Quantitative molecular, morphological

and analytical assessment of a major

blue-green algae bloom event in the

Sydney water supply

Brett Neilan

The University of New South

Wales

National Cyanobacteria Workshop, 2009

Overview

• Introduction• Cyanobacteria and their toxins

• Conventional detection methods

• Toxin gene clusters

• Molecular detection methods• PCR

• Q-PCR

• Microarrays

• Application of molecular methods• Warragamba Dam

• Future applications

cyanobacteria and algae

freshwater cyanotoxins

OMe

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NH

N

NH

NH

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COOH

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NH

NH2

NH

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OMe

Me MeNH

NH

O

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COOH

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NH

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NH

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CH2

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CH2

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Nodularin

R4

N

O

N

R2

R5

R3

OHNH2

H

HN

NH

H2N

R1

Saxitoxin

CH3NH2

O

Anatoxin-a

CH3NH2

O

Homoanatoxin-a

NHN

N

CH3

CH3

O

P

HOO

NH2 O

CH3

Anatoxin-a(S)

N NH NH NH

HN

SO3

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H

H H

OH

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OCylindrospermopsin

Microcystin

Conventional Detection Methods

for Cyanobacteria & their Toxins

• Microscopy• Morphological characteristics don’t correlate with

toxicity

• Bioassays (e.g. mouse)• Sensitive but not selective

• Ethical issues & high cost

• Analytical techniques (HPLC, MALDI-TOF etc.)• Can be sensitive & selective

• Laborious sample preparation protocols

• Expensive machinery

• Require toxin standards

Discovery of Cyanotoxin

Genes• Microcystin

• Tillett et al, 2000

• Nodularin• Moffitt and Neilan, 2004

• Cylindrospermopsin• Schembri et al, 2001; Mihali et al, 2008

• Saxitoxin• Kellmann et al, 2008

0 2000 4000 6000 8000 10000 12000 14000 16000

sxtAsxtB sxtD sxtE sxtF sxtG sxtH sxtIJ sxtK sxtL sxtM sxtNsxtC sxtN

Cyanotoxin Gene Clusters

C. Cylindrospermopsin synthetase C. raciborskii

D. Saxitoxin synthetase C. raciborskii

B. Microcystin synthetase M. aeruginosa

A. Nodularin synthetase N. spumigena

Use of Cyanotoxin Biosynthesis

Genes as Molecular Targets

• Polymerase Chain Reaction (PCR)-

based techniques

• Simple, rapid & cost-effective

• Sensitive, specific & amenable to high-

throughput analysis

Molecular Targets

• Phylogenetically identical species have variable toxicity

• Detection & differentiation of toxigenic cyanobacteria

• Best molecular targets for PCR are genes essential for toxin production and conserved within a range of toxic cyanobacterial genera

• Tailored reactions: Exploit conserved or divergent sequences within target when designing primers (depending on target group of organisms)

• Example: mcyE; Rantala et al, 2006 vs Jungblutt et al, 2006

Method Target locus Primer set Sample

type

Use Reference

PCR 16S rRNA 209F/409R Cultures Specific detection of the potentially

hepatotoxic genus, Microcystis

Neilan et al,

1997PCR/DNA

hybridization

NRPS adenylation

domain homologues

MTF2/MTR Cultures,

environment

al samples

Detection of non-ribosomal peptide

synthetase genes

Neilan et al,

1999

mcyB FAA/RAA Specific detection of hepatotoxigenic

strainsPCR mcyA N-

methyltransferase

domain

MSF/MSR Cultures,

environment

al samples

Specific detection of hepatotoxigenic

strains

Tillett et al,

2001

PCR 16S rRNA NTS/1494R Cultures Specific detection of toxic Nodularia (N.

spumigena ) strains

Moffitt et al,

2001PCR ndaF NPF/NPR Cultures Specific detection of toxigenic Nodularia

(N. spumigena ) strains

Moffitt et al,

2001PCR PKS homologues M4/M5 Cultures Specific detection of toxigenic C.

raciborskii and A. bergii strains

Schembri et

al, 2001NRPS homologues M13/M14

PCR + RFLP

analysis

16S rRNA ITS 23SRITS/16SCITS Cultures,

environment

al samples

Differentiation of potential microcystin-

producing and non-toxic strains

Neilan, 2002

Phycocyanin gene PCbF/PCaR

PCR mcyE/ndaF mcyE-F2/mcyE-R4 Environmen

tal samples

Specific detection of hepatotoxigenic

genera

Vaitomaa et

al, 2003;

Rantala et al,

2006mcyE mcyE-F2/mcyE-12R Specific detection of hepatotoxigenic

Anabaena strainsmcyE mcyE-F2/mcyE-R8 Specific detection of hepatotoxigenic

Microcystis strainsmcyE mcyE-F2/mcyE-plaR3 Specific detection of hepatotoxigenic

Planktothrix strainsPCR mcyE/ndaF

aminotransferase

domain

HEPF/HEPR Cultures,

environment

al samples

Specific detection of hepatotoxigenic

genera

Jungblutt et

al, 2006

Conventional PCR

False Results

• False positives

• Toxigenic but non-toxic (mutants)

• False negatives

• Divergent sequences not detected (new toxins?)

• Minimising false results

• Careful target selection & rigorous testing

• Supplementary toxicity tests (e.g. analytical tests)

Quantitative (q)-PCR

• Amplified DNA is quantified as it

accumulates using fluorescent dyes (e.g.

SYBR green) or oligonucleotide probes

• Detection, differentiation & quantification

of toxigenic cyanobacteria

• Measure gene expression - quantitative

reverse transcription (qRT) PCR

Real-time PCR mcyB MIf/MIr Cultures,

environment

al samples

Specific detection and quantification of

hepatotoxigenic Microcystis strains

Foulds et al,

2002

mcyA MISYf/MISYr

Real-time PCR Phycocyanin ITS 188F/254R Cultures,

environment

al samples

Specific detection and quantification of

cyanobacteria

Kurmayer

and

Kutzenberge

r, 2003Real-time PCR mcyB 30F/108R Specific detection and quantification of

hepatotoxigenic Microcystis strainsReal-time PCR mcyE mcyE-F2/MicmcyE-R8 Cultures,

environment

al samples

Specific detection and quantification of

hepatotoxigenic Microcystis strains

Vaitomaa et

al, 2003;

Rantala et al,

2006mcyE-F2/AnamcyE-12R Specific detection and quantification of

hepatotoxigenic Anabaena strainsReal-time PCR mcyA MSF/MSR-2R Cultures,

environment

al samples

Specific detection and quantification of

hepatotoxigenic strains (potential

microcystin producers)

Furukawa et

al, 2006

Real-time PCR ndaF ndaF8452/ndaF8640 Cultures,

environment

al samples

Specific detection and quantification of

hepatotoxigenic strains (potential nodularin

producers)

Koskenniemi

et al, 2007

Real-time PCR rpoC1 (RNA

polymerase)

various Cultures,

environment

al samples

Detection and quantification of C.

raciborskii

Rasmussen

et al, 2007

aoaA various Detection and quantification of toxigenic

C. raciborskii strains (potential

cylindrospermopsin-producers) aoaB various

aoaC various

Quantitative PCR

DNA Microarrays

• Similar technology to q-PCR

• Probes are hybridised to DNA chip

• High-throughput detection, differentiation

& quantification of toxigenic cyanobacteria

DNA Microarrays

DNA chip 16S rRNA various Cultures,

environment

al samples

Detection, differentiation and qantification

of cyanobacterial genera

Rudi et al,

2000

DNA chip 16S rRNA various Cultures,

environment

al samples

Detection, differentiation and qantification

of cyanobacterial genera

Castiglioni

et al, 2004

DNA chip mcyE/ndaF various Cultures,

environment

al samples

Detection, differentiation and qantification

of hepatotoxigenic genera

Rantala et al,

2008

Application of Technology -

Detection & Quantification of

Cyanobacteria and their Toxins in

Warragamba Dam

• Cyanobacterial bloom observed in August 2007

• Tentatively, M. aeruginosa

Warragamba Dam

• Supplies 80% of Sydney’s water

• 2007 drought saw dam at its lowest in 40 yrs

• High nutrient load, minimal mixing = favorable

conditions for algal growth

Aims

• Assess safety of Sydney’s major drinking

water supply

• Examine bloom population (toxic vs non-toxic

genotypes)

• Determine proportion of toxigenic strains

• Determine toxin type and concentration

Methods- Polyphasic

Approach• Conventional

• Cell counts

• Analytical

• HPLC, MS

• Biological assays

• Protein phosphatase inhibition

• Molecular

• PCR and q-PCR

Molecular Analysis• Water samples collected from 5 sites in dam

over several months

Concentrate cells

(filtration)

Extract DNA

PCR

(16S & mcyE)Sequencing

q-PCR

(16S & mcyB)

Identification of

major species

Quantification of

hepatotoxic species

Molecular targets PCR: mcyE & 16S

rRNA gene

•mcyE

•Mixed PKS/NRPS

•Essential for toxin biosynthesis

•16S rRNA

•Universal gene

•Cyanobacteria-specific primers

Molecular target q-PCR: mcyB

•NRPS

•Essential for toxin biosynthesis

Results: PCR & Sequencing

• Microcystis aeruginosa confirmed

• Toxigenic (mcyE+)

16S mcyE

Microcystis aeruginosa

www-cyanosite.bio.purdue.edu

www-cyanosite.bio.purdue.edu

ABC News Sep. 2007

Results: 16S q-PCR & Cell

counts• Monitoring total bloom population (cell

enumeration)

• Minor fluctuations over the 3 month period at all

sites

• Minimal differences between sites

• Results correlated well with microscopic counts

Sep-Dec 07

Eastern site

Results: mcyE q-PCR

• Toxigenic population• Major fluctuations at all sites

• Major differences between sites

• Important implications for water treatment protocols

Sep-Dec 07

Water Sample Analysis during

2nd Sep 07 - 11th Feb 08

0.00E+00

1.00E+03

2.00E+03

3.00E+03

4.00E+03

5.00E+03

6.00E+03

7.00E+03

8.00E+03

9.00E+03

1.00E+04

12/1

1/07

19/1

1/07

26/1

1/07

14/1

2/07

21/1

/08

28/1

/08

4/2/

08

11/2

/08

Date of the water samples collected

Cell

den

sit

y (

cell

s/u

l)

0.00%

1.00%

2.00%

3.00%

4.00%

5.00%

6.00%

% o

f Toxic

cell

s

Microscope

qRT-PCR

% toxicCollection date

Nov 07 - Feb 08

Analytical Methods

• ESI-MS/MS

• Microcystin isoform [Dha7]MCYST-LR

• LD50 of 250 ug/kg

• HPLC

• Microcystin conc. ~0.5-1 ug/L

• 1.3 ug/L max (WHO, 1992)

• Water “safe” to drink

Conclusions

• qRT-PCR revealed that Warragamba bloom is

dynamic with toxigenic species fluctuating in

time and space

• Important for predicting “times of danger”

• Important for choice of treatment strategy

• Non toxic bloom - filtration or chemical lysis

• Toxic bloom - filtration of cells

- removal of toxins?

Future Applications of Molecular

Detection Methods

• Automated high-throughput analysis

• DNA chips that detect,differentiate, and

quantify all toxigenic species

• Neurotoxin gene clusters

• Saxitoxins (PSP toxins)

• Seafood industry

Acknowledgements

• Sydney Catchment Authority

• Australian Research Council

• Diagnostic Technology

• Jasper Pengelly

• Leanne Pearson

• Alex Roberts