磷浓度是决定太湖有毒微囊藻种群丰度及其比例的关键因子。     

Microcystins in natural blooms and laboratory cultured Microcystis aeruginosa from Laguna de Bay,Philippines

Laguna de Bay, the largest freshwater lake in the Philippines, experiences periodic blooms of the cyanobacteria Microcystis aeruginosa. Blooms of these cyanobacteria in 1996, 1998 and 1999 were sampled. HPLC and MALDI-TOF mass spectrometry were used to analyze for microcystins. A total of 16 structural variants of the toxin were isolated from the samples with microcystin LR (MC-LR) as the most abundant variant in the samples from 1996 and 1999 making up 77 to 85% of the total, respectively. MC-RR was the dominant variant in the 1998 bloom making up 38%. The samples from 1996 had the highest total toxin concentration (4049 microg g(-1)) followed by those from 1998 (1577 microg g(-1)) and 1999 (649 microg g(-1)). A strain of M. aeruginosa previously isolated from the lake was also cultured in the laboratory under different nitrogen concentrations (1, 3 and 6 mg L(-1)) and elevated phosphorus concentration (0.5 mg L(-1)) to determine the influence of these factors on toxin production. A total of 9 different structural variants of microcystin were isolated from the laboratory cultures with MC-LR consisting more than 75% of the total in all treatments. No significant differences in the total toxin concentration as well as the % distribution of the different variants among treatments were observed. However, the strain of M. aeruginosa cultured in the laboratory had from 3 to 20 times higher total microcystin than those harvested from the lake.     

Quantitative Real-Time PCR for Determination of Microcystin Synthetase E Copy Numbers for Microcystis and Anabaena in Lakes

Cyanobacterial mass occurrences in freshwater lakes are generally formed by Anabaena, Microcystis, and Planktothrix, which may produce cyclic heptapeptide hepatotoxins, microcystins. Thus far, identification of the most potent microcystin producer in a lake has not been possible due to a lack of quantitative methods. The aim of this study was to identify the microcystin-producing genera and to determine the copy numbers of microcystin synthetase gene E (mcyE) in Lake Tuusulanjärvi and Lake Hiidenvesi in Finland by quantitative real-time PCR. The microcystin concentrations and cyanobacterial cell densities of these lakes were also determined. The microcystin concentrations correlated positively with the sum of Microcystis and Anabaena mcyE copy numbers from both Lake Tuusulanjärvi and Lake Hiidenvesi, indicating that mcyE gene copy numbers can be used as surrogates for hepatotoxic Microcystis and Anabaena. The main microcystin producer in Lake Tuusulanjärvi was Microcystis spp., since average Microcystis mcyE copy numbers were >30 times more abundant than those of Anabaena. Lake Hiidenvesi seemed to contain both nontoxic and toxic Anabaena as well as toxic Microcystis strains. Identifying the most potent microcystin producer in a lake could be valuable for designing lake restoration strategies, among other uses.     

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 diversity and distribution of toxigenic Microcystis spp. in present day and archived pelagic and sediment samples from Lake Erie

The reoccurrence of significant cyanobacterial blooms in Lake Erie during the last 13 years has raised questions concerning the long-term persistence of microcystin-producing cyanobacteria and the presence of natural sediment reservoirs for potentially toxic cyanobacteria in this large lake system. To address these questions, we analyzed phytoplankton and sediment samples which were collected and preserved in the 1970s as well as samples collected in 2004 from locations within Lake Erie. The identification of microcystin-producing cyanobacteria in Lake Erie was examined via PCR amplification of the mcyA gene fragment. Based on the high % sequence similarity, the mcyA sequences from all 1970s phytoplankton and sediment samples were determined to belong to Microcystis spp., in spite of reports suggesting that Lake Erie was dominated by filamentous cyanobacteria in the 1970s. In sediment samples from 2004, signature genes for Microcystis were distributed and preserved not only in the surface sediments but also up to 10–12 cm in depth. Based on cell quantities determined by a quantitative polymerase chain reaction (qPCR) method, 0.18% of eubacteria in the sediments were Microcystis cells, of which 4.8% were potential microcystin producers. In combination with experiments showing that Microcystis cells can be cultured from Lake Erie surface sediments, this paper demonstrates the potential for these sediments to act as a reservoir for pelagic Microcystis populations and that the composition of the population of microcystin-producing cyanobacteria in Lake Erie has not changed remarkably since the 1970s.