Random Amplified Polymorphic DNA (RAPD) Analyses for Discriminating Genotypes of<i>Microcystis</i>Cyanobacteria

Nishihara, Hirofumi; Miwa, Hiroyoshi; Watanabe, Masayuki; Nagashima, Minoru; Yagi, Osami; Takamura, Yoshichika

doi:10.1271/bbb.61.1067

Cited by 49 publications

(35 citation statements)

References 3 publications

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“…The medium was inoculated with the seed culture to give an OD of 0.02 at 660 nm. The CTmedium contains Ca(NO 3 ) 2 4H 2 0: 150 mg; KNO 3 : 100 mg; MgSO4 7H2O: 40 mg; Na2 glycerophosphate: 50 mg; Vitamin B 12 : 0.1 mg; biotin: 0.1 mg; thiamin: 10 mg; TAPS (N-Tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid): 400 mg and 3 ml of metal solution 18) , all in 1 liter of deionized water, pH 8.2.…”

Section: Organisms and Cultural Conditionsmentioning

confidence: 99%

Growth Inhibition of Microcystis Cyanobacteria by L-Lysine and Disappearance of Natural Microcystis Blooms with Spraying

et al. 2004

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Low concentrations of L-lysine and L-lysine-containing-peptides strongly inhibited the growth of axenic strains of 4 species belonging to the genus Microcystis (cyanobacteria). Inhibitory activities decreased in theThe addition of 50 mM of L-lysine to growing cells of axenic strains of M. novacekii TAC20-1 and M. viridis NIES102 resulted in a loss of buoyancy, a decrease in chlorophyll a content and consequent cell lysis. The cells took up most of the L-lysine added to the culture medium of M. viridis NIES-102 in one day. However, the amino acid was released back into the medium with cell lysis 3 days after the addition. Laboratory results were confirmed in experimental ponds outdoors by spraying L-lysine onto natural Microcystis blooms in the summer of 1999 and 2000. The spraying of 50 mM L-lysine caused Microcystis colonies to vanish from the surface water within two days; there was a dramatic change in the color and transparency of the pond surface water. After the immediate disappearance of Microcystis sp., both Euglena sp. and/or Phormidium tenue appeared, the latter becoming the dominant species in the phytoplankton community of the pond. In the experiment in summer 2000, the L-lysine provided to the pond was assayed. The level decreased gradually and L-lysine was not detected in the pond water after 6 days. The mechanisms of growth inhibition by L-lysine and a possible mitigation of Microcystis blooms by the amino acid are discussed.

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Section: Organisms and Cultural Conditionsmentioning

confidence: 99%

Growth Inhibition of Microcystis Cyanobacteria by L-Lysine and Disappearance of Natural Microcystis Blooms with Spraying

et al. 2004

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“…Previous studies include, among others, studies of random amplified polymorphic DNA (RAPD) (33,35), repetitive DNA elements (3,39), and 16S rRNA genes (34,37,43,44,50). None of these analyses revealed any simple correlation with toxicity, although some recent studies have demonstrated that it may be feasible to generate genetic probes that are indicators of toxicity (4,50).…”

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Natural Variation in the Microcystin Synthetase Operon mcyABC and Impact on Microcystin Production in Microcystis Strains

et al. 2003

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Toxic Microcystis strains often produce several isoforms of the cyclic hepatotoxin microcystin, and more than 65 isoforms are known. This has been attributed to relaxed substrate specificity of the adenylation domain. Our results show that in addition to this, variability is also caused by genetic variation in the microcystin synthetase genes. Genetic characterization of a region of the adenylation domain in module mcyB1 resulted in identification of two groups of genetic variants in closely related Microcystis strains. Sequence analyses suggested that the genetic variation is due to recombination events between mcyB1 and the corresponding domains in mcyC. Each variant could be correlated to a particular microcystin isoform profile, as identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Among the Microcystis species studied, we found 11 strains containing different variants of the mcyABC gene cluster and 7 strains lacking the genes. Furthermore, there is no concordance between the phylogenies generated with mcyB1, 16S ribosomal DNA, and DNA fingerprinting. Collectively, these results suggest that recombination between imperfect repeats, gene loss, and horizontal gene transfer can explain the distribution and variation within the mcyABC operon.Cyanobacteria are phototrophic organisms that often form water blooms in eutrophic or estuarine waters. These water blooms undergo fluctuations and may exhibit toxic states. One common genus in such water blooms, Microcystis, produces the hepatotoxin microcystin (6). There are approximately 65 known isoforms of microcystin, representing a family of cyclic heptapeptides having the common structure cyclo(D-Ala-L-X-D-MeAsp-L-Z-Adda-D-Glu-Mdha), where L-X and L-Z are variable L amino acids, Adda is 3-amino-9-methoxy-2,6,8,-trimethyl-10-phenyl-4,6-decandienoic acid, D-MeAsp is 3-methylaspartic acid, and Mdha is N-methyl-dehydroalanine (Fig.

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“…Several morphological studies (25,54) and molecular studies have attempted to resolve the ambiguous relationship of Microcystis toxigenicity to its population structure. Molecular approaches have included those based on allozyme polymorphisms (23), 16S rRNA genes (28,35,41,42), the phycocyanin intergenic spacer (PC-IGS) region (29), DNA-DNA hybridization (55), nucleotide base composition (13), random amplified polymorphic DNA (27,32), the 16S-23S rRNA internal transcribed spacer region (30,36), the rbcL gene (42), repetitive DNA elements (2,40), and rpoD homologs (44). The results, while suggesting a monophyletic origin of Microcystis, have proven inconsistent with respect to subpopulations that might correlate with toxicity.…”

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Detection of Toxigenicity by a Probe for the Microcystin Synthetase A Gene ( mcyA) of the Cyanobacterial Genus Microcystis : Comparison of Toxicities with 16S rRNA and Phycocyanin Operon (Phycocyanin Intergenic Spacer) Phylogenies

Tillett

Parker

Neilan

2001

Appl Environ Microbiol

229

189

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The relationship between toxigenicity and phylogeny within the cyanobacterial genus Microcystis is unclear. To investigate this issue, we have designed PCR primers for the N-methyltransferase (NMT) domain of the microcystin synthetase gene mcyA and have probed 37 Microcystis sp. cultures as well as several field samples. The NMT region was present in all 18 laboratory strains that gave positive reactions in the protein phosphatase inhibition assay for microcystin but was absent in 17 nontoxic strains. Two other nontoxic strains, one of which had previously been reported to produce microcystin, possessed the NMT region. Detection of NMTspecific DNA in field samples corresponded to periods of toxicity as assessed by protein phosphatase inhibition. The Microcystis strains formed a monophyletic cluster based on 16S rRNA gene sequences but comprised two groups with respect to phycocyanin intergenic spacer (PC-IGS) sequences. Toxic and nontoxic strains appeared to be erratically distributed within the PC-IGS and 16S rRNA trees. Sequence analysis of the NMT domain revealed two coherent groups. The genomic region immediately downstream of the mcyABC cluster in all 20 NMT-positive strains contained an open reading frame of unknown function (uma1) at a conserved distance from mcyC. All nontoxic strains also contained uma1, which is not cotranscribed with mcyABC. The consistent linkage of mcyC to uma1 suggests that mcyC has not been frequently transferred into nontoxic strains via any mechanism involving insertion at random chromosomal locations. These results are discussed with respect to various mechanisms that could explain the patchy distribution of toxigenicity among the various Microcystis clades.Microcystis spp., cyanobacteria that frequently occur as noxious blooms in eutrophic freshwaters, are of major concern because many strains produce cyclic heptapeptide toxins called microcystins (5). The microcystins are members of a family of more than 65 heptapeptides and share the common structure, where L-X and L-Z are variable L-amino acids, Adda is 3-amino-9-methoxy-2,6,8,-trimethyl-10-phenyl-4,6-decadienoic acid, DMeAsp is 3-methyl-aspartic acid, and Mdha is N-methyldehydroalanine (38). Toxicity is mediated through the active transport of microcystin into hepatocytes by the bile acid organic anion transport system, followed by inhibition of eukaryotic serine/threonine protein phosphatases 1 and 2A (12, 17). Acute poisoning, leading to death from massive hepatic hemorrhage, has been reported to occur in both animals and humans (3,19,37). Chronic ingestion of sublethal doses has been demonstrated to induce primary hepatocellular carcinoma in rodents (33) and has been epidemiologically linked to primary liver cancer in humans (56,57).Water resource management has been complicated by the inability to differentiate between toxic and nontoxic Microcystis blooms without isolation and testing for toxin production. Several morphological studies (25, 54) and molecular studies have attempted to resolve the ambiguous relationship...

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Random Amplified Polymorphic DNA (RAPD) Analyses for Discriminating Genotypes ofMicrocystisCyanobacteria

Cited by 49 publications

References 3 publications

Growth Inhibition of Microcystis Cyanobacteria by L-Lysine and Disappearance of Natural Microcystis Blooms with Spraying

Growth Inhibition of Microcystis Cyanobacteria by L-Lysine and Disappearance of Natural Microcystis Blooms with Spraying

Natural Variation in the Microcystin Synthetase Operon mcyABC and Impact on Microcystin Production in Microcystis Strains

Detection of Toxigenicity by a Probe for the Microcystin Synthetase A Gene ( mcyA) of the Cyanobacterial Genus Microcystis : Comparison of Toxicities with 16S rRNA and Phycocyanin Operon (Phycocyanin Intergenic Spacer) Phylogenies

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