Genetic analysis of the microcystin biosynthesis gene cluster in Microcystis strains from four bodies of eutrophic water in Japan

Noguchi, Takehiko; Shinohara, Azusa; Nishizawa, Akito; Asayama, Munehiko; Nakano, Tomoyo; Hasegawa, Masateru; Harada, Kenichi; Nishizawa, Tomoyasu; Shirai, Makoto

doi:10.2323/jgam.55.111

Cited by 23 publications

(22 citation statements)

References 44 publications

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“…Such mutations would also result in an inactive gene cluster and effectively a non-toxic cyanobacterial strain. Other cyanotoxin genes, most notably the microcystin biosynthesis genes are known to occasionally contain mutations rendering the biosynthesis pathway inactive (Christiansen et al, 2006;Noguchi et al, 2009;Ostermaier and Kurmayer, 2009). This suggests that similar mutations might be found also in PST-producing cyanobacteria.…”

Section: Discussionmentioning

confidence: 98%

First report of cyanobacterial paralytic shellfish toxin biosynthesis genes and paralytic shellfish toxin production in Polish freshwater lakes

et al. 2017

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In central and southern Europe, Aphanizomenon spp., A. gracile Lemmermann in particular, have been associated with paralytic shellfish toxin (PST) production. In western Poland, A. gracile is very common, and Cylindrospermopsis raciborskii (Woloszyńska) Seenayya & Subba Raju, another potentially PST-producing species, is often found as well. To date it is, however, unknown if the cyanobacterial populations in this area harbour the genetic capability to produce PSTs, and to what extent toxin biosynthesis occurs. The objective of this study was to survey the prevalence of potentially PST-producing cyanobacteria by measuring paralytic shellfish toxin biosynthesis gene sxtB copy numbers, sxtA, sxtG and sxtS gene presence, and PST concentrations in Polish freshwater lakes. In total, 34 lakes in western Poland were sampled twice during summer 2010. The presence of PST biosynthesis genes sxtA, sxtG and sxtS was determined using conventional qualitative PCR. Quantitative PCR (qPCR) was used to measure sxtB copy numbers, and the samples were analysed for PSTs using ion-pair high performance liquid chromatography with post-column oxidation and fluorescence detection (HPLC-FLD). Cyanobacteria carrying the sxtB gene were present in 23.5% of all samples (n=16) and in 14 lakes of the studied 34. Gene copy numbers ranged from 8.2×104 to 5.1×107 sxtB copies L-1 (mean 3.8×106). The median was 4.5×105 sxtB gene copies L-1 and the majority of results clustered at the lower end of the sxtB qPCR linear range. In 12 out of the 16 samples positive for sxtB the gene co-occurred with the other three targeted PST biosynthesis genes sxtA, sxtG and sxtS. However, five additional samples lacked one or two of the targeted four genes. Thirteen samples contained PSTs, of which 12 samples at levels <0.072 µg L-1, i.e., close to or below the quantitative detection limit of the HPLC-FLD method (0.01 µg L-1). One sample contained 0.57 µg L-1 saxitoxin, co-occurring with all four sxt genes studied. No correlation between PST and sxt gene occurrence or copy numbers was observed. A. gracile and C. raciborskii occurred in 92% and 50% of samples, respectively, containing PSTs, sxt genes or both. In conclusion, the results confirm that potential PST producers constitute an established subpopulation of cyanobacteria in Polish freshwater lakes. However, none of the sxt genes targeted in this study could serve as a reliable marker for active PST biosynthesis.

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Section: Discussionmentioning

confidence: 98%

First report of cyanobacterial paralytic shellfish toxin biosynthesis genes and paralytic shellfish toxin production in Polish freshwater lakes

et al. 2017

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“…Interestingly, we identified the mcyE gene, a glutamate-activating adenylation domain which is part of the microcystin-encoding gene cluster (Tillett et al 2000), not only in the MC-producing strain AB2011/53, but also in eight non-microcystin-producing strains from Hartbeespoort Dam. The presence of the mcyE gene in non-MC-producing cyanobacteria has also been described by Noguchi et al (2009) and this raises a question as to the suitability of the mcyE gene to quantify toxin-producing Microcystis spp. in quantitative PCR investigations.…”

Section: R Curvata From Hartbeespoort Dam Than To Cylindrospermopsismentioning

confidence: 99%

Diversity of cyanobacteria and cyanotoxins in Hartbeespoort Dam, South Africa

Ballot

Sandvik

Rundberget

et al. 2014

Mar. Freshwater Res.

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Abstract. The South African Hartbeespoort Dam is known for the occurrence of heavy Microcystis blooms. Although a few other cyanobacterial genera have been described, no detailed study on those cyanobacteria and their potential toxin production has been conducted. The diversity of cyanobacterial species and toxins is most probably underestimated. To ascertain the cyanobacterial composition and presence of cyanobacterial toxins in Hartbeespoort Dam, water samples were collected in April 2011. In a polyphasic approach, 27 isolated cyanobacterial strains were classified morphologically and phylogenetically and tested for microcystins (MCs), cylindrospermopsin (CYN), saxitoxins (STXs) and anatoxin-a (ATX) by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and screened for toxin-encoding gene fragments. The isolated strains were identified as Sphaerospermopsis reniformis, Sphaerospermopsis aphanizomenoides, Cylindrospermopsis curvispora, Raphidiopsis curvata, Raphidiopsis mediterrranea and Microcystis aeruginosa. Only one of the Microcystis strains (AB2011/53) produced microcystins (35 variants). Forty-one microcystin variants were detected in the environmental sample from Hartbeespoort Dam, suggesting the existence of other microcystin producing strains in Hartbeespoort Dam. All investigated strains tested negative for CYN, STXs and ATX and their encoding genes. The mcyE gene of the microcystin gene cluster was found in the microcystin-producing Microcystis strain AB2011/53 and in eight non-microcystin-producing Microcystis strains, indicating that mcyE is not a good surrogate for microcystin production in environmental samples.

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“…The area differs from other places where unicellular cyanobacteria have been isolated from eutrophic waters of ponds, lakes, and dams. 20,22,33,34) Isolation was done twice at six sites (ABRG1 to 6) in the garden. A total of 16 algal strains were eventually separated from ABRG1 to 3 (May 2007) and ABRG4 to 6 (August 2007).…”

Section: Isolation Of Cyanobacterium Abrg5-3l1mentioning

confidence: 99%

“…Recently, a pour plate method has been established to isolate cyanobacterial Microcystis strains. 33) To separate the ABRG5-3TN1 cells from the contaminants completely, a method of bead-beating (see ''Materials and Methods'') was tried, and ABRG5-3L1 was eventually isolated (Fig. 1B).…”

Section: Isolation Of Cyanobacterium Abrg5-3l1mentioning

confidence: 99%

Isolation and Molecular Characterization of a Multicellular Cyanobacterium, Limnothrix/Pseudanabaena sp. Strain ABRG5-3

Nishizawa

Hanami

Hirano

et al. 2010

Bioscience, Biotechnology, and Biochemistry

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A cyanobacterium, semi-filamentous multicellular strain ABRG5-3, was isolated and its unique nature was characterized. This axenic strain formed colonies and was motile on an agarose plate. The 16S rRNA gene of ABRG5-3 exhibited similarities to those of the Limnothrix and Pseudanabaena strains, which are known as filamentous and nonheterocystous cyanobacteria. Peaks in absorbance for the accumulation of chlorophyll a, phycocyanin, and phycoerythrin were observed in the cell extract. Natural separation of the pigments occurred in the supernatant of the autolysed cells. The cell lysis was promoted by osmotic shocks and lysozyme treatments. Chlorophyll a and total DNA were abundantly recovered from the cells. Analysis of the restriction-modification system for genomic DNA revealed novel diversity. Moreover, we made a successful attempt to create antibiotic-resistant strains by conjugation with a foreign plasmid, which indicates that strain ABRG5-3 is transformable.

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Genetic analysis of the microcystin biosynthesis gene cluster in Microcystis strains from four bodies of eutrophic water in Japan

Cited by 23 publications

References 44 publications

First report of cyanobacterial paralytic shellfish toxin biosynthesis genes and paralytic shellfish toxin production in Polish freshwater lakes

First report of cyanobacterial paralytic shellfish toxin biosynthesis genes and paralytic shellfish toxin production in Polish freshwater lakes

Diversity of cyanobacteria and cyanotoxins in Hartbeespoort Dam, South Africa

Isolation and Molecular Characterization of a Multicellular Cyanobacterium, Limnothrix/Pseudanabaena sp. Strain ABRG5-3

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