No abstract

The distribution and genotypic variation of potential microcystin (MC) producers along the southern and eastern shores of Lake Ontario in 2001 and 2003 were examined using a suite of PCR primers. Cyanobacterial, Microcystis sp., and Microcystis-specific toxin primer sets identified shoreline distribution of cyanobacterial DNA (in 97% of the stations) and MC synthetase genes (in 50% of the stations). Sequence analysis of a partial mcyA amplicon targeting Microcystis, Anabaena, and Planktothrix species indicated that the Microcystis sp. genotype was the dominant MC genotype present and revealed a novel Microcystis-like sequence containing a 6-bp insert. Analysis of the same samples with genus-specific mcyE primers confirmed that the Microcystis sp. genotype was the dominant potential MC producer. Genotype compositions within embayments were relatively homogenous compared to those for shoreline and tributary samples. MC concentrations along the shoreline exhibited both temporal and spatial differences as evidenced by the protein phosphatase inhibition assay, at times exceeding the World Health Organization guideline value for drinking water of 1.0 g MC-LR eq liter ؊1 . MC genotypes are widespread along the New York State shoreline of Lake Ontario, appear to originate nearshore, and can be carried through the lake via wind and surface water current patterns.

A three-step medium sequence was developed for rooting microcuttings of American chestnut [Castanea dentata (Marsh.) Borkh.]. First, individual shoots or clumps of shoots were cultivated on shoot-elongation medium for 4--8 wk until shoots were 2-3 cm long. The medium consisted of modified Woody Plant Medium, 500 mg/l polyvinylpyrrolidone (MW 40,000), and 0.89 pM benzyladenine. Microcuttings were then excised, vertically split at the base to approximately 2 mm through the pith, dipped in 5 or 10 mM indolebutyric acid for 1 min, and cultivated on half-strength Murashige and Skoog basal medium plus 0.2 g/l charcoal for 2 wk. During that time, roots were induced and became visible. Finally, the microcuttings were transferred back to shoot-elongation medium and cultivated for 3 wk, allowing growth of both roots and shoots. Using this protocol with 3 genotypes derived from one mature tree and two 1-yr-old seedlings, 57 to 73% rooting was obtained with less than 23% shoot-tip necrosis.

Several marine algae of the class Pelagophyceae produce the unusual marine sterol 24-propylidenecholesterol, mainly as the (24E)-isomer. The (24Z)-isomer had previously been considered as a specific biomarker for Aureococcus anophagefferens, the 'brown tide' alga of the Northeast coast of the USA. To test this hypothesis and to generate chemotaxonomic information, the sterol compositions of 42 strains of pelagophyte algae including 17 strains of Aureococcus anophagefferens were determined by GC analysis. A more comprehensive sterol analysis by HPLC and (1)H-NMR was obtained for 17 selected pelagophyte strains. All strains analyzed contained 24-propylidenecholesterol. In all strains belonging to the order Sarcinochrysidales, this sterol was found only as the (E)-isomer, while all strains in the order Pelagomonadales contained the (Z)-isomer, either alone or together with the (E)-isomer. The occurrence of Delta(22) and 24alpha-sterols was limited to the Sarcinochrysidales. The first occurrence of Delta(22)-24-propylcholesterol in an alga, CCMP 1410, was reported. Traces of the rare sterol 26,26-dimethyl-24-methylenecholesterol were detected in Aureococcus anophagefferens, and the (25R)-configuration was proposed, based on biosynthetic considerations. Traces of a novel sterol, 24-propylidenecholesta-5,25-dien-3beta-ol, were detected in several species.

No abstract

No abstract

While multiple phylogenetic markers have been used in the culture-independent study of microcystinproducing cyanobacteria, in only a few instances have multiple markers been studied within individual cells, and in all cases these studies have been conducted with cultured isolates. Here, we isolate and evaluate large DNA fragments (>6 kb) encompassing two genes involved in microcystin biosynthesis (mcyA2 and mcyB1) and use them to identify the source of gene fragments found in water samples. Further investigation of these gene loci from individual cyanobacterial cells allowed for improved analysis of the genetic diversity within microcystin producers as well as a method to predict microcystin variants for individuals. These efforts have also identified the source of the novel mcyA genotype previously termed Microcystis-like that is pervasive in the Laurentian Great Lakes and they predict the microcystin variant(s) that it produces.Microcystin-producing cyanobacteria are common nuisance organisms in harmful algal blooms in freshwaters around the world (4). This genetically diverse group (based on 16S rRNA, mcyA, mcyD, and mcyE gene sequences [6,10,15,16,22]) ranges in morphology from unicellular and colonial cocci to large filamentous strands. Many species can produce a variety of secondary metabolites that can act as hepatatoxins upon ingestion by animals (e.g., variants of microcystin) (4, 33). Microcystin production reduces the water quality in reservoirs used by human populations and fishery resources, and production of these toxins by this group of cyanobacteria makes them important organisms for continued observation and study (4,33,36). Much effort has been expended over the past 15 years to characterize the genomic and structural components of the microcystin (mcy) synthetase operon responsible for the production of microcystins. Several complete DNA sequences of the mcy synthetase operon are currently available in GenBank (3,11,29,31).Although the mechanisms of microcystin production are now better understood, recent analyses of mcyA gene fragments from Lakes Erie and Ontario indicated a microcystin toxin producer of unknown phylogeny (7,28). This discrepancy suggested a need for improved molecular characterization of naturally occurring microcystin producers, which spurred our research to identify the source of several unusual mcyA fragments from the cyanobacterial community (7, 28). It was apparent from initial sequence data that these mcyA gene fragments, termed Microcystis-like, were highly similar to those from Microcystis spp. (colonial or unicellular cocci). However, they contained a 6-nucleotide insert consistent with mcyA genes from filamentous cyanobacteria (e.g., Anabaena, Nostoc, and Planktothrix) (28). These preliminary findings suggested that these unusual mcyA fragments either came from (i) a novel species or strain, (ii) an ancestral Microcystis, (iii) the highly unlikely hybridization of colonial cocci and filamentous cyanobacteria, or (iv) a chimera of cocci and filamentous PCR products....

No abstract