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Coding functions

DNA

ReplicationTranscription

Gene expression

RNA

Translation

Proteins

Reproduction (growth)

Enzymes: metabolic catalysts

Metabolism: generationof precursors of macro-molecules (sugars, aminoacids, fatty acids, etc.)

ADP + Pi ATPEnergy conservation:

Machine functions

TOXINSGenes fortoxinbiosynthesis

EnzymesNRPS

INTRODUCTION

Book ontoxins

Book onmoleculardetectionmethods

Toxic cyanobacteria

Most blooms are mixturesof different species/generaToxin producers?

Culture collections ofCyanobacteria

We learned which organisms aretoxic

We realized that strains of the samespecies differ (toxic and non-toxicstrains)

Which toxins are produced?

Material for method development andtesting

Molecular tools, genomics ....

Axenic strains, clonal isolates

Molecular methods -> improvedidentification of cyanobacteria

• Based on genes (geneticpotential of the organism)

• Allows phylogeneticcomparison of organisms

• Reveals genetic andphenotypic diversity

Toxic and non-toxic cyanobacteria cannot bedifferentiated by the microscopy ormolecular taxonomy (e.g. 16S rRNA)

Anabaena/DolichospermumAphanizomenon

Planktothrix

Microcystis

Lyra et al. 2001, IJSEM 51:513-526

Ribosomal pathway

+ post-translational

modifications

Nonribosomal pathway

Biosynthesis of bioactive compounds

Unusual non-proteinogenicamino acids

Adda

D-Glu Mdha

D-Ala

Leu

D-Me-AspArg

Gene clusters of the major producers ofmicrocystins and nodularin

peptide synthetasepolyketide synthaseaspartate racemasedehydrogenaseO-methyltransferaseABC transporterthioesterase

Microcystis PCC 7806 mcy gene cluster (Tillett et al., 2000)

A B CH G E DJ FI

Anabaena sp. 90 mcy gene cluster (Rouhiainen et al., 2004)

A B CH GE DJFI

G

Planktothrix CYA 126/8 mcy gene cluster (Christiansen et al., 2003)

A B C JHEDT

Nodularia NSOR10 nda gene cluster (Moffitt and Neilan, 2004)

A BI CF DEH G

Microcystis PCC 7806 mcy gene cluster (Tillett et al., 2000)

A B CH G E DJ FI

Anabaena sp. 90 mcy gene cluster (Rouhiainen et al., 2004)

A B CH GE DJFI

G

Planktothrix CYA 126/8 mcy gene cluster (Christiansen et al., 2003)

A B C JHEDT

Nodularia NSOR10 nda gene cluster (Moffitt and Neilan, 2004)

A BI CF DEH G

• Enzymes encoded by the microcystin (mcy) and nodularin synthetase(nda) genes

molecular methods for detecting toxin producers

Nostoc, Hapaloshiphon, Fisherella …

mcyH

CM KSKSACPKSACP KR ACP KR

TE

N-MT

M

MM

MM

MM

MM

M

Adda

MM

M

M

D-Glu

ATDH

KRAT T AKSATACPCMAMTC T A

DHCM

ATA T C A T EP C A T C A T C A T

mcyImcyF

mcyE mcyJ mcyD mcyG mcyA mcyB mcyC

0 10 20 30 40 50 55 kb

C

MMM

M

Mdha

M

M

MM

D-AlaM

MM

M

Leu

MM

M

M

D-Me-Asp

MM

M

M

Arg

Microcystin synthesis in Anabaena

Adda

D-Glu Mdha

D-Ala

Leu

D-Me-AspArg

Rouhiainen et al., 2004. AEM 70:686-692.

Detection methods for toxiccyanobacteria

• mcyE gene chosen as target forprimer/probe design– both Glu and Adda important for

toxicity– they are present in every variant

of microcystins and nodularins– enough differences:® possible to design genus-

specific probes/primers(conventional and real-timePCR applications)

® microarray to detect toxinproducing genera

HI F E J D G A B C

mcyABCMcyIFEJDGMcyHAdda

D-Glu Mdha

D-Ala

LeuD-Me-Asp

Arg

Nostoc (1)

Anabaena (9)

Microcystis(10)

Planktothrix (7)

Nodularia (3)

Samples were collected mostly in July 2001 (bloom forming season inFinland August September):– General mcyE-PCR 84% of the lakes contained potential

microcystin producers– Genus specific mcyE-PCR:

• 70% toxic Microcystis spp.• 63% toxic Planktothrix spp.• 37% toxic Anabaena spp.

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

2SH01

2TU022K

J03

2SJ0

4

2KN05

2VD06

2PN07

2KE08

2RN09

2LA10

2KY112P

H12

2VU13

2SJ1

4

2VM15

2IL16

2HZ17

2TT18

2LA192P

Q202T

I21

2VE22

2JT2

3

2MY24

2VA25

2VJ2

62IA

27

2SS28

2SN29

2PY30

2PU31

2HK32

2KL33

2IJ34

2KH35

2IK36

2YL37

2HV38

2PO39

2LK402L

O41

2KM42

2HK43

2RJ4

4

2TO45

2TA46

2NJ4

72IR

48

2HH49

2PJ5

0

2PP51

2HI52

2KK53

2KS54

2KU55

2PK56

2VS57

2MA58

2IH59

2SG60

2IV612P

E62

2PV63

2KR64

2SD65

2MK66

2UR67

2RA68

2EN69

2KI70

2VG71

samples

band

surf

ace Plank_mcyE

Ana_mcyEMic_mcyEgeneral_mcyE

Occurrence of mcyE genes in water samplesfrom 70 Finnish Lakes

Proportion of microcystin producersat different trophic levels

• Oligotrophic lakes (TP < 10 µg/l): theoccurrence of only one MC producer wasmost common

• Mesotrophic lakes (TP 10- 34 µg/l): thewidest distribution of different MCproducer combinations

• Eutrophic (TP 35 – 100 µg/l), andhypertrophic (TP > 100 µg/l) lakes:Microcystis, Anabaena, and Planktothrixwas the most common combination

• The proportion of the three-producerlakes increased clearly towards morenutrient-rich conditions

Rantala et al. 2006. AEM, 72:6101-6110.

Oligotrophic (n = 19) Mesotrophic (n = 30)

Eutrophic (n = 17) Hypertrophic (n = 4)

no MC producers

only Microcystis

only Planktothrix

Microcystis and Anabaena

Microcystis and Planktothrix

Anabaena and Planktothrix

Microcystis, Anabaena andPlanktothrix

Real-time qPCR analysis of toxiccyanobacteria in L. Tuusulanjärvi

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

0tu1

80tu2

10tu2

30tu2

40tu2

50tu2

60tu2

70tu2

80tu2

90tu3

00tu3

10tu3

20tu3

30tu3

40tu3

50tu3

60tu3

70tu3

80tu3

90tu4

30tu4

60tu5

01tu3

1tu71tu1

11tu1

41tu1

81tu2

11tu2

31tu2

41tu2

51tu2

61tu2

71tu2

81tu2

91tu3

01tu3

11tu3

21tu3

31tu3

41tu3

51tu3

61tu3

71tu3

81tu3

91tu4

41tu4

62tu3

2tu72tu1

12tu1

5

samples

gene

copi

es/m

lwat

er

Ana-mcyEMic-mcyE

2000 2001

• Toxic blooms were more intense in 2001 than 2000• Toxic Microcystis spp. were present almost all year around• Toxic Anabaena spp. more prevalent in late summer

DNA-chip (Microarray)

•Probes on a DNA-chip•Large amounts of sequence datais needed for probe design

DNA extracted fromenvironmental samples(PCR product) reactswith the probes on thechips

Chips are scanned with laser and results analyzed with computerØ Genetic identification of cyanobacteria present in the sample (16S rRNA)Ø Presence of cyanobacteria with microcystin synthetase genes (mcyE)ØExcellent monitoring tool Castiglione et al., 2004 AEM 70:7161-7172

Rantala et al. 2008 Environ.Microbiol. 10(3): 653-664.

DNA

Chips designedby CNR-ITBA &

Univ. Helsinki chip center

Chip assay – Lake Tuusulanjärvi sample

• Signals from 16S rRNA spotscorresponding to– Microcystis– Synechococcus– several

Anabaena/Aphanizomenongroups

• The same groups weredetected with othermethods (microscopy,cloning, DGGE).

• Signals from spotscorresponding to– toxic Anabaena group (16S

rRNA)– Anabaena-mcyE– Microcystis-mcyE

(Castiglioni et al., 2004. AEM 70:7161-7172)Rantala et al., 2008. Env. Microbiol. 10:653-664.

Development of chip assay to detectmcyE gene expression

· qPCR was used to verify chip assay results· New application for mcy expression profiling in environmental samples

Sipari et al. 2010. Appl. Environ. Microbiol. 76(12): 3797-3805.

Spots corresponding to probe pairs: Anab.+Aph. 16S rRNA (38); Anabaena-mcyE(39); Microcystis-mcyE (40); Microcystis 16S rRNA (1B) (+controls)

Hepatotoxic blooms in the Baltic SeaCyanobacterial blooms in the Baltic Sea

Mass occurrences ofcyanobacteria in theopen Baltic Sea arefrequently hepatotoxic

Correlation between nodularin synthetase (ndaF)gene copy numbers (determined by real-time PCR)and nodularin concentrations (LC/MS) in the Gulfof Finland (Baltic Sea) in late July 2004Koskenniemi et al., 2007. AEM 73:2173-2179.

y = 0,7846x - 1,0697R2 = 0,7244

-1

0

1

2

3

1 2 3 4 5

log

(ng

nodu

larin

/mlw

ater

)log ( copies/ ml water)ndaF

1

Real-Time PCR

§ DGGE analysis based on mcy/nda genes:- Anabaena mcy genotypes were found in theBaltic Sea in 2003 and 2004- They were more diverse than nda genotypes ofNodularia

Hepatotoxic Anabaena in the Baltic Sea(culture independent method to find diversity)

Fewer et al. 2009. Env. Microbiol. 11:855-866.

Early evolution of microcystinsynthetase genes

•The maximun likelihood trees of microcystin synthetase and housekeepinggenes are congruent, which shows that microcystin synthetase genes have co-evolved with housekeeping genes and are ancient.•The genes coding nodularin (a pentapeptide hepatotoxin)synthetase are recently derived from microcystin synthetase.

Rantala et al., 2004. PNAS 101:568-573; Oksanen et al., 2004. AEM 70:5756-5763

Detection of anatoxin-a producers• Anatoxin-a producing Oscillatoria (Cadel-Six et al., 2009 AEM; Mejean et al.,

2009)• Anabaena 37 genome project – biosynthetic genes (Rantala-Ylinen et al. 2011.

Appl. Environ. Microbiol. 77:7271-7278.)

DH

acyl-CoAsynthetaseAMP-binding

acyl carrierprotein

PKS 1 PKS 2 PKS 3

20.3 kb 7 kb

thioesterasecyclase

hyphyp

• Detection methods for anatoxin-a producers

Detection methods for potentialtoxin producers

• Microcystins +nodularin

• Anatoxin-a• Saxitoxins• Cylindrospermopsins• ... Anatoxin-a(S)-> New producers –new primers?

Adda

D-Glu Mdha

D-Ala

Leu

D-Me-AspArg

Detection methods for toxic cyanoacteria(DNA & RNA)

• Conventional PCR – easy and cheap,allows analysis of large data sets

• Quantitative real-time PCR –quantitative results, identifies the maintoxin producer

• Chip assay – simultaneous detection ofthe toxin producers (mcy-probes) orcyanobacterial community (16S probes)– excellent monitoring tool

• DGGE on mcy genes – unknowndiversity can be detected

• These methods reveal only potential -Inactive gene clusters occur (generallyminority in the population)

NostocAnabaena

Microcystis

Planktothrix

Nodularia

mcyE sequences

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

0tu1

80tu2

10tu2

30tu2

40tu2

50tu2

60tu2

70tu2

80tu2

90tu3

00tu3

10tu3

20tu3

30tu3

40tu3

50tu3

60tu3

70tu3

80tu3

90tu4

30tu4

60tu5

01tu3

1tu71tu1

11tu1

41tu1

81tu2

11tu2

31tu2

41tu2

51tu2

61tu2

71tu2

81tu2

91tu3

01tu3

11tu3

21tu3

31tu3

41tu3

51tu3

61tu3

71tu3

81tu3

91tu4

41tu4

62tu3

2tu72tu1

12tu1

5

samples

gene

copi

es/m

lwat

er

Ana-mcyEMic-mcyE

12

1. Microcystin (hybrid NRPS-PKS)2. Anabaenopeptin (NRPS)3. Anabaenopeptilide (NRPS)4. Hassallidin (NRPS)

5-11. Bacteriocin gene clusters12. Anacyclamide (cyanobactin)

1

2

4

5

6

7

8

9

10

11

3

~ 250 kb

GENOMES

Anabaena sp. 90genome shows thechromosomallocation ofbiosynthetic geneclusters

Wang et al.2012. BMCGenomics13(1): 613.

Shih et al. 2013. PNAS 110(3): 1053-1058.

Only few compoundsor biosynthetic enzymes

are characterized

Cyanobacterial species tree and distribution of peptide biosynthesis

• 30 cyanobacterial genomes out of 116 strains studied had gene cluster to code for 4-methylproline

• 116 cyanobacteria strains screened by PCR and LC-MS found Nostoc as the mainproducer

• Two new groups of peptides were identified.

R3

R4

R5

R6

NH

O

O N

O NH

O

NH

O

HN

O

HO

HN

O OH

N

ON

O

N

O

O

N

R2

R1

Nostoweipeptin W1-W6

H

O

O

R1

R2

N

OO

HN

O NH

O

N

OHN

O

NH

OHN

O

O

NH2

N

OO

Nostopeptolide L1-L4

Combination of research on genomics andchemistry – new discoveries

Liu et al. 2014. ACSChemical Biology 9(11):2646-2655.

Nostoweipeptins6 variants

Nostopeptolides4 variants

Molecular studies on mechanism of action:4-methylproline containing compounds preventapoptosis caused by cyanobacterial hepatotoxins

Liu et al. 2014. ACS Chemical Biology 9(11): 2646-2655.

Hepatocyte toxicity cell assays may not give accurate answerin case there are these antitoxins present

New toxins

• Benthic cyanobacteria produceanabaenolysins

• New mechanism – lysing theeukaryotic membranes

• Knowledge of biosynthesis helpsto get information of producersand occurrence

Oftendal et al. 2012. Biochim. Biophys. Acta, 1818:3000–3009.Shishido et al. 2015. PNAS 112(44):13669-13674

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• Methodology will develop and improvefurther -> anatoxin-a(S) biosynthesis

• More genomes – genome comparisons:differences between the toxin producersand non-producers, regulationmechanisms …

• Metagenomics will yield valuableinformation what happens in nature