Diversity of coexisting <Emphasis Type="Italic">Planktothrix</Emphasis> (Cyanobacteria) chemotypes deduced by mass spectral analysis of microystins and other oligopeptides

Cyanobacteria are reported to produce secondary metabolites of which toxic and bioactive peptides are of scientific and public interest. Many peptides are synthesized by the non-ribosomal peptide synthesis pathway and their presence is a stable feature of individual clones. We isolated 18 clonal strains of Planktothrix from a single water sample from lake Maxsee near Berlin and analyzed them by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, HPLC, and PCR for their production of peptides and the presence of microcystin synthetase genes. Microcystins could be detected in seven of the strains with considerable variability of contents and numbers of structural variants. Other known peptides like anabaenopeptins B and E/F, microviridin I, and prenylagaramide B and new variants of known peptide classes like aeruginosins and cyanopeptolins were detected in some strains while lacking in others. The 18 strains represented 15 chemotypes with respect to their peptide patterns. In contrast, all strains were morphologically very similar with respect to cell dimensions and pigmentation. Given the diversity of chemotypes among the randomly selected isolates, an immense diversity of chemotypes in the entire population can be assumed.     

Heterogeneity of S-layer proteins from aggregating and non-aggregating <Emphasis Type="Italic">Lactobacillus kefir</Emphasis> strains

Since the presence of S-layer protein conditioned the autoaggregation capacity of some strains of Lactobacillus kefir, S-layer proteins from aggregating and non-aggregating L. kefir strains were characterized by immunochemical reactivity, MALDI-TOF spectrometry and glycosylation analysis. Two anti-S-layer monoclonal antibodies (Mab5F8 and Mab1F8) were produced; in an indirect enzyme-linked immunosorbent assay Mab1F8 recognized S-layer proteins from all L. kefir tested while Mab5F8 recognized only S-layer proteins from aggregating strains. Periodic Acid-Schiff staining of proteins after polyacrylamide gel electrophoresis under denaturing conditions revealed that all L. kefir S-layer proteins tested were glycosylated. Growth of bacteria in the presence of the N-glycosylation inhibitor tunicamycin suggested the presence of glycosydic chains O-linked to the protein backbone. MALDI-TOF peptide map fingerprint for S-layer proteins from 12 L. kefir strains showed very similar patterns for the aggregating strains, different from those for the non-aggregating ones. No positive match with other protein spectra in MSDB Database was found. Our results revealed a high heterogeneity among S-layer proteins from different L. kefir strains but also suggested a correlation between the structure of these S-layer glycoproteins and the aggregation properties of whole bacterial cells.     

Detection of potential microcystin-producing cyanobacteria in Brazilian reservoirs with a mcyB molecular marker

One of the most serious problems related to water eutrophication is the occurrence of increasingly frequent blooms of toxic cyanobacteria in freshwater ecosystems. Microcystin (MCYST) molecular markers may be used for the detection of toxic cyanobacteria, both cultivated strains and environmental samples, independently of their taxonomic category and production of the toxin at the moment of analysis. Sixty Microcystis spp. strains from 15 water reservoirs of south, southeastern and northeastern Brazil were analyzed by polymerase chain reaction (PCR) with oligonucleotide primers for mcyB gene of the operon that encodes a microcystin synthetase. It was found out that the presence of a unique amplified product of approximately 780 bp in 18 strains, indicated the presence of the microcystin-producing genotype. There was correspondence between the presence of the mcyB gene and microcystin determined by ELISA. Eight reservoirs contained toxic strains, two of these reservoirs being used mainly for public water supply. The coexistence of a mixture of toxic and non-toxic genotypes in populations of several reservoirs was found. Thus, it is evident that Microcystis populations present in blooms compose a mosaic, with genetically different individuals within the same population, each one, possibly, with its own tolerance to environmental factors and with distinct toxicity potential.     

广东省水库微囊藻的产毒特征和ITS序列的遗传多样性分析

为了解广东省水库微囊藻的产毒特征和ITS 序列的遗传多样性,从广东省供水水库中分离得到28 株微囊藻(Microcystisspp.),对它们的产毒特征和15 株微囊藻的ITS 序列进行了分析.高效液相色谱(HPLC)和微囊藻毒素合成酶基因mcyE 的检测结果表明,广东省水库中的微囊藻以产毒藻株占优势,微囊藻毒素的主要类型为MC-RR.广东省15 株藻株的ITS 序列相似性大于93.2%,在用相邻法(NJ)构建的系统树上,不同形态的种和不同地理区域的藻株没有区分开,产毒和非产毒藻株没有形成独立分支.这说明微囊藻ITS 序列的遗传多样性较低,ITS 序列和mcyE 存在没有相关性,表型不能够反映藻株的进化关系.因此,有必要将藻类传统分类方法与分子方法结合起来对蓝藻进行重新分类.     

The gene <Emphasis Type="Italic">bap</Emphasis>, involved in biofilm production,is present in <Emphasis Type="Italic">Staphylococcus</Emphasis> spp. strains from nosocomial infections

This study analyzed ten strains of coagulase-negative staphylococci (CNS) involved in nosocomial infections in three Brazilian hospitals. Their antibiotic susceptibility profile showed that most strains exhibited multiple antibiotic resistance and possessed the mecA gene. The ability of these strains to adhere to polystyrene microtiter plates was also tested and nine of them proved to be biofilm producers at least in one of the three conditions tested: growth in TSB, in TSB supplemented with NaCl, or in TSB supplemented with glucose. The presence of the bap gene, which codes for the biofilm-associated protein (Bap), was investigated in all ten strains by PCR. AU strains were bop-positive and DNA sequencing experiments confirmed that the fragments amplified were indeed part of a bap gene. The presence of the icaA gene, one of the genes involved in polysaccharide intercellular adhesin (PIA) formation, was also detected by PCR in eight of the ten strains tested. The two icaA-negative strains were either weak biofilm producer or no biofilm producer, although they were bop-positive. To our knowledge, this is the first report demonstrating the presence of the bap gene in nosocomial isolates of CNS, being also the first report on the presence of this gene in Staphylococcus haemolyticus and S. cohnii. Electronic Supplementary Material  Supplementary material is available for this article at and is accessible for authorized users.     

Influence of <Emphasis Type="Italic">Daphnia</Emphasis> infochemicals on functional traits of <Emphasis Type="Italic">Microcystis</Emphasis> strains (Cyanobacteria)

We conducted a laboratory experiment to investigate the influence of Daphnia infochemicals on growth rate, microcystin production, colony formation and cell size of eight Microcystis strains isolated from two lakes. The strains were characterized genetically by their 16S-23S rDNA ITS sequence. The experiment was composed of four treatments: (1) a control using filtered WC medium, (2) addition of Scenedesmus obliquus culture medium filtrate, (3) addition of Daphnia magna culture medium filtrate and (4) addition of sodium octyl sulphate, a commercially available Daphnia infochemical. Our results showed that sympatric strains differed strongly for the measured functional traits, while no correlations between traits were found. Between-strain differences in growth rate, microcystin production, colony formation and cell size were generally larger than the differences in phenotypes observed between treatments. Despite this, several strains reacted to the infochemicals by changing functional trait values. Daphnia culture medium filtrate and, to a lesser extent, sodium octyl sulphate had a negative influence on the growth rate of half of the strains and stimulated microcystin production in one strain, but the latter effect was not Daphnia-specific as Scenedesmus culture medium filtrate had the same effect. Daphnia culture medium filtrate also induced colony formation in one strain. Our data suggest that Daphnia infochemicals generally have a weak influence on growth rate, microcystin production and colony formation of Microcystis strains as compared to the inter-strain variability, while existing inducible effects are highly strain-specific.     

Characterization of freshwater benthic biofilm‐forming Hydrocoryne (Cyanobacteria) isolates from Antarctica

The aims of this work were to study cyanobacterial isolates resembling the genus Hydrocoryne using a combination of morphology and phylogeny of 16S rRNA and nifH sequences and to investigate genes involved in cyanotoxin and protease inhibitor production. Four new cyanobacterial strains, isolated from biofilm samples collected from King George Island, Antarctica, were studied. In terms of morphology, these new strains share traits similar to true Anabaena morphotypes (benthic ones), whereas phylogenetic analysis of their 16S rRNA gene sequences grouped them with the sequence of the type species Hydrocoryne spongiosa (H. Schwabe ex Bornet and Flahault 1886–1888), but not with sequences of the type species from the genus Anabaena. This cluster is the sister group of Anabaena morphotypes isolated only from the Gulf of Finland. In addition, this cluster is related to two other clusters formed by sequences of Anabaena isolated from different sites. Partial nifH genes were sequenced from two strains and the phylogenetic tree revealed that the Antarctic nifH sequences clustered with sequences from Anabaena. Furthermore, two strains were tested, using PCR with specific primers, for the presence of genes involved in cyanotoxins (microcystin and saxitoxin) and protease inhibitor (aeruginosin, and cyanopeptolin). Only cyanopeptolin was amplified using PCR. These four Hydrocoryne strains are the first to be isolated and sequenced from Antarctica, which improves our knowledge on this poorly defined cyanobacterial genus.     

Toxin production of cyanobacteria is increased by exposure to zooplankton

1. Cyanobacterial toxin production in response to direct and indirect zooplankton feeding activity was examined using four strains of Microcystis aeruginosa, of which three were previously reported to be toxic to zooplankton and one non‐toxic. Direct (Microcystis cultured with zooplankton) and indirect effects (Microcystis cultured with filtered zooplankton culture media, ZCMF) were tested for the zooplankton species, Moina macrocopa, Daphnia magna or D. pulex. 2. With direct exposure to zooplankton, increased mass‐specific microcystin productions occurred in all Microcystis strains, with mean microcystin concentrations up to five times greater (61.5–177.3 μg g^?1 dry cell) than the controls. 3. With indirect exposure, mass‐specific microcystin production increased over controls in three strains of M. aeruginosa. Mean maximum concentrations of microcystin during the experiment were 92.6–125.7 μg g^?1 dry cell. 4. These results suggest that several strains of Microcystis aeruginosa increased toxin production in response to direct and indirect exposure to herbivorous zooplankton of several species, and support the hypothesis that this response is an induced defence mediated by the release of info‐chemicals from zooplankton.     

Proteomic analysis of phytopathogenic fungus <Emphasis Type="Italic">Botrytis cinerea</Emphasis> as a potential tool for identifying pathogenicity factors,therapeutic targets and for basic research

Botrytis cinerea is a phytopathogenic fungus causing disease in a substantial number of economically important crops. In an attempt to identify putative fungal virulence factors, the two-dimensional gel electrophoresis (2-DE) protein profile from two B. cinerea strains differing in virulence and toxin production were compared. Protein extracts from fungal mycelium obtained by tissue homogenization were analyzed. The mycelial 2-DE protein profile revealed the existence of qualitative and quantitative differences between the analyzed strains. The lack of genomic data from B. cinerea required the use of peptide fragmentation data from MALDI-TOF/TOF and ESI ion trap for protein identification, resulting in the identification of 27 protein spots. A significant number of spots were identified as malate dehydrogenase (MDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The different expression patterns revealed by some of the identified proteins could be ascribed to differences in virulence between strains. Our results indicate that proteomic analysis are becoming an important tool to be used as a starting point for identifying new pathogenicity factors, therapeutic targets and for basic research on this plant pathogen in the postgenomic era.     

Differentiation of commercial strains of <Emphasis Type="Italic">Agaricus</Emphasis> species in China with inter-simple sequence repeat marker

In recent years, Agaricus mushroom production has undergone an important increase in the world. However, there is no consensus over the taxonomy of some commercial strains. So rapid and reliable methods to identify Agaricus strains are urgently needed. In this paper, six inter-simple sequence repeat (ISSR) primers were screened from 20 primers to analyse the genetic diversities of the 12 main commercial strains of Agaricus in China. The results showed that a total of 124 bands were produced, among which 80.6% was polymorphic, the similarity coefficients ranged from 0.69 to 0.98. Twelve tested strains were divided into 2 groups on the similarity coefficient of 0.75. Three Agaricus bitorquis strains were clustered in one group, and seven Agaricus bisporus strains were clustered with Agaricus strains S1 and S2 in the other group. As comparison with ISSR, intergenic spacers combined with restriction fragment length polymorphisms (IGS-RFLP) was employed to analyse genetic diversities of the tested strains by digesting IGS amplified products with restriction enzymes Hae III, Alu I, Rsa I, respectively, and IGS-RFLP cluster results were similar with those of ISSR. However, ISSR was superior to IGS-RFLP in differentiating closely related strains. It is suggested that the ISSR marker is an alternative method to differentiate Agaricus strains.     

Cloning of milk-derived bioactive peptides in <Emphasis Type="Italic">Streptococcus thermophilus</Emphasis>

The enzymatic breakdown of milk proteins releases bioactive peptides. Two such peptides are the 11-residue antimicrobial peptide from bovine lactoferrin (BL-11) and the 12-residue hypotensive peptide from α_s1-casein (C-12). These two peptides have now been cloned in Streptococcus thermophilus to develop strains that enhance the functionality and nutritional value of dairy food products. Nucleic acid sequences encoding the peptides were generated by overlapping PCR and were subsequently cloned into a new expression vector under control of the ST₂₂₀₁ promoter. S. thermophilus transformants were successfully identified using GFP as a selectable marker. The presence of the synthetic gene constructs in S. thermophilus was confirmed by PCR. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">In vitro</Emphasis> biocontrol analysis of <Emphasis Type="Italic">Alternaria alternata</Emphasis> (Fr.) Keissler under different environmental conditions

The species Trichoderma harzianum was analyzed as possible biocontrol agent of Alternaria alternata under different environmental conditions (water activity and temperature). The strains were analyzed macroscopically to obtain the Index of Dominance. The analysis was completed using two microscopic techniques. T. harzianum showed dominance on contact over A. alternata at all testing temperatures and water activities tested except at 0.95 a _w and 15 °C, at which T. harzianum inhibited A. alternata at a distance. Biocontrol was governed by different mechanisms such as competition for space and nutrients, mycoparasitism, and possible antibiosis. Temperature and water activity significantly influenced fungal growth rate.     

A PCR‐BASED TEST TO ASSESS THE POTENTIAL FOR MICROCYSTIN OCCURRENCE IN CHANNEL CATFISH PRODUCTION PONDS1,2

Microcystis aeruginosa is a common form of cyanobacteria (blue‐green algae) capable of forming toxic heptapeptides (microcystins) that can cause illness or death. Occasionally, blooms of cyanobacteria have caused toxic fish‐kills in catfish production ponds. We have developed a PCR test that will detect the presence of microcystin‐producing cyanobacteria. Microcystin producers are detected by the presence of the microcystin peptide synthetase B gene (an obligate enzyme in the microcystin pathway), which appears to be present only in toxin‐producing cyanobacteria. These PCR amplifications can be performed in multiplex using purified DNA from pond waters or by two‐stage amplification from native water samples. A synoptic survey of 476 channel catfish production ponds from four states in the southeastern United States revealed that 31% of the ponds have the genetic potential to produce microcystins by toxic algae.     

Dynamics of microcystin-degrading bacteria in mucilage of <Emphasis Type="Italic">Microcystis</Emphasis>

To reveal the process of degradation of hepatotoxic microcystin produced in Microcystis cells during the Microcystis bloom period, we used fluorescence in situ hybridization (FISH) to analyze the population dynamics of microcystin-degrading bacteria in Microcystis mucilage. We designed and applied an oligonucleotide probe targeted to the 16S rRNA sequence of strain Y2 of a microcystin-degrading bacterium (MCD-bacterium), which was isolated from Lake Suwa, Japan. In both the 1998 and 1999 tests, FISH clearly showed that MCD-bacteria existed in the mucilage and that, when a high concentration of cell-bound microcystin was detected, MCD-bacteria exceeded 10% of the sum of bacteria hybridized with group-specific probes. The concentration of MCD-bacteria was highest in summer 1998, when a toxic species, M. viridis, was dominant. There was a high correlation between the number of MCD-bacteria in the mucilage and the concentration of cell-bound microcystin in the lake. Our results suggest that MCD-bacteria responded to changes in the concentration of microcystin and degraded the microcystin when it was released from Microcystis cells. We also analyzed changes in the bacterial community structure associated with the Microcystis colonies by using domain- and group-specific oligonucleotide probes. Changes in the concentrations of the Cytophaga/Flavobacterium group and -Proteobacteria, which can degrade macromolecules derived from Microcystis cells, were synchronized with changes in the concentration of Microcystis. The results not only suggest the significant role of MCD-bacteria in detoxification, but also demonstrate a possible sequence of degradation from Microcystis cells to microcystin maintained in the cell, which is then carried out by bacterial consortia in the mucilage.     

Comparative analysis of <Emphasis Type="Italic">Pasteurella pneumotropica</Emphasis> isolates from laboratory mice and rats

Selected biochemical and genetic characteristics of the wild-type strains of Pasteurella pneumotropica isolated from mice and rats were investigated and compared in order to determine the significant differences among the isolates. The isolates were divided into six groups on the basis of the patterns of carbon source utilization in the host rodents. The genome sizes were determined by electrophoretic analysis, and the mean genome size of the isolates from mice was larger than that of the isolates from rats (P < 0.05). Cluster analysis of the rpoB sequences discriminated five clusters; the differences might have correlated with the host associations. Principal component analysis (PCA) based on both the biochemical and genetic characteristics revealed total 44 strains discriminated into three groups comprising the host-dependent and host-independent groups. Although the P. pneumotropica isolates were mainly classified on the basis of the host rodents by the examinations, the existence of isolates that could not be discriminated on the basis of the host rodents alone was confirmed by the PCA. These results indicated that the P. pneumotropica isolates could be further classified by taxonomic analysis and also suggested the existence of a host-independent group in addition to the host-dependent groups.     

Genetic variation in cultivated strains of <Emphasis Type="Italic">Agaricus blazei</Emphasis>

Genetic differences among Agaricus blazei strains were investigated using somatic incompatibility testing, isozyme analysis, restriction fragment length polymorphism (RFLP) analysis of mitochondrial DNA (mtDNA), and random amplified polymorphic DNA (RAPD) analysis. Eight strains, one cultivated strain from Brazil and seven from Japan, were used in this study. Somatic incompatibility interactions were observed between the Brazilian cultivated strain and the Japanese strains. The Brazilian cultivated strain had its own distinct patterns of esterase isozyme and mtDNA RFLP, but all seven Japanese cultivated strains showed identical patterns. When the RAPD patterns, obtained using eight primers, were compared the eight strains had their own distinct RAPD profiles. Distance values were calculated between all pairs of the strains based on presence or absence of individual RAPD bands, and a dendrogram was constructed by unweighted pair-group method with arithmetic clustering (UPGMA) analysis. Seven Japanese cultivated strains were grouped to each other, and this group was finally linked to the Brazilian cultivated strain. Based on these results, the degree of genetic variation among the A. blazei strains used is discussed.     

Genetic variability of the invasive cyanobacteria <Emphasis Type="Italic">Cylindrospermopsis raciborskii </Emphasis>from Bir M’cherga reservoir (Tunisia)

Cylindrospermopsis raciborskii is an invasive freshwater cyanobacteria of tropical origin, also found in temperate regions. Due to its known ability to produce potent toxins, such as cylindrospermopsin and the paralytic shellfish poisoning, this species is of major concern from a water quality perspective. This study presents a genetic characterization of four C. raciborskii strains isolated from the Bir M’cherga Tunisian reservoir. The toxicity assessment was investigated via molecular biology tools, which suggested that all the isolated strains were not producing cylindrospermopsin, saxitoxin, or microcystin. This result was further confirmed by HPLC and MALDI-TOF analyses. However, we report for the first time in C. raciborskii the presence of mcyA and mcyE, two segments of the microcystin synthetase mcy cluster. All the strains were identified taxonomically based on the 16S rRNA sequences, and their phylogenetic relationships were assessed using the rpoC1 region. Tunisian strains formed a distinct clade separated from the other African strains.     

Toxicity to Daphnia of a compound extracted from laboratory and natural Microcystis spp., and the role of microcystins

1. The microcystin content of a variety of Microcystis spp., from both laboratory strains and natural blooms, was analysed by HPLC. The microcystin content of laboratory strains ranged from 1.6 to 4.3μgmg^?1 dry weight. Yearly and seasonal variation was detected in an analysis of bloom material collected from Bautzen Reservoir over a 3-year period. The microcystin concentration in bloom material ranged from undetectable to 1.16 μg ml^?1 dry weight. 2. Toxicity of laboratory and natural Microcystis to Daphnia pulicaria was determined using an established LC₅₀ technique. Partially purified water extracts from different Microcystis samples exhibited a wide range of toxicity. The highest activity was found in natural Microcystis samples, with an LC₅₀ of 36 μgm^?1 dry weight of Microcystis, whereas one strain did not appear toxic at 1600 μg ml^?1. 3. No correlation was found between the concentrations of microcystins of different laboratory and natural Microcystis strains and the toxicity of extracts to Daphnia pulicaria from the same strains. Therefore, we discriminated between hepatotoxic microcystins and the compound(s) that is toxic to Daphnia, here termed DTC (Daphnia-toxic compound), which is independent of microcystins.     

Accumulation and depuration of microcystin produced by the cyanobacterium <Emphasis Type="Italic">Microcystis</Emphasis> in a freshwater snail

Seasonal changes in microcystin concentrations in a resident snail (Sinotaia histrica) and an edible clam (Corbicula sandai) in Lake Biwa were surveyed. To clarify both the accumulation and depuration of microcystins, experimental studies with microcystin were also carried out on the snail. In the field investigation, microcystin was detected from the hepatopancreas and intestine of S. histrica (up to 3.2µgg^–1 dry weight and 19.5µgg^–1 dry weight, respectively); however, no microcystin was detected in the hepatopancreas of C. sandai. In the laboratory experiment, the microcystin-LR concentration in the hepatopancreas of S. histrica reached a value of 436µgg^–1 dry weight on day 10 of 15 days of uptake, and a high value persisted despite a depuration period of 15 days. The depuration rate constant of microcystin and its biological half-life were 0.0828 day^–1 and 8.4 days, respectively. These results indicate that S. histrica has a high ability to accumulate microcystin in its tissue. Because S. histrica is predated by fish and water fowl, it is likely to play an important role as a vector for microcystin in lakes with dense blooms of toxic cyanobacteria.