Molecular Tools for the Detection and Quantification of Toxigenic Cyanobacteria E-Book
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- Herausgeber: John Wiley & Sons
- Kategorie: Wissenschaft und neue Technologien
- Sprache: Englisch
A guide to state-of-the-art molecular tools for monitoring and managing the toxigenicity of cyanobacteria Runaway eutrophication and climate change has made the monitoring and management of toxigenic organisms in the world's bodies of water more urgent than ever. In order to influence public policy regarding the detection and quantification of those organisms, it is incumbent upon scientists to raise the awareness of policy makers concerning the increased occurrence of toxigenic cyanobacteria and the threats they pose. As molecular methods can handle many samples in short time and help identify toxigenic organisms, they are reliable, cost-effective tools available for tracking toxigenic cyanobacteria worldwide. This volume arms scientists with the tools they need to track toxigenicity in surface waters and food supplies and, hopefully, to develop new techniques for managing the spread of toxic cyanobacteria. This handbook offers the first comprehensive treatment of molecular tools for monitoring toxigenic cyanobacteria. Growing out of the findings of the landmark European Cooperation in Science and Technology Cyanobacteria project (CYANOCOST), it provides detailed, practical coverage of the full array of available molecular tools and protocols, from water sampling, nucleic acid extraction, and downstream analysis--including PCR and qPCR based methods--to genotyping (DGGE), diagnostic microarrays, and community characterization using next-gen sequencing techniques. * Offers an overview of the latest trends in the field, while providing a foundation for understanding and applying the tools and techniques described * Provides detailed coverage of the full range of molecular tools currently available, with expert guidance on the analysis and interpretation of results * Includes step-by-step guidance on standard operational procedures, including molecular tests used in environmental monitoring, with individual chapters devoted to each procedure * Complements the published Handbook of Cyanobacterial Monitoring and Cyanotoxin Analysis from the CyanoCOST project This handbook is an indispensable working resource for scientists, lab technicians, and water management professionals and an excellent text/reference for graduate students and supervisors who use molecular tools. It will also be of great value to environmental health and protection officials and policy makers.
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Table of Contents
Cover
Title Page
Copyright
Hans C.P. Matthijs (1951–2016): A passion for light and life
List of Contributors
About the Editors
Dr. Rainer Kurmayer, PhD, Assoc. Prof
Dr. Kaarina Sivonen, PhD, Prof
Dr. Annick Wilmotte, PhD
Dr. Nico Salmaso, PhD
About the Book
Preface
Acknowledgments
Chapter 1: Introduction
1.1 A Brief Historical Overview
1.2 The Genetic Basis of Toxin Production
1.3 Application of Molecular Tools
1.4 Laboratory Safety Issues
1.5 References
Chapter 2: Sampling and Metadata
2.1 Introduction
2.2 Handling of Samples
2.3 Sample Contamination
2.4 Sampling
2.5 Subsampling Food Supplement Samples
2.6 Sampling of Nucleic Acids
2.7 General Conclusions
2.8 References
SOP 2.1 Sampling and Filtration (DNA)
SOP 2.2 Sampling of Benthic Cyanobacteria
SOP 2.3 Isolation of Single Cyanobacteria Colonies/Filaments
SOP 2.4 Sampling Food Supplements
SOP 2.5 Sampling and Filtration (RNA)
SOP 2.6 Sampling of Abiotic and Biotic Data and Recording Metadata
Chapter 3: Isolation, Purification, and Cultivation of Toxigenic Cyanobacteria
3.1 Introduction
3.2 Methodical Principles for Cyanobacterial Isolation, Purification, and Cultivation
3.3 General Conclusions
3.4 References
SOP 3.1 Isolation, Purification, and Clonal Isolate Testing
SOP 3.2 Isolation of Picocyanobacterial Cells by Flow Cytometer (FCM) Sorting
SOP 3.3 Axenization
SOP 3.4 Culture Media (Solid and Liquid)
SOP 3.5 Strain Maintenance (Living Cultures)
SOP 3.6 Cryopreservation and Recovery
Chapter 4: Taxonomic Identification of Cyanobacteria by a Polyphasic Approach
4.1 Introduction
4.2 Nomenclature and Classification of Cyanobacteria
4.3 Microscopy
4.4 Molecular Markers: Single Loci
4.5 Molecular Markers: Multiple Loci
4.6 Molecular Typing Methods Based on Gel Electrophoresis
4.7 Denaturing Gradient Gel Electrophoresis (DGGE)
4.8 Taxonomic and Molecular Databases
4.9 The Polyphasic Approach
4.10 Final Considerations
4.11 References
SOP 4.1 Taxonomic Identification by Light Microscopy
SOP 4.2 Polyphasic Approach on Cyanobacterial Strains
Chapter 5: Nucleic Acid Extraction
5.1 Introduction
5.2 Specific Extraction Procedures and Storage
5.3 References
SOP 5.1 Standard DNA Isolation Technique for Cyanobacteria
SOP 5.2 DNA Isolation Protocol for Cyanobacteria with Extensive Mucilage
SOP 5.3 Quantitative DNA Isolation from Filters
SOP 5.4 Genomic DNA Extraction from Single Filaments/Colonies for Multiple PCR Analyses
SOP 5.5 Whole Genome Amplification Using Bacteriophage Phi29 DNA Polymerase
SOP 5.6 DNA Extraction from Food Supplements
SOP 5.7 RNA Extraction from Cyanobacteria
SOP 5.8 cDNA Synthesis
Chapter 6: Conventional PCR
6.1 Introduction
6.2 Principle of PCR and Available Enzymes
6.3 Special Notes
6.4 References
SOP 6.1 PCR Detection of Microcystin Biosynthesis Genes Combined with RFLP Differentiation of the Producing Genus
SOP 6.2 PCR Detection of Microcystin and Nodularin Biosynthesis Genes in the Cyanobacterial Orders Oscillatoriales, Chroococcales, Stigonematales, and Nostocales
SOP 6.3 Genus-Specific PCR Detection of Microcystin Biosynthesis Genes in
Anabaena
/
Nodularia
and
Microcystis
and
Planktothrix
, Respectively
SOP 6.4 PCR Detection of Anatoxin Biosynthesis Genes Combined with RFLP Differentiation of the Producing Genus
SOP 6.5 PCR Detection of the Saxitoxin Biosynthesis Genes,
sxt
A,
sxt
X,
sxt
H,
sxt
G, and
sxt
I
SOP 6.6 PCR Detection of the Cylindrospermopsin Biosynthesis Gene
cyr
J
SOP 6.7 PCR from Single Filament of Toxigenic
Planktothrix
SOP 6.8 Analysis of Microcystin Biosynthesis Gene Subpopulation Variability in
Planktothrix
SOP 6.9 PCR Detection of Microcystin Biosynthesis Genes from Food Supplements
Chapter 7: Quantitative PCR
7.1 Introduction
7.2 Primer/Probe Design
7.3 Optimization
7.4 Absolute Quantification
7.5 Relative Quantification
7.6 Calibration of qPCR Results
7.7 General Conclusions
7.8 References
SOP 7.1 Optimization of qPCR Assays
SOP 7.2 Calibration of qPCR Results
SOP 7.3 Quantification of Potentially Microcystin/Nodularin-Producing
Anabaena
,
Microcystis, Planktothrix
, and
Nodularia
SOP 7.4 Relative Quantification of
Microcystis
or
Planktothrix mcy
Genotypes Using qPCR
SOP 7.5 Quantification of Transcript Amounts of
mcy
Genes in
Planktothrix
SOP 7.6 Quantification of Potentially Cylindrospermopsin-Producing
Chrysosporum ovalisporum
SOP 7.7 qPCR Detection of the Paralytic Shellfish Toxin Biosynthesis Gene
sxt
B
SOP 7.8 Application of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) Guidelines to Quantitative Analysis of Toxic Cyanobacteria
Chapter 8: DNA (Diagnostic) and cDNA Microarray
8.1 DNA (Diagnostic) Microarray
8.2 cDNA Microarray for Cyanobacteria
SOP 8.1 DNA-Chip Detection of Potential Microcystin and Nodularin Producing Cyanobacteria in Environmental Water Samples
SOP 8.2 cDNA Microarrays for Cyanobacteria
Chapter 9: Analysis of Toxigenic Cyanobacterial Communities through Denaturing Gradient Gel Electrophoresis
9.1 Introduction
9.2 Main Applications of the Method
9.3 Possible Applications
9.4 DGGE Procedure
9.5 General Conclusions Including Pros and Cons of the Method
9.6 Optimization of the Method and Troubleshooting
9.7 References
SOP 9.1 DGGE-
mcy
A Conditions
Chapter 10: Monitoring of Toxigenic Cyanobacteria Using Next-Generation Sequencing Techniques
10.1 Introduction
10.2 Specific Procedures
10.3 Bioinformatic Processing of Amplicon Sequencing Datasets
10.4 References
SOP 10.1 Standard Technique to Generating 16S rRNA PCR Amplicons for NGS
SOP 10.2 Bioinformatics Analysis for NGS Amplicon Sequencing
Chapter 11: Application of Molecular Tools in Monitoring Cyanobacteria and Their Potential Toxin Production
11.1 Introduction
11.2 Possible Applications
11.3 Checklist of Publications, Applications and Lessons from Practice
11.4 General Conclusions
11.5 Acknowledgments
11.6 References
Appendix: Supplementary Tables
Chapter 6
References Table
S6.1
References Table
S6.2
Chapter 7
References, Table S7.1
Chapter 11
References, Table S11.1
References, Table
S11.2
Cyanobacterial Species Cited in the Book
References
Glossary
Index
End User License Agreement
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Guide
Cover
Table of Contents
Preface
Begin Reading
List of Illustrations
Chapter 1: Introduction
Figure 1.1 Scheme of the genetic basis of microcystin/nodularin synthesis in sequenced cyanobacterial genera. Arrows mark the bi-directional promotor region (from Tillett
et al.
, 2000; Christiansen
et al.
, 2003; Moffitt and Neilan, 2004; Rouhiainen
et al.
, 2004; Fewer
et al.
, 2013; Shih
et al.
, 2013).
Figure 1.2 Scheme of the genetic basis of cylindrospermopsin synthesis in various cyanobacterial genera (from Mihali
et al.
, 2008; Mazmouz
et al.
, 2010; Stüken and Jakobsen 2010; Jiang
et al.
, 2014).
Figure 1.3 Scheme of the genetic basis of saxitoxin synthesis in various cyanobacterial genera (from Kellmann
et al.
, 2008b; Mihali
et al.
, 2009; Stucken
et al.
, 2010; Murray
et al.
, 2011).
Figure 1.4 Scheme of the genetic basis of (homo)anatoxin-a synthesis in various cyanobacterial genera (from Méjean
et al.
, 2009; Rantala-Ylinen
et al.
, 2011; Calteau
et al.
, 2014).
Figure 1.5 Scheme of the handbook's structure, individual chapters, and linked SOPs. SOPs from individual chapters but highlighted by the same background are directly related.
Chapter 2: Sampling and Metadata
Figure 2.1 Sampling of toxigenic cyanobacterial communities in lakes and reservoirs from the boat: (A) pulling a plankton net at the surface, (B) grabbing a sample from the surface, (C) pulling a plankton net vertically, (D) using a self-closing water sampling to sample metalimnetic layers, (E) using a gravity corer to sample cyanobacteria settled to the sediment.
Figure 2.2 Flowchart showing the processing of water samples for sample filtration from (A) samples collected using a plankton net, (B) depth-integrated samples collected through a water column (see SOP 2.1). Gray shading marks processing of samples in the laboratory.
Figure 2.3 Flow diagram outlining the steps for sampling and sample preparation of food supplements (see SOP 2.4).
Chapter 3: Isolation, Purification, and Cultivation of Toxigenic Cyanobacteria
Figure 3.1 Schematic overview of protocols (see SOPs 3.1–3.6) described in this chapter.
Figure 3.2 Flowchart showing the methods of isolation presented in SOP 3.1.
Figure 3.3 Front panel of FACSCalibur.
Figure 3.4 Gating strategy for sorting. G1 gate for PC cells; G2 gate for PE cells.
Figure 3.5 Flowchart showing the method for flow cytometer sorting presented in SOP 3.2.
Figure 3.6 Wire loops and spatulas that can be sterilized in a flame.
Figure 3.7 Spread and streak on an agar plate a drop of a young impure cyanobacterial culture. (A) Principle of spread and streak of an impure colony on an agar plate within four areas. Area 1: For spreading on 1/3 of the plate; Area 2: To streak from Area 1; Area 3: To streak through Area 2; Area 4: To streak through Area 3 without reaching Area 1. (B) Example of a well-grown axenization plate, from the most concentrated Area 1 to the most diluted one (Area 4). Other examples for spread and streak on an agar plate are presented in Rippka (1988).
Figure 3.8 Flowchart showing the method for axenization presented in SOP 3.3.
Figure 3.9 Flowchart showing the method for preparation of culture medium Z8.
Figure 3.10 Some equipment for the transfer of liquid cultures.
Figure 3.11 Flowchart showing the methods for maintenance of living cultures presented in SOP 3.5.
Figure 3.12 All the equipment needed to prepare the tube for the cryopreservation of a healthy cyanobacterial culture under the hood.
Figure 3.13 Near the hood: vortex, a handy reservoir of liquid nitrogen, gloves and face protection for the manipulator, the clamps, the cryotube rack, and the cryocanes.
Figure 3.14 Flowchart showing the methods for cryopreservation and recovery presented in SOP 3.6.
Chapter 4: Taxonomic Identification of Cyanobacteria by a Polyphasic Approach
Figure 4.1 (A) Bloom of
Microcystis aeruginosa
in a lake of the Institute of Botany of São Paulo (photo: A. Tucci). (B) Yellow coloration from a bloom of
Cylindrospermopsis raciborskii
(photo: H.D. Laughinghouse IV). (C) Bloom of
Planktothrix rubescens
; Lake Ledro (photo: A. Boscaini). (D) Bloom of
Planktothrix rubescens
, showing a particular and enlarged section of Lake Ledro (photo: A. Boscaini). (E) Bloom of
Dolichospermum lemmermannii
; Lake Garda (photo: N. Salmaso). (F) Bundles of
Aphanizomenon flos-aquae
looking like larch needles in Bergnappweiher, a small pond used for bathing in Bavaria (photo: K. Teubner). (G) Emus crossing a mixed bloom of cyanobacteria and euglenophytes at the Zoo of Rio Grande do Sul (photo: H.D. Laughinghouse IV and V.R. Werner). (H)
Microcystis aeruginosa
, Lake Garda, net sample, magnification 100× (photo: N. Salmaso). (I)
Cylindrospermopsis raciborskii
, scale bar 10 μm (photo: V.R. Werner). (J)
Planktothrix rubescens
, Lake Como, scale bar 10 μm (photo: A. Boscaini). (K)
Dolichospermum lemmermannii
with attached vorticellids, Lake Garda, scale bar 40 μm (photo: S. Shams). (L) Cluster of akinetes of
Dolichospermum lemmermannii
, Lake Garda, scale bar 40 μm (photo: N. Salmaso). (M) Aggregate of
Aphanizomenon flos-aquae
filaments, Lake Winnipeg; flakes are between 3 and up to 10–20 mm (photo: H.J. Kling). (N)
Dolichospermum crassum
(planktic), scale bar 50 μm (photo: H.D. Laughinghouse IV). (O)
Tychonema bourrellyi
, Lake Garda, scale bar 20 μm (photo: N. Salmaso). Source: Courtesy of Tucci, Boscaini, Teubner, Werner, Shams, Kling.
Figure 4.2 Identification of cyanobacteria in a bloom sample from a freshwater lake (Etagnac, Poitou-Charentes, France, 10/08/2000) by phase contrast (A, C) and autofluorescence (B, D) microscopy. A green filter (Zeiss 15, excitation band path 546/12 nm, beam splitter 580 nm, long path emission 590 nm) was used for fluorescence detection. Bar markers: 10 μm. In (B) note the red fluorescence of “picocyanobacteria” of various different cell dimensions, in addition to the filamentous cyanobacterium of unknown taxonomic position. The presence of the unicellular cyanobacteria cannot be differentiated from other bacteria within and around the mineral precipitate and cellular debris in the corresponding phase contrast image (A). In the absence of the autofluorescence image (B), even the identification of the filamentous cyanobacterium containing large light refractile inclusions (most likely polyalkanoate) would have been difficult, given that morphologically similar non-cyanobacterial taxa exist. The phase contrast image (C) shows a colony of
Woronichinia
, and some individual cells of this cyanobacterial genus. Note the doublets of lengthwise dividing cells and light refractile gas vesicle clusters (“aerotopes”) typical of this planktonic taxon. Gas vesicle collapse resulting from the pressure exerted by the cover slip gives some of the cells a darker appearance. Note also the mucilaginous appendages interconnecting the
Woronichinia
cells within the colony, and associated thin rod-shaped cells. From the corresponding autofluorescent image (D), it is clear that in this field of view
Woronichinia
is the only representative of the cyanobacterial lineage. Differences in the intensity of red fluorescence may be attributable to partial loss of phycocyanin, and/or the fact that not all
Woronichinia
cells are located in the same plane of observation (photos: R. Rippka).
Figure 4.3 PCR products of
rpo
B analyzed by electrophoresis on agarose gel, and staining with ethidium bromide. The amplicons were obtained with primers
rpo
BanaF and
rpo
BanaR (Table 4.2). The bands refer to strains isolated from samples collected in lakes Como (October 2014), Maggiore (November 2014) and Garda (November 2014). “+” positive (
Anabaena
strain 37 UHCC) and “–” negative controls. The first line reports the bands obtained from the DNA ladder; the size of the ladder (left scale) is in base pairs; the white arrow indicates the direction of movement of the ladder DNA. The
rpo
B amplicons are all located at a level corresponding to the region between the molecular marker bands of 500 and 750 bp. The sequencing of the PCR products and phylogenetic analyses allowed associating the
rpo
B amplicons to
Dolichospermum lemmermannii
(Salmaso
et al.
2015b).
Figure 4.4 Maximum likelihood (ML) rooted (
M. aeruginosa
) tree topology of
Dolichospermum lemmermannii
strains isolated from water samples collected in Lake Garda (LN871475, May 2014; LN871471, November 2013) and resurrected from akinetes isolated from the deep sediments of Lake Garda (1989–2012) (highlighted in blue). The other two
D. lemmermannii
strains were isolated from Finnish water bodies. The
Dolichospermum
species included in the analysis are identified by names and accession numbers. The tree is based on the alignment of the
rpo
B gene using R 3.3.0 and PhyML 3.1 (cf. SOP 4.2). The analysis of DNA substitution models indicated in the K80+G the best-fitting evolutionary model. Branch support aLRT-SH-like option values < 0.7 were not shown; the selection threshold for SH-like supports should be in the 0.8–0.9 range (Guindon
et al.
2010).
Figure 4.5 Polyphasic approach for the detection and identification of cyanobacteria from environmental samples based on the analysis of strains obtained by culture-dependent methods. The gray boxes include the criteria used in the identification of cyanobacteria. The determination of secondary toxic (and nontoxic) metabolites and the analysis of genes encoding cyanotoxin synthesis (dashed box) add further information on the characteristics of species and their toxigenic potential.
Chapter 5: Nucleic Acid Extraction
Figure 5.1 Schematic overview of the protocols described in this chapter.
Figure 5.2 Genomic DNA extracted from
Chamaesiphon
strain PCC 6505 and separated by agarose gel electrophoresis (0.8%, w/v) using ethidium bromide staining. Bands 1, 2, 3, and 4 were loaded with DNA extracts from different culture flasks (1 μg of DNA). M, λPstI marker (in bp).
Figure 5.3 Flow diagram showing steps of DNA extraction from cells collected on glass fiber filters (see SOP 5.3).
Figure 5.4 Flow diagram showing steps of individual filament/colony isolation, purification by washing in medium and DNA extraction using sonification.
Figure 5.5 Flow diagram showing steps related to DNA extraction from food supplements.
Figure 5.6 Flow diagram showing steps of RNA extraction from cells collected on filters.
Figure 5.7 Flow diagram showing steps of cDNA synthesis in SOP 5.8 from RNA extracted as described in SOP 5.7.
Chapter 8: DNA (Diagnostic) and cDNA Microarray
Figure 8.1 Workflow for two color DNA microarray experiments.
Chapter 9: Analysis of Toxigenic Cyanobacterial Communities through Denaturing Gradient Gel Electrophoresis
Figure 9.1 Scheme showing the principle of DGGE analysis with two main applications: clustering analyses and sequencing of separated bands.
Figure 9.2 DGGE machine with equipment. 1, lid; 2, buffer tank; 3, inner core; 4, casting stand; 5, gradient maker; 6, spacers; 7, comb, 8, glass plates; 9, clamps.
Chapter 10: Monitoring of Toxigenic Cyanobacteria Using Next-Generation Sequencing Techniques
Figure 10.1 Standard workflow for the NGS of 16S rRNA amplicons for monitoring toxic cyanobacteria (see SOP 5.3 for quantitative DNA extraction from environmental samples).
Figure 10.2 Standard workflow for processing NGS data with USEARCH. Raw 454 data can be converted to a FASTQ format, and join at Step 2. This, however, comes at the cost of quality information loss, and specialized quality filtering algorithms and programs for pyrosequencing data (e.g. MOTHUR) that output FASTA files will yield better results. These, as well as other quality filtered FASTA, can start at Step 5.
Chapter 11: Application of Molecular Tools in Monitoring Cyanobacteria and Their Potential Toxin Production
Figure 11.1 Evolution of the number of new sequences, deposited in the GenBank nucleotide database, for genes involved in the biosynthesis of cyanotoxins. Example of a Boolean search string used: (((microcystin[Title]) OR
mcy
*[Title]) AND cyanobacter*[Organism]) AND (“1997”[Publication Date] : “1997”[Publication Date]). The first (capitalized) letter of each toxin name indicates the release date of the first annotated gene sequence for this toxin (see text to an accession number and publication reference).
Figure 11.2 Percentage of publications on toxic cyanobacteria that used molecular methods, until mid-2016; by cyanotoxin. Example of a Boolean search string used: TITLE-ABS-KEY ((cylindrospermopsin* AND (cyr* OR aoa*)).
Figure 11.3 Overview of the main applications of molecular-based methods for cyanobacterial (and/or their toxins) detection and identification. (A) General flow diagram showing main stages. (B) Methodological notes or applications description; in bold are possible outcomes or achievements, at this particular stage. (C) Examples of relevant literature for topics identified in (B), within square brackets. * gDNA and eDNA regard to isolate genomic DNA and environmental DNA, respectively; ** in the scarce studies on cyanobacteria, hybridization is preceded by PCR (see stage 4).
List of Tables
Chapter 4: Taxonomic Identification of Cyanobacteria by a Polyphasic Approach
Table 4.1 Selection of Web databases and sites relevant for the taxonomic classification and nomenclature of toxigenic cyanobacteria
Table 4.2 Selection of genetic markers and primers used for the identification of cyanobacteria and phylogenetic studies. The list is not exhaustive and includes only some of the most frequently targeted genes and loci. The primer specificity quoted for cyanobacteria in general, or particular groups/taxa is based on the currently available references. Some primers target a locus in all bacteria but may be used for PCR amplification, if the second primer is specific for the cyanobacterial phylum, or specific lineages therein. The primer sequences, PCR conditions, and procedure details are reported in the cited references, which should be consulted
Table 4.3 Examples of taxonomic identification of cyanobacteria using the polyphasic approach
Chapter 6: Conventional PCR
Table 6.1 Commonly used primers for the detection and differentiation of cyanotoxin biosynthesis genes and for general detection of cyanobacterial DNA
Table 6.2 Troubleshooting guide for the interpretation of PCR results
Chapter 7: Quantitative PCR
Table 7.1 Troubleshooting qPCR results
Chapter 9: Analysis of Toxigenic Cyanobacterial Communities through Denaturing Gradient Gel Electrophoresis
Table 9.1 Loci used in DGGE analysis
Chapter 10: Monitoring of Toxigenic Cyanobacteria Using Next-Generation Sequencing Techniques
Table 10.1 Overview of sequencing platforms, comparison of the different technologies and platforms currently available for amplicon sequencing
Chapter 11: Application of Molecular Tools in Monitoring Cyanobacteria and Their Potential Toxin Production
Table 11.1 List of studies showing the main applications (and some methodological features) in which the detection/identification of tox(igen)ic cyanobacteria have been pursued, by molecular-based method used
Table 11.2 Examples of other applications/studies where molecular methods were applied for the detection/identification of cyanobacteria (including potentially toxic or harmful taxa) and of the microbial communities related to them
Appendix: Supplementary Tables
Table S6.1 Reference strains: positive and negative controls for PCR performed in SOPs 6.1–6.9
Table S6.2 Oligonucleotides to observe the mutations occurring within the
mcy
gene cluster of
Planktothrix
sp. Chen
et al.
(2016), see SOP 6.7
Table S7.1 Examples of quantitative PCR assays to detect and quantify microcystin-, nodularin-, cylindrospermopsin-, and saxitoxin-producing cyanobacteria
Table S11.1 List of studies showing the main different applications (and some methodological features) in which the detection/identification of tox(igen)ic cyanobacteria have been pursued, by molecular-based method used. Note: this is the full form of Table 11.1
Molecular Tools for the Detection and Quantification of Toxigenic Cyanobacteria
Edited by
RAINER KURMAYER
Research Institute for Limnology, University of Innsbruck, Austria
KAARINA SIVONEN
Department of Food and Environmental Sciences, Division of Microbiology and Biotechnology, University of Helsinki, Finland
ANNICK WILMOTTE
InBios - Center for Protein Engineering, University of Liège, Belgium
NICO SALMASO
Research and Innovation Centre, Fondazione Edmund Mach - Istituto Agrario di S. Michele all'Adige, Italy
This edition first published 2017
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Library of Congress Cataloging-in-Publication Data Applied for
ISBN: 9781119332107
Cover image:
Main image:
Bloom of the filamentous cyanobacterium Planktothrix rubescens (“Burgunderblutalge”) in Mindelsee (near Constance, Germany) in December 2006 (Photocredit: Guntram Christiansen). The surface water sample contained the hepatotoxin microcystin.
Inserts:
Top left: cyanoHAB occurrence (Photocredit: Elke Dittmann), (further three inserts courtesy of the editors). Second top left: Biomass used as food supplement, third top left: Single cyanobacteria filaments from Planktothrix rubescens in the light microscope (400x magnification), bottom left: the essence of molecular tools' application (DNA extraction, (q)PCR amplification, sequencing of PCR products).
Cover design by Wiley
Hans C.P. Matthijs (1951–2016): A passion for light and life
We are honored that the editors wish to dedicate this book to the memory of our dear colleague Dr. Hans C.P. Matthijs. Hans was an inspirational biochemist, who was fascinated by the life of cyanobacteria. He contributed nearly 40 years of groundbreaking research on their physiology and biochemistry. After many years of primarily fundamental research, his last years were dedicated to research with a distinctive societal relevance.
This book has been initiated and supported by the CYANOCOST action in which Hans has been very active. Besides contributing to the handbooks prepared in this action, Hans organized a training school on the effects of hydrogen peroxide on cyanobacteria in 2014.
Hans studied chemistry at the University of Amsterdam (UvA), the Netherlands, and obtained his PhD degree on the energy metabolism of the cyanobacterium Plectonema boryanum at the Vrije Universiteit Amsterdam (VU) in 1983. Afterward, Hans travelled abroad and worked at Brookhaven National Laboratory near New York in the United States, the University of the Mediterranean Aix-Marseille II, and the Washington University of St. Louis back in the United States. In 1991, Hans returned to the University of Amsterdam, where he was appointed Assistant Professor in the research group of Professor Luuc Mur. In 2001, he was promoted to Associate Professor and together with Professor Jef Huisman and Dr. Petra Visser he formed the scientific core of the research group Aquatic Microbiology of the new Institute for Biodiversity and Ecosystem Dynamics. During this time, Hans made significant contributions to fundamental new insights into cyclic electron transport around photosystem I and to structural and regulatory changes in photosynthesis due to stress induced by, for example, nitrogen and iron limitation.
In recent years, Hans explored the possibilities of LED lighting to enhance biomass production and save energy in algal biotechnology and horticulture. Employing the flashing light effect, a study he had performed in 1996, he hypothesized that photosynthesis could be just as efficient with a lot less light input if only one offered cells the right color of light at the right time. Together with a PhD student and a post-doc, the culture rooms were transformed into colorful labs with advanced LED lighting to strengthen his hypothesis.
Another recent topic of Hans' interest was the termination of harmful cyanobacterial blooms with hydrogen peroxide. Owing to a key difference in the Mehler reaction at photosystem I, cyanobacteria are much more sensitive to hydrogen peroxide than eukaryotic algae. Several lakes were treated with great success and two PhD students and a post-doc looked to optimize the procedure and to fully understand the implications of the treatment.
Additionally, Hans was interested in the effects of rising levels of CO2 on the CCM of cyanobacteria. In April 2016, one of his students defended a PhD thesis on this topic. Although Hans was already seriously ill at that point, he insisted on joining the defense ceremony. Hans presented a very inspirational laudation, one that we will never forget. It was his last public appearance. Hans passed away on April 17, 2016, due to the result of pancreatic cancer. While Hans is no longer with us, his creative insights and ideas for research will live on.
Jef Huisman, Petra Visser, and Merijn SchuurmansDepartment of Aquatic Microbiology, University of Amsterdam, The Netherlands
List of Contributors
Andreas Ballot
Norwegian Institute for Water Research, Oslo, Norway
Stephan Blank
Research Institute for Limnology, University of Innsbruck, Mondsee, Austria
Alexandra Bukowska
Department of Microbial Ecology and Environmental Biotechnology, Faculty of Biology, University of Warsaw, Poland
Camilla Capelli
Research and Innovation Centre, Fondazione Edmund Mach – Istituto Agrario di S. Michele all'Adige, Italy
Qin Chen
Research Institute for Limnology, University of Innsbruck, Mondsee, Austria, and, College of Natural Resources and Environment, Northwest A & F University, Yangling, P. R. China
Guntram Christiansen
Research Institute for Limnology, University of Innsbruck, Mondsee, Austria, and Miti Biosystems GmbH, Max F. Perutz Laboratories, Vienna, Austria
Samuel Cirés
Department of Biology, Autonomous University of Madrid, Spain
Li Deng
Institute of Virology, Helmholtz Zentrum Munich, Munich, Germany, and Research Institute for Limnology, University of Innsbruck, Mondsee, Austria
Elke Dittmann
Institute of Biochemistry and Biology, University of Potsdam, Germany
Rehab El-Shehawy
Institute IMDEA Water, Alcalá de Henares (Madrid), Spain
Elisabeth Entfellner
Research Institute for Limnology, University of Innsbruck, Mondsee, Austria
David P. Fewer
Department of Food and Environmental Sciences, Division of Microbiology and Biotechnology, University of Helsinki, Finland
Ilona Gągała
European Regional Centre for Ecohydrology of the Polish Academy of Sciences, Łódź, Poland
Muriel Gugger
Collection of Cyanobacteria, Institut Pasteur, Paris, France
Sigrid Haande
Norwegian Institute for Water Research, Oslo, Norway
Camilla H.C. Hagman
Norwegian Institute for Water Research, Oslo, Norway
Kaisa Haukka
Department of Food and Environmental Sciences, Division of Microbiology and Biotechnology, University of Helsinki, Finland
Jean-Francois Humbert
Institute of Ecology and Environmental Sciences, UPMC, Paris, France
Iwona Jasser
Department of Microbial Ecology and Environmental Biotechnology, Faculty of Biology, University of Warsaw, Poland
Konstantinos Kormas
Department of Ichthyology and Aquatic Environment, University of Thessaly, Volos, Greece
Ewa Kozłowska
Department of Immunology, Faculty of Biology, University of Warsaw, Poland
Rainer Kurmayer
Research Institute for Limnology, University of Innsbruck, Mondsee, Austria
H. Dail Laughinghouse IV
Fort Lauderdale Research and Education Center, University of Florida/IFAS, Davie, United States of America, and Department of Botany, MRC-166, National Museum of Natural History – Smithsonian Institution, Washington, United States of America
Joanna Mankiewicz-Boczek
European Regional Centre for Ecohydrology of the Polish Academy of Sciences, Łódź, Poland, and Department of Applied Ecology, Faculty of Biology and Environmental Protection, University of Lodz, Poland
Hans C.P. Matthijs
Department of Aquatic Microbiology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, The Netherlands
Cristiana Moreira
Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), University of Porto, Matosinhos, Portugal
Dagmar Obbels
Laboratory of Protistology and Aquatic Ecology, Department of Biology, Ghent University, Belgium
Igor S. Pessi
InBios – Center for Protein Engineering, University of Liège, Belgium
Antonio Quesada
Department of Biology, Autonomous University of Madrid, Spain
Vitor Ramos
Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), University of Porto, Matosinhos, Portugal, and Faculty of Sciences, University of Porto, Portugal
Anne Rantala-Ylinen
Department of Food and Environmental Sciences, Division of Microbiology and Biotechnology, University of Helsinki, Finland
Rosmarie Rippka
Institut Pasteur, Unité des Cyanobactéries, Centre National de la Recherche Scientifique (CNRS) Unité de Recherche Associé (URA) 2172, Paris, France
Martin Saker
Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), University of Porto, Matosinhos, Portugal, and Alpha Environmental Solutions, Dubai, United Arab Emirates
Nico Salmaso
Research and Innovation Centre, Fondazione Edmund Mach – Istituto Agrario di S. Michele all'Adige, Italy
Henna Savela
Department of Biochemistry/Biotechnology, University of Turku, Turku, Finland
J. Merijn Schuurmans
Department of Aquatic Microbiology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, The Netherlands
Kaarina Sivonen
Department of Food and Environmental Sciences, Division of Microbiology and Biotechnology, University of Helsinki, Finland
Maxime Sweetlove
Laboratory of Protistology and Aquatic Ecology, Department of Biology, Ghent University, Belgium
Vitor Vasconcelos
Faculty of Sciences, University of Porto, Porto, Portugal, and Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), University of Porto, Matosinhos, Portugal
Elie Verleyen
Laboratory of Protistology and Aquatic Ecology, Department of Biology, Ghent University, Belgium
Wim Vyverman
Laboratory of Protistology and Aquatic Ecology, Department of Biology, Ghent University, Belgium
Annick Wilmotte
InBios – Center for Protein Engineering, University of Liège, Belgium
About the Editors
Dr. Rainer Kurmayer, PhD, Assoc. Prof
Research Institute for Limnology, University of Innsbruck, Mondsee, Austria
Professional Career
2014–present: Associate Professor, Research Institute for Limnology, University of Innsbruck, University Doctorate, University of Vienna; 2007–2014: Senior Scientist Institute for Limnology, Austrian Academy of Sciences (ÖAW) and University of Innsbruck; 2001–2007, Junior Scientist Institute for Limnology, ÖAW; 1999–2001: post-doctorate position at the Federal Environmental Agency, Berlin, Germany. Training and Mobility of Researchers network “Toxin Production in Cyanobacteria” (TOPIC) within the 4th EU framework program; 1997–1999: PhD fellowship ÖAW “Effects of Cyanobacteria on Zooplankton.”
Research
Molecular ecology and evolution of toxin-producing algae (cyanobacteria); consequences of toxins produced by algal blooms; alpine lakes as in situ observatories for climate change effects.
Publications
Fifty international peer-reviewed publications, more than 20 popular scientific contributions (newspapers, magazines, TV).
Dr. Kaarina Sivonen, PhD, Prof
Department of Food and Environmental Sciences, Division of Microbiology and Biotechnology, University of Helsinki, Helsinki, Finland
Professional Career
2010–present: Professor in Microbiology at University of Helsinki; 2000–2010: Academy Professor, Junior (1990–1996) and Senior Scientist (1997–2000) of the Academy of Finland, Research scientist, project leader (1996–1997) at the Helsinki University. Visiting Research Associate at the University of Chicago, Department of Molecular Genetics and Cell Biology, Chicago, Illinois, USA (17.7.1991–31.12.1992). Visiting Scientist at Wright State University, Department of Biological Sciences, Dayton, Ohio, USA (15.11.1990–15.07.1991).
Research
Over 30 years of research on toxic and bioactive compound-producing cyanobacteria in Finnish freshwater and in the Baltic Sea. Maintaining culture collection of 1200 cyanobacteria. Current research covers ecology, physiology, genomics, post-genomics, biosynthesis, method development, structures, and bioactivities of new compounds.
Publications
One hundred and ninety-seven international peer-reviewed publications major as lead and/or corresponding author, number of popular scientific contributions (newspapers, magazines, TV).
Dr. Annick Wilmotte, PhD
InBios – Center for Protein Engineering, University of Liège, Liège, Belgium
Professional Career
1996–present: Research Associate of the FRS-FNRS (Belgian Funds for Scientific Research), University of Liège (eq. Principal Investigator); 1989–1996: Post-doctorate projects at University of Antwerp (BE), Department of Biochemistry (Molecular Biology Group), and the Flemish Institute of Biotechnology (BE) (Genetics and Biotechnology Lab) concerning cyanobacterial and fungal phylogeny, and gene transfers in soil bacteria, respectively; 1983–1989: PhD fellowship, University of Liège on the taxonomy of cyanobacteria; 1982–1983: Research at the University of Groningen (NL), “Plant Systematics” (Prof. Van Den Hoek & Stam). Coordinator of two EC projects on the molecular diversity of Antarctic microbial mats (MICROMAT) and the detection of planktonic cyanobacteria by DNA-chips (MIDI-CHIP) plus national projects on toxic cyanobacterial blooms in Belgium and the molecular diversity and biogeography of cyanobacteria in Polar Regions. Promotor of the BCCM/ULC public culture collection of cyanobacteria.
Research
Biodiversity, taxonomy, evolution, and molecular ecology and biogeography of cyanobacteria. Cultivation and cryopreservation of cyanobacterial strains.
Publications
Seventy-three international peer-reviewed papers (40 as first or last author), regular outreach conferences and expertise missions for the environmental protection of Antarctica.
Dr. Nico Salmaso, PhD
Research and Innovation Centre, Fondazione Edmund Mach – Istituto Agrario di S. Michele all'Adige, S. Michele all'Adige, Italy
Professional Career
2016–present: Head of the Hydrobiology Unit at the Istituto Agrario di S. Michele All'Adige, Fondazione E. Mach (FEM); 2011–2015: Head of the Limnology and River Ecology group at FEM; 2009–2010: Head of the Biocomplexity and Ecosystem Dynamics group at FEM; 2005–2008: Head of the Limnology and Fish Ecology group (FEM); 2007–2008: Deputy Coordinator of the Natural Resources Department (FEM); 1995–2004: Research Assistant at the University of Padua; 31/01/2014: National Scientific Habilitation 05/C1 – II Fascia, Ecology; 06/04/2017: National Scientific Habilitation 05/A1 – I Fascia, Botany. Responsible for several National and European (Central Europe Programme) projects.
Editorial activity
Editor-in-Chief of Advances in Oceanography and Limnology. Associate Editor of Cryptogamie-Algologie.
Research
Ecology of phytoplankton and cyanobacteria. Environmental and biotic factors promoting the development of toxic cyanobacteria. Detection of toxic genotypes. Invasive cyanobacteria.
Publications
Sixty international peer-reviewed publications, more than 75% as lead and/or corresponding author. More than 30 contributions on international non-ISI and national journals and book chapters.
About the Book
The strong interest in participating in the EU-COST Action CYANOCOST throughout Europe reflects: (1) the increasing global occurrence of cyanobacterial blooms; (2) associated adverse health, environmental, and economic impacts throughout the world; (3) the roles of international and national health and environmental agencies in establishing national monitoring and analysis strategies; and (4) the continuing growth of research at the international level on cyanobacterial blooms and cyanotoxins. Therefore, it was decided during the second project meeting in Madrid (November 2–4, 2012) that, in addition to the handbook on analytical methods edited by Jussi Meriluoto, Geoffrey A. Codd, and Lisa Spoof, a handbook on the various molecular detection methods for toxigenic cyanobacteria would be compiled. Rainer Kurmayer, Nico Salmaso, Kaarina Sivonen, and Annick Wilmotte were elected editors.
This book is designed as a handbook describing the molecular monitoring of diversity and toxigenicity of cyanobacteria (blue–green algae) in surface waters including lakes, rivers, drinking water reservoirs, and food supplements. In water bodies, cyanobacteria mass developments = cyanobacteria harmful algal blooms (cHAB) are highly favored by eutrophication and global temperature rise.
Chapters include a series of standard operational procedures (SOPs). The list of SOPs contains the molecular tests that could be routinely used in environmental monitoring. The introductory chapters are concise papers written according to an a priori given outline, covering the purpose, methodological details, and advantages and disadvantages of the various tools. Although each introductory chapter refers to the individual SOPs, each SOP should be considered an independent unit with its own (essential) references section.
The introductory chapter gives an overview of the current knowledge on the genetic basis of cyanotoxin synthesis in cyanobacteria (Chapter 1). In particular it provides up-to-date overviews plus the necessary foundation for the subsequent use of molecular tools, analysis, and the interpretation of the results. The other chapters are dedicated to the individual steps from water (food supplement) sampling (Chapter 2), nucleic acid extraction (Chapter 5), and downstream analysis, including PCR- (Chapter 6) and qPCR- (Chapter 7), based methods but also more traditional tools of genotyping (Chapter 9), diagnostic microarrays (Chapter 8), and community characterization by next-generation sequencing techniques (Chapter 10). One chapter is dedicated to the isolation of cyanobacteria strains from water samples (Chapter 3) and another contains guidelines to taxonomic assignment (Chapter 4). The last chapter is dedicated to a review of the application of molecular tools (Chapter 11). The practical chapters (SOPs) contain the necessary protocol details to enable trained operators to perform the described application. All published SOPs have been used in the lab of the respective authors for years and are considered robust. They are based on reference strains which are reliably available from international culture collections.
The book is intended to be used by trained professionals analyzing cyanobacterial diversity and toxigenicity in water samples in the laboratory in both academic and governmental institutions, as well as technical offices and agencies which are in charge of waterbody surveillance and monitoring. Students will learn important methods' standards of essential protocols including steps from sampling to results evaluation. More generally, the book will benefit the reader by (1) increasing the knowledge of the currently available molecular toolbox and (2) enabling them to carry out state-of-the-art analyses on the toxigenicity of cyanobacteria in surface water.
Preface
Cyanobacteria (blue–green algae) are among the oldest organisms on earth, and fossil records of cyanobacteria have been described from stromatolites dating back billions of years. Probably because of their long evolutionary history, cyanobacteria are highly successful and colonize all available habitats. Perhaps unsurprisingly, cyanobacteria also frequently dominate aquatic communities and can form mass accumulations that often appear on the water surface, so-called algal blooms. These algal blooms can cover hundreds of square meters or even square kilometers on the surface of the water and substantially deteriorate water quality and threaten the environment and humans by a number of problems, not least because of the production of poisonous compounds, so-called cyanotoxins.
During the 1970s and 1980s, major progress was made in the identification of the source organisms and the symptoms of toxification. The first chemical structures of toxins were derived during the 1970s and 1980s, for example microcystin-LA and anatoxin-a. The number of fully characterized toxin variants substantially increased during the 1990s as well as the description of cyanobacterial taxa producing the toxin variants. Further major progress was achieved in the detection and quantification of cyanobacterial toxins in the environment as well as in the knowledge concerning their global distribution and occurrence.
In general the cyanobacterial toxins can be classified into three functional groups: hepatotoxins (e.g. microcystin/nodularin, cylindrospermopsin), neurotoxins (e.g. saxitoxin, anatoxin-a), and irritant-dermal toxins (e.g. lipopolysaccharides). Typically, these toxins are inside the cells, but can suddenly be released into the water during cell lysis, for example following algaecide treatment. In 1997, the World Health Organization (WHO) published a guideline value on the hepatotoxic microcystin-LR in drinking water. In the last revision on guidelines for drinking water quality performed by the WHO, in 2004, cyanobacteria were considered a relevant source of organic toxic compounds deteriorating drinking water quality worldwide.
In the last two decades, major progress has been made in elucidating the genetic basis of toxin production in cyanobacteria. In particular the biosynthesis of the abundant cyanobacterial toxins has been discovered: microcystin, nodularin, cylindrospermopsin, saxitoxin, and anatoxin-a. Advances in this field have made it possible to develop detection methods to study producers of these toxins. Since the toxin-producing and non-producing strains cannot be distinguished under the microscope, these molecular tools provide shortcut methods for detection.
Although the knowledge to apply genetic techniques has increased significantly, the use of those techniques for environmental studies, water management, and risk assessment is still in its infancy. We think it would be worthwhile to make an effort to ensure that molecular genetic discoveries are translated into tools that can be used in field and laboratory monitoring. This book, the first of its kind, was written as a handbook for the molecular monitoring of toxigenicity of cyanobacteria in surface waters including lakes, rivers, drinking water reservoirs, and food supplements.
The book is structured into two main parts. Each chapter is linked to a variable number of specific protocols (called standard operational procedures, SOPs). We hope that this book will contribute to a greater acceptability and use of molecular tools in monitoring of potential cyanotoxicity worldwide. We further hope that this book will be useful for graduate students, laboratory technicians, professionals, and supervisors applying molecular tools.
Rainer Kurmayer, Kaarina Sivonen, Annick Wilmotte and Nico SalmasoMondsee, August 2016
Acknowledgments
The book is a key output of a major European Cooperation in Science and Technology (COST) Action (ES1105; 2012–2016), www.cyanocost.com. This action, “Cyanobacterial Blooms and Toxins in Water Resources: Occurrence, Impacts and Management” included over 100 active participants from 33 countries and served to a large extent the transfer of know-how between institutions on the topic. The proposal and the coordination of this activity by Triantafyllos Kaloudis, Athens Water Supply and Sewerage Company, Greece and Ludek Blaha, RECETOX, Faculty of Sciences, Czech Republic is gratefully acknowledged.
Beside the many authors, the colleagues who contributed to this handbook are gratefully acknowledged. In particular, as a leader of the relevant Work Package 1 (Occurrence of cyanobacteria and cyanotoxins), Jussi Meriluoto, Åbo Akademi University, Finland is appreciated for his guidance and support toward finalization of this demanding book project.
Many thanks to the team from John Wiley & Sons, Ltd, Chichester, UK for their assistance in contract preparation (Jenny Cossham and Emma Strickland) and project editing (Ashmita Thomas Rajaprathapan and Tim Bettsworth).
COST is a pan-European intergovernmental framework. Its mission is to enable breakthrough scientific and technological developments leading to new concepts and products strengthening Europe's research and innovation capacities. It allows researchers, engineers, and scholars to jointly develop their own ideas and take new initiatives across all fields of science and technology, while promoting multi- and interdisciplinary approaches. COST aims to foster a better integration of less research-intensive countries to the knowledge hubs of the European Research Area. The COST Association, an international not-for-profit association under Belgian law, integrates all management, governing, and administrative functions necessary for the operation of the framework. The COST Association currently has 36 member countries (www.cost.eu).
COST is supported by the EU Framework Programme
Horizon 2020
List of other research projects to be acknowledged (Principal Investigators in Alphabetical Order)
Principal Investigator
Project (Title)
Grant Number
Organization
Ilona Gągała, Joanna Mankiewicz-Boczek
Explanation of cause-effect relationships between the occurrence of toxinogenic cyanobacterial blooms, and abiotic and biotic factors with particular emphasis on the role of viruses and bacteria
N N305 096439
National Science Centre
Muriel Gugger
Cyanobacterial toxin production and Photoprotection processes in a changing
ANR-15-CE34-002
Agence Nationale de la Recherche (ANR)
EnviRonment (CYPHER)
Rainer Kurmayer
Mobilomics of toxin production in cyanobacteria
P24070
Austrian Science Fund (FWF)
Vitor Ramos
fellowship
SFRH/BD/80153/2011
Fundação para a Ciência e a Tecnologia (FCT
Henna Savela (Timo Lövgren)
nucleoTracker
40013/10
Finnish Funding Agency for Innovation
Vitor Vasconcelos
Innovation and Sustainability in the Management and Exploitation of Marine Resources (INNOVMAR)
NORTE-01-0145-FEDER-000035, Research Line NOVELMAR
Northern Regional Operational Program (NORTE2020), European Regional Development Fund (ERDF)
Elie Verleyen, Annick Wilmotte
Saving Freshwater biodiversity REsearch Data (SAFRED)
BR/154/A6/SAFRED
Belgian Science Policy Office
Wim Vyverman
Manscape
EV/29
Belgian Science Policy Office
Wim Vyverman
Pondscape
SD/BD/02A and SD/BD/02B
Belgian Science Policy Office
Wim Vyverman, Annick Wilmotte
Climate Change and Antarctic Microbial Biodiversity (CCAMBIO)
SD/BA/03A
Belgian Science Policy Office
Wim Vyverman, Annick Wilmotte
Preservation of microalgae in BCCM collections (PRESPHOTO)
BR/132/A6
Belgian Science Policy Office
Wim Vyverman, Annick Wilmotte
Cyanobacterial blooms: toxicity, diversity, modelling and management (B-BLOOMS2)
SD/TE/01A
Belgian Science Policy Office
Wim Vyverman, Annick Wilmotte
Cyanobacterial blooms: toxicity, diversity, modelling and management (B-BLOOMS1)
EV/34
Belgian Science Policy Office
Annick Wilmotte
High-throughput pyrosequencing of cyanobacterial populations (PYROCYANO)
CR.CH.10-11-1.5139.11
FRS-FNRS
Annick Wilmotte
BIPOLES : Geographical and ecological distribution of cyanobacteria from Antarctica and the Arctic
FRFC-2.4570.09
FRS-FNRS
Chapter 1Introduction
Rainer Kurmayer1*, Kaarina Sivonen2 and Nico Salmaso3
1Research Institute for Limnology, University of Innsbruck, Mondsee, Austria
2Department of Food and Environmental Sciences, Division of Microbiology and Biotechnology, University of Helsinki, Helsinki, Finland
3Research and Innovation Centre, Fondazione Edmund Mach – Istituto Agrario di S. Michele all'Adige, S. Michele all'Adige, Italy
* Corresponding author: [email protected]
1.1 A Brief Historical Overview
During the last two decades, genetic methods have significantly increased our understanding of the distribution of genes involved in the production of toxins within the phylum of cyanobacteria (e.g. Sivonen and Börner, 2008; Dittmann et al., 2013; Méjean and Ploux, 2013). Early on the synthesis pathways of microcystin in the three genera Microcystis, Planktothrix, and Anabaena (Tillett et al., 2000; Christiansen et al., 2003; Rouhiainen et al., 2004) and of the closely related nodularin have been elucidated (Moffitt and Neilan, 2004). Further, the elucidation of the genes involved in cyanotoxin synthesis increased the understanding of its inheritance and evolution, (e.g. the phylogenetically derived conclusion on the evolutionary age of the microcystin/nodularin synthesis pathway) (Rantala et al., 2004) implying that potentially all cyanobacteria are able to produce microcystins, and, indeed, the number of cyanobacterial genera discovered to produce microcystins is consistently increasing (e.g. Calteau et al., 2014).
Subsequently, the elucidation of the synthesis pathways of other toxins has been achieved, that is first results suggested the involvement of polyketide synthases (PKS) and an amidinotransferase in the synthesis of cylindrospermopsin in Aphanizomenon (Shalev-Alon et al., 2002; Kellmann et al., 2006) which then led to the identification of the first putative cylindrospermopsin gene cluster (cyr) in Cylindrospermopsis (Mihali et al., 2008). Other cylindrospermopsin synthesis gene clusters followed, in particular for Oscillatoria (Mazmouz et al., 2010), for Aphanizomenon (Stüken and Jakobsen, 2010), Raphidiopsis curvata, and Cylindrospermopsis raciborskii (Jiang et al., 2014). In general, compared with mcy genes (encoding the synthesis of microcystins), there is more shuffling of genes, and eleven genes cyrA–K are thought to make the core of the cyr gene cluster.
Similarly, candidate genes for saxitoxin biosynthesis have been isolated and the sequence of the complete putative saxitoxin biosynthetic gene cluster (sxt) was obtained (Kellmann et al., 2008a,b). This work started with screening of putative saxitoxin biosynthetic enzymes in cyanobacterial isolates, using a degenerate PCR approach, resulting in identification of an O-carbamoyltransferase that was proposed to carbamoylate the hydroxymethyl side chain of saxitoxin precursor. Orthologues of sxt1 were exclusively present in paralytic shellfish poisoning (PSP) strains of cyanobacteria and had a high sequence similarity to each other (Kellmann et al., 2008a). The first sxt gene cluster was sequenced from Cylindrospermopsis, and orthologous gene clusters from Anabaena, Aphanizomenon, Raphidiopsis, and Lyngbya followed (Murray et al., 2011). Genetic proof (e.g. by experimental gene inactivation) for the role of this gene cluster in saxitoxin biosynthesis is lacking. However, in the absence of suitable tools of genetic transformation, the functions of the ORF (open reading frame) were bioinformatically inferred, and this prediction was combined with the liquid chromatography-tandem mass spectrometry analysis of the biosynthetic intermediates (Kellmann and Neilan, 2007; Kellmann et al., 2008b).
The first anatoxin-a synthesis gene cluster (ana) was sequenced from Oscillatoria (Méjean et al., 2009). Subsequently anatoxin-a gene clusters were described from Anabaena (Rantala-Ylinen et al., 2011) and Cylindrospermum (Calteau et al., 2014). In the following, the genetic basis of microcystin/nodularin, cylindrospermopsin, saxitoxin, and anatoxin synthesis is described in more detail.
1.2 The Genetic Basis of Toxin Production
1.2.1 Microcystin and Nodularin
Microcystins are produced by planktonic freshwater genera Microcystis, Planktothrix, Dolichospermum, Nostoc, and Fischerella (Dittmann et al., 2013). Early studies, however, also documented microcystin production in a broader range of terrestrial genera, for example in Hapalosiphon (Prinsep et al., 1992) and later in Nostoc symbionts associated with fungi (Oksanen et al., 2004). In addition numerous freshwater and brackish water genera (e.g. Arthrospira, Oscillatoria, Phormidium, Pseudanabaena, Synechococcus, Synechocystis) have been reported to produce microcystins (Sivonen and Börner, 2008; Fiore et al., 2009; Bernard et al., 2017). In contrast, the closely related nodularin has been characterized from the brackish water species Nodularia spumigena and Nostoc (Bernard et al., 2017), while in the marine sponge, Theonella swinhoei, a nodularin analogue called motuporin has been found (de Silva et al., 1992). The sponge is known to harbor cyanobacterial symbionts. Microcystins are known for their toxicity because of the inhibition of eukaryotic protein phosphatases 1 and 2A resulting in the hyperphosphorylation and breakdown of the structural protein skeleton (Carmichael, 1994). Not at least because of the interference with eukaryotic signaling cascades, microcystins are considered tumor promotors under sublethal exposure conditions (Zhou et al., 2002).
Microcystins are cyclic heptapeptides and share the common structure cyclo (- D-Ala(1) - X(2) - D-MAsp(3) - Z(4) - Adda(5) - D-Glu(6) - Mdha(7)), where X and Z are variable L-amino acids (e.g., microcystin (MC)-LR refers to leucine and arginine in the variable positions), D-MAsp is D-erythro-ß-iso-aspartic acid, Adda is (2S, 3S, 8S, 9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid, and Mdha is N-methyl-dehydroalanine (Carmichael et al., 1988). Considerable structural variation has been reported, most frequently in positions 2, 4, and 7 of the molecule, and a large number of structural variants have been characterized (molecular weight 909 – 1115 Da), either from field samples or from isolated strains (e.g. Diehnelt et al., 2006; Spoof and Catherine, 2017). Nodularin (824 Da) and motuporin (812 Da) are both pentapeptides containing N-methyl-dehydrobutyrine (Mdhb) instead of Mdha(7) and lack D-Ala(1) and X(2) when compared with microcystin. Nodularin differs from motuporin due to the substitution of L-Arg(4) by L-Val(4) (Fig. 1.1).
Figure 1.1Scheme of the genetic basis of microcystin/nodularin synthesis in sequenced cyanobacterial genera. Arrows mark the bi-directional promotor region (from Tillett et al., 2000; Christiansen et al., 2003; Moffitt and Neilan, 2004; Rouhiainen et al., 2004; Fewer et al., 2013; Shih et al., 2013).
The biosynthesis of microcystin is catalyzed by nonribosomal peptide synthesis (NRPS) via the thio-template mechanism. This biosynthetic pathway has been intensively investigated in different bacteria and fungi, as their end products are often of great pharmaceutical value (Fischbach and Walsh, 2006). At present six gene clusters from five genera (Microcystis, Planktothrix, Anabaena, Nodularia, Fischerella) responsible for the biosynthesis of microcystin have been sequenced (Tillett et al., 2000; Christiansen et al., 2003; Rouhiainen et al., 2004; Moffitt and Neilan, 2004; Fewer et al., 2013; Shi et al., 2013) and the involvement in the production of microcystins could be proven by genetic manipulation in Microcystis and Planktothrix (Dittmann et al., 1997; Christiansen et al., 2003). The whole mcy gene cluster comprises a minimum of nine genes (ca. 55 kb) consisting of PKS, nonribosomal peptide synthetases (NRPS), and tailoring enzymes. It has a modular structure (Fig. 1.1), each module containing specific functional domains for activation (aminoacyl adenylation (A)-domains), thioesterification (thiolation domains) of the amino acid substrate and for the elongation (condensation (C)-domains) of the growing peptide. McyD, McyE, and McyG are responsible for the production of the amino acid Adda and the activation and condensation of D-glutamate. McyA, McyB, and McyC are NRPS and responsible for the incorporation of the other five amino acids in positions 7, 1, 2, 3, and 4 of the molecule (Tillett et al., 2000). Synthesis is thought to start with activation of phenyllactate through the adenylation domain of McyG (Hicks et al., 2006) followed by extension of the polyketide through McyD and McyE. The polyketide is then condensed with D-glutamate through McyE forming the core of the microcystin peptide (comprising the Adda side chain and the Glutamate). The residual amino acids are then condensed through McyA, B, C proteins and finally the peptide is cyclized through a dedicated type I thioesterase (Tillett et al., 2000). Several tailoring enzymes modify the growing peptide molecule, i.e. McyF (an aspartate racemase; Sielaff et al., 2003), McyI (a dehydrogenase involved in the production of the methyl aspartate unit MeAsp(3); Pearson et al., 2007), McyJ (an O-methyltransferase; Christiansen et al., 2003), McyH (a putative ATP binding cassette (ABC) transporter; Pearson et al., 2004), and McyT (a type II thioesterase; Christiansen et al., 2008).
Phylogenetic analyses relatively early lead to the conclusion that microcystin synthesis is an evolutionary old feature that has been lost repeatedly during the evolution of cyanobacteria (Rantala et al., 2004). The nda genetic cluster involved in the synthesis of nodularin was probably derived from the genes encoding microcystin synthesis via a gene deletion event (Moffitt and Neilan, 2004). The theory that cyanobacteria share a common microcystin-producing ancestor implies that
