climate change and regulation of hepatotoxin production in ... · climate change and regulation of...

M IN I R E V I EW

Climate change and regulation of hepatotoxin production inCyanobacteria

Michelle M. Gehringer1 & Nicola Wannicke2,3

1Department of Plant Ecology and Systematics, Technical University of Kaiserslautern, Kaiserslautern, Germany; 2Leibniz-Institute for Baltic Sea

Research, Rostock, Germany; and 3Leibniz-Institute of Freshwater Ecology and Inland Fishery, Berlin, Germany

Correspondence: Michelle M. Gehringer,

Department of Plant Ecology and

Systematics, Technical University of

Kaiserslautern, Gottlieb-Daimler Str. Geb.13,

67663 Kaiserslautern, Germany. Tel.: +49 (0)

631 68031144; fax: +49 (0)631 2052998;

e-mail: [email protected]

Received 4 October 2013; revised 20 January

2014; accepted 20 January 2014. Final

version published online 3 March 2014.

DOI: 10.1111/1574-6941.12291

Editor: Gerard Muyzer

Keywords

nodularin; microcystin; CO2; nodularia;

microcystis; climate change.

Abstract

Harmful, bloom-forming cyanobacteria (CyanoHABs) are occurring with

increasing regularity in freshwater and marine ecosystems. The most commonly

occurring cyanobacterial toxins are the hepatotoxic microcystin and nodularin.

These cyclic hepta- and pentapeptides are synthesised nonribosomally by the

gene products of the toxin gene clusters mcy and nda, respectively. Understand-

ing of the regulation of hepatotoxin production is incomplete, although there is

strong evidence supporting the roles of iron, light, higher nitrate availability and

inorganic carbon in modulating microcystin levels. The majority of these studies

have focused on the unicellular freshwater, microcystin-producing strain of Mi-

crocystis aeruginosa, with little attention being paid to terrestrial or marine toxin

producers. This review intends to investigate the regulation of microcystin and

nodularin production in unicellular and filamentous diazotrophic cyanobacteria

against the background of changing climate conditions. Special focus is given to

diazotrophic filamentous cyanobacteria, for example Nodularia spumigena, capa-

ble of regulating their nitrogen levels by actively fixing dinitrogen. By combining

data from significant studies, an overall scheme of the regulation of toxin pro-

duction is presented, focussing specifically on nodularin production in diazo-

trophs against the background of increasing carbon dioxide concentrations and

temperatures envisaged under current climate change models. Furthermore, the

risk of sustaining and spreading CyanoHABs in the future ocean is evaluated.

Introduction

Cyanobacteria are phototrophic autotrophs that date back

to c. 3.5 billion years ago (Schopf & Packer, 1987). They

exhibit an array of innovative adaptations to ensure sur-

vival in a range of environments, including some of the

most extreme niches. As primary producers, Cyanobacte-

ria harvest sunlight during photosynthesis, are competent

in the uptake of nutrients such as nitrogen (N), phospho-

rous (P), iron (Fe) and other trace metals, and, in some

cases, are able to fix dinitrogen from the atmosphere, a

nitrogen source unavailable to the majority of other

organisms. Of concern is the ability and tendency of these

organisms to form cyanobacterial harmful blooms

(CyanoHABs) in freshwater, brackish and marine waters

when conditions are suitable (El-Shehawy et al., 2012;

Paerl & Paul, 2012). The hepatotoxins microcystin and

nodularin are commonly produced by bloom-forming

bacteria and pose a threat to human health by virtue of

their ability to strongly inhibit protein phosphatases

1 and 2A in vitro (Honkanen et al., 1990, 1991) and

in vivo (Runnegar et al., 1993). Microcystins are sus-

pected tumour promoters and use of water containing

these toxins can be fatal, as demonstrated by the death of

60 patients in Brazil receiving renal dialysis (Jochimsen

et al., 1998). Over 100 variants of the heptapeptide micr-

ocystin exist and are synthesised by a wide range of cy-

anobacterial species such as Microcystis, Anabaena, Nostoc,

Phormidium, Oscillatoria and Planktothrix (Neilan et al.,

2008).Transcription of the microcystin gene cluster (mcy)

is not constitutive, as demonstrated by significant changes

in mcyE transcript levels monitored in a Microcystis

bloom (Wood et al., 2012). Only eight variants of nodul-

arin have been identified (Mazur-Marzec et al., 2006),

and production was thought to occur only in the

bloom-forming Nodularia spumigena or benthic Nodularia

FEMS Microbiol Ecol 88 (2014) 1–25 ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

MIC

ROBI

OLO

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sphaerocarpa. However, nodularin production was

recently identified by terrestrial Nostoc species acting as

endosymbionts of cycads (Gehringer et al., 2012) as well

as in Nostoc containing lichens (Kaasalainen et al., 2012,

2013). A summary of lake modelling research papers sug-

gested a general trend towards increased cyanobacterial

blooms with increasing nutrient loads, decreased flushing

and raised temperatures (Elliott, 2012). In certain cases

where inorganic nitrogen such as nitrate and nitrite were

depleted, diazotrophic species were advantaged. Increased

anthropomorphic nutrient loading on both freshwater

and marine water bodies has led to an increase in the

occurrence of CyanoHABs (Elliott, 2012; Paerl & Paul,

2012; Suikkanen et al., 2013). Of increasing concern are

the observations that increasing CO2 levels and tempera-

tures may result in competitive dominance of blooms by

toxin producers (Kleinteich et al., 2012; O’Neil et al.,

2012), although dominance of toxic blooms under carbon

limitation and low light has also been reported (Van De

Waal et al., 2011). Stratification of water is increased

under higher temperatures, thereby extending the bloom

periods as the warm stratified layers are maintained over

longer periods (Paerl & Huisman, 2009; Huber et al.,

2012). Cyanobacteria rarely occur as unialgal cultures with

blooms usually comprised of a range of different species

including toxin and nontoxin producers as well as nitro-

gen and non-nitrogen-fixing species (Kurmayer, 2011).

Nitrogen pulses released from diazotrophs may stimulate

and sustain nondiazotrophic species, including toxin pro-

ducers and higher trophic levels (Ohlendieck et al., 2007;

Beversdorf et al., 2013; Wannicke et al., 2013).

The factors responsible for increasing toxin production

and the increase in the occurrence of toxin-producing

species are still unclear. Evidence would suggest that even

under nutrient limiting conditions, increased tempera-

tures favour the growth of toxin producers, as seen for

polar cyanobacterial mat samples grown at increased tem-

peratures (Kleinteich et al., 2012). Increased growth of

toxin-producing Microcystis spp. was seen in the USA

(Davis et al., 2009) and China (Wilhelm et al., 2013).

This is supported by recent research suggesting that toxin

producers are better able to withstand stress conditions

(Zilliges et al., 2011) and increase toxin production under

stress conditions (Kurmayer, 2011). Microcystin produc-

tion has been clearly demonstrated in desert soil crusts

(Metcalf et al., 2012). Very few detailed ecological studies

exist investigating the effects of ecological changes on ter-

restrial cyanobacteria and their adaptive responses,

including toxin production, or on their potentially altered

biodiversity (Mollenhauer et al., 1999). The intention of

this review is to: (1) present the current stage of knowl-

edge concerning the gene regulation of hepatotoxin pro-

duction in unicellular and filamentous cyanobacteria in

the context of the general metabolism; (2) describe how

factors associated with climate change, especially

increased temperature and CO2 affect gene regulation;

and finally (3) propose tentative risks of increased

CyanoHABs in the future ocean and limnic habitats on

the basis of current scientific knowledge.

The biological function of the hepatotoxins is not fully

understood. As the synthesis of these secondary metabo-

lites is energy and resource demanding, it is considered

that their synthesis offers some ecological or physiological

advantage over nontoxin producers. Insights into the

potential advantages offered by microcystin or nodularin

production have been obtained in numerous studies pre-

sented in Table 1. We have attempted to include all semi-

nal studies pertaining to the regulation of hepatotoxin

synthesis in cyanobacteria since the year 2000. Due to the

wide scope of this research area and in an attempt not to

distract from the focus of this review, we restricted the

references used in Table 1 to those with defined experi-

mental conditions that measured either toxin gene

expression or synthesis, or were directly related to chang-

ing climatic conditions. Noninclusion of a scientific work

means it did not fit within the parameters of our review.

Sivonen and Jones (1999) provide a comprehensive sum-

mary of studies prior to 1999, although they may have to

be revised in the light of the recent discovery that a large

amount of toxin remains covalently bound to protein

and would not have been detected in the studies measur-

ing toxin in methanol extracts (Meissner et al., 2013).

These authors discovered that microcystin and nodularin

synthesised in response to high light stress is bound rap-

idly and covalently to proteins, thereby making them

undetectable using the standard methanol extraction tech-

niques (Meissner et al., 2013). While this does not affect

studies measuring extracellular toxin levels in water

bodies, it does affect the interpretation of studies measur-

ing the effects of environmental conditions on the global

expression of the toxin gene clusters against the total

amount of toxin produced.

Regulation of hepatotoxin synthesis incontext of cell metabolic processes

The regulation of cyanobacterial hepatotoxin production

has focussed on the synthesis of microcystin in the fresh-

water bloom-forming species Microcystis aeruginosa

PCC7806 (Kaplan et al., 2012) with one study using four

different Microcystis species (Alexova et al., 2011a, b).

Nodularin production studies are limited to a handful

and focusing only on Nodularia spumigena. These toxins

are synthesised nonribosomally by enzymes encoded on

the microcystin (mcy) or nodularin (nda) gene clusters

(Fig. 1). The nda cluster is proposed to have arisen from

FEMS Microbiol Ecol 88 (2014) 1–25ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

2 M.M. Gehringer & N. Wannicke

Table

1.Table

highlightingthepublished

research

ontheregulationoftoxinproductionin

Cyanobacteria

Strain

Temp.°C

Lightlmol

photons

m�2s�

1N

C/CO2latm

Fe3+

Phosphate

Toxin

Other

Referen

ce

M.aeruginosa

CYA

228/1

(toxic)*

22

20–7

5

lEm

�2s�

1

^2.5

fold

at40lE

m�2s�

1

Utkilenan

d

Gjølm

e(1992a)

M.aeruginosa

CYA

228/1

(toxic)

M.aeruginosa

CYA43(non-toxic)

*

22

0,20,

54lE

m�2s�

1

Limiting

Limiting

Limiting

^toxin

with^N

^toxin

with^Fe

Utkilenan

d

Gjølm

e(1995a)

M.aeruginosa

MASH

01-A19&8isolates

(toxic)

20

38

0.0118–1

.18

mM

nitrate

MC

production

correlates

tocellular

growth

rate

Orr

andJones

(1998)†

M.aeruginosa

PCC7806

23

30continuous

16,31,68,

400red,blue

^mcyD

expression

&^mcyB

expression

athighlight

andredlight

Kaebernicket

al.

(2000)†

M.aeruginosa

PCC7806(toxic)

M.aeruginosa

PCC7806mcyA�&mcyB�

(non-toxic)

23

5,16,68

blue,

red

MrpA

iden

tified

inPC

C7806

andnotin

mutants

Dittm

annet

al.

(2001)

M.aeruginosa

PCC7806(toxic)

M.aeruginosa

PCC7806mcyB�

(non-toxic)*

20

high12:12

light:dark

ND

chlorosisin

mutantunder

highlight

Hesse

etal.(2001)

M.aeruginosa

MASH

01-A19

(toxic)

*

26

40

0.02–0

.2

mM

nitrate

CellularMC

levelscorrelate

togrowth

rate

Longet

al.(2001)†

M.aeruginosa

PCC7806(toxic)

25

31continuous

16,31,68

ND

-254mcyD

TSPused

under

low

light

-206mcyD

TSPwith

NtcA

recognition

site

usedunder

high

light

Kaebernicket

al.

(2002)


Microcystin/nodularin occurrence under climate change 3

Table

1.Continued

Strain

Temp.°C

Lightlmol

photons

m�2s�

1N

C/CO2latm

Fe3+

Phosphate

Toxin

Other

Referen

ce

M.aeruginosa

2toxicstrains

2non-toxicstrains

20

25continuous

Limit

Excess

Limit

Excess

Toxicstrains

advantaged

under

Nexcess

Non-toxic

strains

advantaged

under

N

limitation

Vezie

etal.(2002)†

M.aeruginosa

PCC7806(toxic)*

22

10–4

03

12:12light:

dark

^MC

per

cellwith^

lightto

40.

vMC

with

higher

light

Wiedner

etal.

(2003)

M.aeruginosa

PCC7806&

UV027(toxic)

23

140continuous

1.18–2

1.2

mM

nitrate

0.067–0

.175

mM

^toxin

at^N

Max.toxin

&protein

synthesis

atmax.

growth

rate

Downinget

al.

(2005a)

†

M.aeruginosa

PCC7806&

UV027(toxic)

23

51continuous

0.125,7,10,

15,18mM

nitrate

^toxin

with^N

Toxinlevels

neg

atively

relatedto

Cfixation

Downinget

al.

(2005b)†

Microcystis

aeruginosa

V163,CYA140

and

isolates(toxic)

M.aeruginosa

V163&CYA43(nontoxic)*

20

25

vMC

levels

withtime

incompetition

studies

Non-toxic

strainsout

competed

toxicstrains

Kardinaal&

Visser(2005)†

Plan

ktothrix

arghardii(toxic)*

23

25,110

6-112

12:12

light:dark

^lightinduced

increasedMC-LR

&vMC-RR

synthesis

^mcyA

tran

scripts

with^light

to60,then

vover100

Tonket

al.(2005)†

Microcystis

aeruginosa

V163,CYA140

and

isolates(toxic)

M.aeruginosa

V163&CYA43(nontoxic)*

20

25continuous

vin

MC

levels

reflecting

declinein

toxic

strain

in

competitive

experim

ents

Non-toxic

strains

outcompeted

toxicstrains

Kardinaalet

al.

(2007)†



Table

1.Continued

Strain

Temp.°C

Lightlmol

photons

m�2s�

1N

C/CO2latm

Fe3+

Phosphate

Toxin

Other

Referen

ce

M.aeruginosa

PCC7806(toxic)

M.aeruginosa

PCC7005

(non-toxic)

25

16continuous

Limit

^MC

levels

withFe

limitation

^mcyD

expression

withFe

limitation.

Toxicstrain

overcam

eFe

defi

cien

cybetter

than

non-toxic

strain.TSP’s

unchan

ged

Sevilla

etal.(2008)†

M.aeruginosa

HUB5-2-4

(toxic)*

23

50continuous

Excess

Excess

^totalMC

production,

specifically

in^MC-RR

Stongcorrelation

betweenN

:

Can

dNlevels

Van

deWaalet

al.

(2009)†

M.aeruginosa

UTC

C299(toxic)

24

24,56,136,

272,820

16:8

light:

dark

Initial^

MC/cell

at56followed

byavMC/cell

withincreased

light

MC

contentlinked

toChlacontent.

MC

inversely

correlated

tocell

divisionrate

Deb

lois&Juneau

(2010)†

M.aeruginosa

PCC7806(toxic)

––

Excess,

limit,starved

^NtcA

&mcyB

expressionunder

Nlim

itation&

starvation

Ginnet

al.(2010)

M.aeruginosa

PCC7806(toxic)

25

16continuous

0.2,2,

20mM

nitrate

increased

Toxin/cell

constan

t

^growth

rate

with^N.mcyB

tran

script:toxin

constan

t

Sevilla

etal.(2010)†

M.aeruginosa

CYA140&

PCC7806(toxic)

M.aeruginosa

CYA43&PC

C7806mcyB�

(non-toxic)*

21.5

50,25

continuous

200ppm

1000–1

250

ppm

MC

levels

correlated

tolevelsof

toxicstrain

present

Toxicstrain

dominan

t

atlow

CO2levels.

Non-toxic

strain

dominan

t

inlow

light

andhighCO2

levels

Van

deWaalet

al.

(2011)†

M.aeruginosa

MASH

-01A19

(toxic)

15,26,30

8,40,65

continuous

0.03,

0,20,200

0.1,0.3,1,6uM

vMC

with^

lightan

dP.

v

MC

withvS

Variable

Stested

Jahnichen

etal.

(2011)†



Table

1.Continued

Strain

Temp.°C

Lightlmol

photons

m�2s�

1N

C/CO2latm

Fe3+

Phosphate

Toxin

Other

Referen

ce

Microbialmats

(Arctican

d

Antarctic)

4,8,16,23

23

16:8

light:

dark

limit

^cyan

obacterial

toxinlevelsin

matsat

8&

16°C

^cyan

obacterial

diversity

in

matsat

8&

16°C

Kleinteichet

al.

(2012)†

M.aeruginosa

PCC7806(toxic)

M.aeruginosa

PCC7005,PC

C7806

mcyH�(non-toxic)

27

90

14:10light:dark

10uM,

1000,

100,

10nM

^cellularMC

contentin

10nM

Fe

starvedcultures

^in

isiA

tran

scripts

in

10nM

Festarved

cultures

Alexova

etal.

(2011a)

†

M.aeruginosa

PCC7806,UWOCC

MRC,UWOCC

MRD

(toxic)

M.aeruginosa

UWOCC

CBS,

PCC7005,HUB5.3

(non-toxic)

28

25continuous

P II&NdhK

proteinsincreased

while

NrtA,Ccm

K3,

Ccm

L&TrxM

decreased

innon-toxic

strains.

NtcA

tran

scriptionincreased

innon-toxicstrains.

Alexova

etal.

(2011b)†

M.aeruginosa

PCC7806(toxic)

25-30

25continuous

replete

^2OG

replete

^NtcA

bindingto

mcyA

promoter

Kuniyoshiet

al.

(2011)

M.aeruginosa

HUB524,W333,

PCC7806(toxic)

HUB018,P4

61,

PCC7806mcyB�

mutant(non’toxic)

3fieldsamples

(Boulder

Lake,USA

;

Innen

alster,German

y;Lake

Taihu,China)

20

26

32

72

16:8

light:dark

MC-LR

levels

increased

withrising

temperatures

inaxen

ic

strains

producing

both

MC-YR

&MC-LR

Overall,

the

concentration

ofmicrocystins

rose

with

increasing

temperature

Therewas

ashifttoward

toxicstrains

athigher

temperatures.

Heterotrophic

bacteriaaffected

thequan

tity

and

qualityof

cyan

obacterial

toxinsproduced.

Dziallas&

Grossart

(2011)†



Table

1.Continued

Strain

Temp.°C

Lightlmol

photons

m�2s�

1N

C/CO2latm

Fe3+

Phosphate

Toxin

Other

Referen

ce

M.aeruginosa

PCC7806(toxic)

M.aeruginosa

PCC7806

mcyA�(non-toxic)

High37

Low

8&17

continuous

varying

MC

levels

correlated

togrowth

rates.

Toxicstrain

advantage

under

high

light.

Nostrain

advantageat

lower

light.

Phelan

&Downing(2011)†

M.aeruginosa

PCC7806

M.aeruginosa

PCC7806

mcyB�&mcyH�

(non-toxic)

23

16continuous

70,300,700

limit

MC

bindsprotein

under

highlight

andoxidative

stress

conditions.

Zilliges

etal.(2011)

M.aeruginosa

PCC7806

25

28,50,109

continuous

0

PIIinhibit

^toxinwith

^mcyD

expression

^mcyD

expressionwith

increasedlight.v

mcyD

expression

under

Plim

itation

andin

darkn

ess.

Sevilla

etal.(2012)†

M.aeruginosa

PCC7806

M.aeruginosa

PCC7806mcyB�

(non-toxic)

22

5&39

12:12light:

dark

0.036&

9

mM

nitrate

Themutant

strain

outcompeted

toxinproducer

under

favourable

conditions.

Brian

det

al.(2012)†

M.aeruginosa

PCC7806(toxic)

25

16continuous

0.05,0.2,

2mM

phosphate

^MC

content

withreduced

phosphate

^mcyD

tran

scripts

withreduced

phosphate

Kuniyoshiet

al.

(2013)†

M.aeruginosa

PCC7806,M.

aeruginosa

NIES8

43,Plan

ktothrix

agardhiiNIVA-CYA

126(toxic)

M.aeruginosa

PCC7806mcyB�

(non-toxic)

23

8continuous

250

^protein

boundMC

under

high

lightan

d

oxidativestress.

MC

bindsproteins

under

highlight

inducedoxidative

stress.

Meissner

etal.

(2013)



Table

1.Continued

Strain

Temp.°C

Lightlmol

photons

m�2s�

1N

C/CO2latm

Fe3+

Phosphate

Toxin

Other

Referen

ce

M.aeruginosa

PCC7941,CYA140

Plan

ktothrixag

ardhii

CYA126(toxic)

Plan

ktothrix

agardhiiCYA116

Synechococcuselongates

PCC6301(non-toxic)

20-35

80

16:8

light:dark

Meanoptimal

growth

temp.

of29.5

°C.

Allgrew

well

at35°C

Lurlinget

al.(2013)

Nodulariaspumigen

a

(2isolates)

10–3

025,50,80

continuous

0.3,0.6,1

^NOD

at20°C

lightchan

ges

had

noeffect

lowesttoxin

at0.3

P

Lehtimakiet

al.

(1994)†

Nodulariaspumigen

a

BY1(toxic)

20

7-28

25

2-155

continuous

0.02–4

2

mgL�

1

0.2–5

.5^NOD

at

bestgrowth

conditions:

highlight,

temp.&

nitrogen

fixation

rates

vNOD

with

highan

d

low

salinities

andhigh

inorgan

icN

Lehtimakiet

al.

(1997)†

Nostocsp.IO-102-I

7(toxic)

21

20–2

7

continuous

free

6MC

varian

ts

detected

Oksan

enet

al.

(2004)‡

Nodulariaspumigen

a

CCY9414(toxic)

16

85

14:10light:dark

free

Gradientfrom

153to

731

5.4

lM

ND

HighCO2caused

decreasein

growth,elevation

ofC

:Nan

dN

:

Pratio,slight

decreasein

N2fixation

Czernyet

al.(2009)

Nodulariaspumigen

aAV1

(toxic)*

20

45

16:8

light:dark

NH4

addition

limit

Novariation

inNOD

detected

^ndaexpression

withvP

vndaexpression

with^NH4

Jonassonet

al.

(2008)†

Nodulariaspumigen

a

BA-15(toxic)

15,30

10,150,290

16:8

light:dark

ND

Tolerantofhigh

lightat

low

temperature

Jodlowska&Latala

(2010)



Table

1.Continued

Strain

Temp.°C

Lightlmol

photons

m�2s�

1N

C/CO2latm

Fe3+

Phosphate

Toxin

Other

Referen

ce

NostocPC

C9237(originally

Nostocsp.152)(toxic)

12&20

1&50

continuous

0&

5.88

mM

nitrate

0.14vs.

144lM

Max.MC

detected

under

low

lightin

replete

med

ium

at

12°C

Max.intracellular

MC

inPreduced

cultures

Kurm

ayer

(2011)†

Nodulariaspumigen

a

CCY9414(toxic)

23

200

16:8

light:

dark

free

315

398

548

0.3

uM

ND

HighCO2caused

increase

ingrowth,

Can

dN2fixation,

elevationofC:P

andN:P

ratio

Wan

nicke

etal

(2012)

Nostocstrains

(2isolates)

22

27

16:8

light:

dark

NOD

and[L-Har

2]-

NOD

produced

Geh

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a deletion event spanning the mcyA-A2 to the McyB-C2

domain (Moffitt & Neilan, 2004; Rouhiainen et al., 2004;

Gehringer et al., 2012). Expression of the hepatotoxin

gene clusters is regulated by the region spanning the

mcyD and mcyA genes for mcy (Kaebernick et al., 2002),

and ndaA and ndaC for nda (Moffitt & Neilan, 2004).

While microcystin is synthesised in both diazotrophs

and nondiazotrophs, nodularin production is restricted to

nitrogen-fixing species of Nodularia and Nostoc. Biological

nitrogen fixation (BNF) is dependent on the activity of

the enzyme nitrogenase to catalyse the energy-demanding

reaction generating ammonia from N2. Photosystem

I (PSI) provides the reduced ferredoxin required for the

conversion of N2 to ammonia during biological nitrogen

fixation (BNF; Bothe et al., 2010). The molybdenum-con-

taining nitrogenase found in Nostoc and Nodularia species

is encoded by the genes nifHDK, of which nifH is the

most conserved, and hence commonly used for diversity

studies (Severin & Stal, 2010a, b; Severin et al., 2012). No

fixed correlations have been found between nitrogenase

activity and expression of nifH (Severin & Stal, 2010a, b;

Severin et al., 2012), making uptake studies using the sta-

ble isotope 15N2 or acetylene (C2H2) still the most reliable

means of assessing active dinitrogen fixation.

Cyanobacteria are known to perceive nitrogen levels by

means of 2-oxoglutarate (2-OG) sensing (Muro-Pastor

et al., 2001). 2-OG), previously known as 2-ketoglutarate

or a-ketoglutarate is the essential carbon skeleton for the

incorporation of N into the cell via the action of gluta-

mine synthetase (GS), encoded by glnA, and glutamate

synthase (GOGAT) in cyanobacteria (Zhao et al., 2010;

Kuniyoshi et al., 2011). The GS-GOGAT cycle assimilates

ammonia by the ATP-dependent amination of glutamate

to yield glutamine. The amide group is then transferred,

in a reaction catalysed by GOGAT, to 2-OG to generate

two molecules of glutamate. Accumulation of 2-OG in

the cell as the result of reduced N availability acts as a

signal of nitrogen deprivation and an indicator of the

intracellular carbon balance with respect to N content

(Ermilova & Forchhammer, 2013 for a review).

The signal transduction protein, PII, encoded by glnB,

is responsive to cellular nitrogen status by its binding of

2-OG, reducing nitrate uptake when CO2 fixation rates

drop. It is considered a suitable candidate for the signal

for the N/C balance (Luque et al., 2004) and (Forchham-

mer, 2008) for a review. In cells of Synechococcus elonga-

tus growing in ammonia, the PII homotrimer is almost

completely dephosphorylated, a situation also observed if

the CO2 supply to the cells is limited, or CO2 fixation

inhibited. Under conditions of higher levels of intracellu-

lar nitrogen, dephosphorylated PII is bound to another

regulatory protein, PipX (PII-interacting protein X). PII is

maximally phosphorylated in nitrogen starved cells,

resulting in the release of PipX (Forchhammer, 2008).

Intermediate levels of PII phosphorylation are observed in

cells provided with nitrate as the N source, and this phos-

phorylation increases with increasing CO2 concentrations

(Forchhammer, 2008). Nitrogenase activity may also be

stopped under the regulation of PII (Forchhammer, 2008

for a review). PII may also respond to the redox state of

the cell as it immediately becomes dephosphorylated

under oxidative stress conditions (Hisbergues et al.,

1999).

Microcystin synthesis

The bidirectional promotor for mcy carries recognition

sites for the global nitrogen regulator protein, NtcA

(Ginn et al., 2010), as well as the ferric uptake regulator,

FurA (Kaebernick et al., 2002), a ferric uptake regulator,

and the redox-active photoreceptor protein RcaA (Mar-

tin-Luna et al., 2006; Alexova et al., 2011a, b). We were

unable to find any published research on RcaA binding to

the mcy promotor region.

FurA is a dimeric transcriptional regulator that binds

to its DNA recognition sequences (iron boxes) in the

Microcys n (mcy) gene cluster: Microcys s aeruginosa PCC7806, 55 kb.

Nodularin (nda) gene cluster: Nodularia spumigena NSOR10, 48 kb.

Fig. 1. The diagrammatic representation of the gene clusters, mcy and nda, responsible for the synthesis of microcystin and nodularin, of

Microcystis aeruginosa PCC7806 and Nodularia spumigena NSOR10, respectively (Gehringer et al., 2012). The promoter regions are indicated by

the thickened black lines between mcyA and mcyD, as well as ndaA and ndaC. The ORF2 gene found on the nda cluster is coloured black. The

proposed deletion of mcyA and mcyB modules to generate ndaA is indicated (Moffitt & Neilan, 2004).



presence of ferrous iron (Fe2+) to repress expression of

the genes it regulates (Alexova et al., 2011a, b). In vitro

DNA binding assays demonstrated that Microcystis

PCC7806 derived Fur protein binds to so-called iron

boxes in the promoter region of the mcy cluster (Martin-

Luna et al., 2006). Iron limitation resulted in increased

toxin production and transcription of mcyD (Sevilla

et al., 2008) and isiA (iron stress-induced protein; Alex-

ova et al., 2011a, b) supporting the putative role of FurA

in regulating toxin production. Transcript levels of mcyA

did not match changes in MC levels in Fe-starved cul-

tures while FurA transcription was reduced (Alexova

et al., 2011a, b). In contrast to this, another group identi-

fied an increase in microcystin production with increasing

iron availability (Utkilen & Gjølme, 1995). The discrepan-

cies may be due to methanol extracts being used for toxin

determination, with protein-bound toxin not being mea-

sured. Further work is required to determine the regula-

tion of microcystin production by FurA.

The autoregulatory transcription factor, NtcA, acts as

the global prokaryote regulator of nitrogen metabolism in

both diazotrophic and nondiazotrophic cyanobacteria.

This protein dimer acts either as a transcriptional activa-

tor or as an inhibitor of nitrogen response genes by selec-

tively binding a recognition sequence on the DNA

upstream of the genes it regulates. Under conditions of

high nitrogen availability, PipX, required for NtcA activa-

tion, is tightly bound to the dephosphorylated PII. When

nitrogen levels decrease, cellular 2-OG levels increase and

PII is phosphorylated, releasing PipX to form a complex

with NtcA and 2-OG [(Ermilova & Forchhammer, 2013)

for a review]. Binding of 2-OG to NtcA enhances the

DNA binding activity of an NtcA dimer to its recognition

sequence (Zhao et al., 2010).

The NtcA binding sequence shows great variability but

commonly exhibits the structure 50 GTA N8 TAC 30

(Luque et al., 2004) with the first and last two bases

being the most highly conserved (Su et al., 2005). The

sequence is usually flanked by an A/T base pairing while

the intervening spacer nucleotides may vary in number

(Ramasubramanian et al., 1996), thereby either positively

or negatively affecting protein binding and hence regula-

tion. The consensus sequence is usually found c. 22 base

pairs upstream of the -10 sigma hexameric signal for ini-

tiation of transcription (Luque et al., 2004). NtcA regu-

lates its own expression and is a transcriptional activator

of the genes involved in the differentiation of heterocytes

(nitrogen-fixing cells) in Anabaena PCC7120 upon nitro-

gen starvation, triggered by 2-OG accumulation and

binding. It also activates genes involved in nitrate/nitrite

uptake and reduction (nirA, nirB- ntcB operon), uptake

and fixation such as, narB, amt1, glnA and glnB (Luque

et al., 2004; Kleinteich et al., 2012). NtcA also represses

transcription of gifA and gif B, inhibitors of glnA under

conditions of low ammonia availability (Jiang et al.,

2000). Some genes not involved in nitrogen assimilation

are repressed by NtcA binding, such as rbcL, which

encodes the large subunit of RuBisCo (Ramasubramanian

et al., 1996) and gor (glutathione reductase; Jiang et al.,

1995) while NtcA binding both positively and negatively

regulates sucrose metabolism (Marcozzi et al., 2009).

NtcA levels should decrease under conditions of

increased nitrogen content, and the enhancement of NtcA

dimer binding to its DNA recognition sequences under

the influence of 2-OG would be expected to be reduced.

The observed fourfold increase in the expression of ntcA

that occurred in microcystin-producing M. aeruginosa

PCC7806 under nitrogen limiting conditions when com-

pared to expression in cells grown under excess N (Ginn

et al., 2010) was expected. Of interest was the significant

increase in the expression of mcyB under nitrogen-limit-

ing conditions that was 14 times greater than under

nitrogen-replete conditions, suggesting strong upregula-

tion of the mcy by NtcA binding. Additional NtcA bind-

ing sites were recently identified in the mcy promoter

region (Kuniyoshi et al., 2011). The affinity of NtcA

binding to its mcyA promoter region was increased 2.5

times in the presence of 2-OG at 25 lmol pho-

tons m�2 s�1, thereby supporting the idea that the 2-OG

level regulates microcystin synthesis via NtcA binding.

Weak binding of NtcA to the longer recognition

sequence (50 GTA N9 TAC 30) observed in the mcyABC

promoter was detected in gel shift assays (Ginn et al.,

2010). One of the NtcA recognition sequences identified,

50GTT N7 TAC 30, (Ginn et al., 2010) is similar to the

repressor binding site for gor in Anabaena PCC7120 but

with a retained TAC motif (Jiang et al., 1995). It overlaps

the -10 Pribnow box at the mcyABC transcription start

point (TSP) identified 206 bases upstream of the transla-

tion start site for mcyA (Kaebernick et al., 2002), suggest-

ing a potential means of negative regulation by NtcA

(Herrero et al., 2001). This mcyA TSP was transcribed

under high light conditions (68 lmol photons m�2 s�1)

while the TSP at 254 bp upstream was used under low

(16 lmol photons m�2 s�1) light conditions and seemed

not to be associated with any NtcA binding site (Kaeber-

nick et al., 2002). Different TSPs for mcyA were identified

at 162 and 168 bp upstream of the translational start

codon but were not associated with an NtcA binding site

(Sevilla et al., 2008).

The availability of nitrogen is important for cyanobac-

teria and is the most common limiting factor for growth.

The effects of nitrate concentrations on microcystin pro-

duction are conflicting with some studies indicating an

overall increase in toxin production with increasing

nitrate availability in the unicellular, non-nitrogen-fixing



cyanobacterial species Microcystis aeruginosa (Utkilen &

Gjølme, 1995; Downing et al., 2005a, b), while others

demonstrate an increase under nitrogen limitation (Long

et al., 2001). The tight correlation of microcystin and

protein concentration in M. aeruginosa suggested that the

growth media N : P ratio and associated nitrogen assimi-

lation played an important role in protein, and hence

microcystin, production (Downing et al., 2005a, b).

Adjusting the N : P ratio to 40 : 1 increased both the

mcyD transcript levels and cellular MC content (Kuniyo-

shi et al., 2013). Nontoxin-producing M. aeruginosa

strains grew better than toxin producers under low nutri-

ent conditions. Under high nutrient load, the toxin pro-

ducers increased their biomass to a larger degree than

nontoxin producers (V�ezie et al., 2002). Increased nitrate

availability resulted in an increase in overall growth rate

of M. aeruginosa PCC7806; however, the levels of micro-

cystin per cell remained constant and correlated with the

expression of mcyD (Sevilla et al., 2010) .

Variations in light intensity and composition result in

changes in the transcription start sight (Kaebernick et al.,

2000). Transcript levels of mcyD show an initial increase

in cultures of M. aeruginosa PCC7806 exposed to

increased light intensities of 28 and 109 lmol pho-

tons m�2 s�1 (Sevilla et al., 2012) or 30 lmol pho-

tons m�2 s�1 (Kaebernick et al., 2000). The levels fell

shortly afterwards as the cells became stressed. Toxin lev-

els showed conflicting correlations to mcyD transcription

levels (Kaebernick et al., 2000; Sevilla et al., 2010, 2012).

However, as methanol toxin extracts were used, addi-

tional protein-bound toxin in the cell debris may not

have been detected and could account for the discrepan-

cies (Meissner et al., 2013). Interestingly, there was no

change in the transcription start point for mcyD, nor

mcyB, under different nitrogen levels in the media at

16 lmol photons m�2 s�1 (Sevilla et al., 2010), confirm-

ing that light intensity has a greater influence than nitro-

gen availability on mcy expression. Media composition

providing varying N/C ratios had no significant effect on

the survival or toxin content of microcystin-producing

strain at high light conditions (Phelan & Downing, 2011).

Microcystin-producing M. aeruginosa PCC7806 was

advantaged over a nonmicrocystin-producing mutant at

high (37 lmol photons m�2 s�1) light exposure while no

advantage was observed at lower light intensities (17 and

8 lmol photons m�2 s�1). Microcystin levels in methanol

extracts of the toxin-producing strain strongly correlated

to growth rate (Phelan & Downing, 2011). Moreover, this

study suggested that microcystin synthesis offers protec-

tion during high light stress by preventing chlorosis, as

previously suggested (Hesse et al., 2001). Microcystis aeru-

ginosa mutants unable to produce microcystin, contained

less pigments in the form of chlorophyll a, b-carotene,

zeaxanthin and echinenone when compared to the micro-

cystin-producing wildtype grown under high light condi-

tions (Hesse et al., 2001).

The relationship between microcystin production and

photosynthesis was highlighted by Deblois and Juneau,

2010 who demonstrated an initial increase in microcystin

content per cell of M. aeruginosa UTCC299 when

increasing the light intensity from 24 to 56 lmol pho-

tons m�2 s�1 followed by a reduction in toxin levels at

high photon irradiances of 136, 272 and 820 lmol pho-

tons m�2 s�1. At these three high levels of irradiance,

there was a clear correlation between cellular microcystin

and chlorophyll a levels with increasing photoirradiance.

The relative electron transport rate was inversely related

to microcystin cellular content, determined from soluble

methanol extracts, as was the redox state of the photosyn-

thetic apparatus. Inhibition of the electron transfer chain

of photosystem II resulted in a decrease in mcyD tran-

scripts similar to that seen in the dark, indicating that

microcystin synthesis requires active photosynthesis

(Sevilla et al., 2012). Additional studies imply a stabilising

role for microcystin in cells undergoing oxidative stress

induced by high light exposure (Kaplan et al., 2012). Spe-

cifically, the large subunit of RuBisCo, RbcL, was pro-

tected from protease digestion by the binding of

microcystin under conditions of light-induced oxidative

stress at 70 lmol photons m�2 s�1 (Zilliges et al., 2011).

The fact that NtcA binding regulates both the expression

of the mcy gene cluster and rbcL suggests that toxin pro-

duction is linked to C cycling within the cell, especially

under conditions of oxidative stress.

A recent study investigated the relationship between

light, nitrogen availability and microcystin production in

M. aeruginosa PCC7806 and a nonmicrocystin-producing

mutant (Briand et al., 2012). While no differences in

growth rates were observed in the exponential phase for

monocultures, the nontoxin producer outcompeted the

microcystin producer in competition studies at 5 and

39 lmol photons m�2 s�1. The investigators did not look

at growth reactions under a higher light exposure where

toxin production appears to provide and advantage over

nontoxin producers as demonstrated at 70 lmol pho-

tons m�2 s�1 (Zilliges et al., 2011). Of interest is the

observed increase in microcystin cellular content, deter-

mined from methanol extracts, of the toxin producer in

co-culture and under low light conditions (Briand et al.,

2012).

Being a nitrogen-rich compound, microcystin produc-

tion may be dependent on nitrogen availability and play

a part in maintaining the N/C balance in the cell. The

addition of leucine, a N-poor amino acid, to the culture

medium of Planktothrix agardhii strain 126/3 resulted in

an increase in [Asp3] microcystin LR in relation to



microcystin-RR (Tonk et al., 2008). Addition of the N-

rich amino acid arginine reduced the [Asp3] microcystin

LR/RR ratio. The authors suggested that under conditions

of low N availability, leucine was preferably synthesised

and incorporated into microcystin whereas the N-rich

arginine was utilised under conditions of high nitrogen

availability. They did not however notice changes in the

leucine/arginine ration, nor the [Asp3] microcystin LR/RR

ratio when a culture of Planktothrix agardhii was shifted

from N-replete to N-limited conditions suggesting that

regulation of the mcy gene cluster is more complex than

anticipated.

The role of N in regulating microcystin synthesis has

been suggested in previous studies on Microcystis (Luque

et al., 2004; Downing et al., 2005a, b; Zilliges et al., 2011)

and is supported by the observed increase in the synthesis

of a nitrogen-rich microcystin variant in Microcystis aeru-

ginosa cells supplied with excess N and CO2 (Van de

Waal et al., 2009). The strain was capable of synthesising

several microcystin variants of differing N/C stoichiome-

tries, but under increased C availability, opted to increase

levels of the N-rich microcystin-RR (MCRR). The

increase in the N/C ratio and MCRR levels observed in

culture was also seen in blooms in Microcystis-dominated

lakes suggesting that increased CO2 levels might result in

an increase in the occurrence of microcystin-producing

Microcystis blooms in nitrogen-rich waters (Van de Waal

et al., 2009). While variations in the cellular N/C ratios

were observed under different treatments of N, C and

light availabilities, the differences were not a reflection of

the cellular C content, that is that there was a very strong

correlation between cellular N/C ratios and N content

(Van de Waal et al., 2009). In contrast, recent study of

Microcystis aeruginosa strains found an increase in growth

rate of nonmicrocystin-producing strains over microcy-

stin-producing strains under increased CO2 levels (Van

De Waal et al., 2011). The toxic strain Microcystis

CYA140 showed a lower half-saturation constant for CO2

and a lower minimum C content than the nontoxic

Microcystis CYA43 strain. Microcystin production has

been studied in Nostoc sp. 152, a diazotroph isolated from

Lake S€a€aksj€arvi, Finland (Kurmayer, 2011). He identified

a strong correlation between microcystin production and

cell division rate with the highest levels of cellular toxin

being produced under P limitation and low irradiance

(1 lmol photons m�2 s�1). This is in contrast to the

increased microcystin produced under high light in

M. aeruginosa (Meissner et al., 2013); however, it could

potentially be explained by the varying levels of stress the

organisms were experiencing. In addition, Kurmayer

(2011) investigated a diazotrophic strain of cyanobacteria,

which may have a different stress control regulatory

mechanism of toxin production than M. aeruginosa

strains. Again, it is important to note that in this study,

only the methanol-extracted toxin was quantified and

could explain part of the contradictions occurring

between the two studies.

Cyanobacteria possess a carbon dioxide-concentrating

mechanism (CCM) (Badger & Price, 2003) that increases

the concentration of CO2 around the active site of ribu-

lose-1,5-biphosphate carboxylase–oxygenase (RuBisCo),

the rate limiting enzyme in photosynthesis (Badger et al.,

1993). Carboxysomes, the specialised compartments in

which carbon fixation occurs, are composed of an icosa-

hedral protein shell of repeat hexamers of the carbon-

concentrating mechanism proteins CcmK1-4, with CcmL

forming the pore carrying pentamer at the vertices of the

shell (Tanaka et al., 2008). Comparing the proteome of

toxic and nontoxic M. aeruginosa strains, nine proteins

were found to be differentially expressed including two

proteins involved in nitrogen uptake and metabolism (PIIand NrtA), components of the carboxysome (CcmK3 and

CcmL) and two proteins involved in regulating the redox

balance within the cell (NdhK and TrxM; Alexova et al.,

2011a, b). NrtA is inhibited by high levels of nitrates,

while PII, encoded by glnB, is known to regulate carbon

transport and regulate the fixation rate of carbon in Syn-

echocystis (Forchhammer, 2004). PII reduces nitrate

uptake as soon as a reduction in CO2 fixation occurs.

Toxic strains exhibited higher NrtA and lower PII protein

levels compared with nontoxic strains which suggested a

high 2-OG level resulting from a high C : N metabolic

ratio (Alexova et al., 2011a, b). These expression patterns

were consistent with high NtcA activity (low nitrogen

availability); however, expression levels of NtcA were

below the levels of detection of the method used. Micro-

cystin synthesis was demonstrated not to be a static trait,

in that a previously nontoxin-producing strain of

M. aeruginosa UWOCC MRC, carrying the mcy cluster,

commenced toxin production (Alexova et al., 2011a, b).

The complete sequence of this cluster was not character-

ised prior to the conversion, nor afterwards, making any

conclusions about the mechanism of toxin re/activation

speculative. Very little commonality exists between the

proteomes of the toxin-producing and the nontoxin-pro-

ducing strains of M. aeruginosa investigated, with only 82

of the compiled proteins occurring in all strains (Alexova

et al., 2011a, b). This agreed with a study that demon-

strated a great variability between the genomes of the two

microcystin-producing strains of M. aeruginosa PCC7806

and NIES-843 (Frangeul et al., 2008).

Nodularin synthesis

A schematic overview on the current knowledge of

regulation of nodularin production in dinitrogen-fixing



cyanobacteria forming heterocysts is presented in Fig. 2.

The nda cluster in N. spumigena NSOR10 is transcribed

as two polycistronic mRNAs: ndaAB, ORF1, ORF2 and

ndaC (Moffitt & Neilan, 2004). Preliminary analysis iden-

tified three potential NtcA binding sequences in the nda

promoter region of N. spumigena NSOR10 and N. spumi-

gena CCY9414, upstream of ndaA (Supporting Informa-

tion, Fig. S1), suggesting a role for NtcA in the regulation

of toxin production in N. spumigena NSOR10. A similar

recognition sequence (50 GTC N8 TGC 30) was shown to

bind NtcA and regulate the expression of genes involved

in heterocyst development in Anabaena PCC7120 (Cam-

argo et al., 2012). This raises the question of whether

NtcA, and hence nitrogen levels, is involved in the regula-

tion of nodularin production in dinitrogen-fixing cyano-

bacterial species?

While expression of the nda cluster appears to be

constitutive, phosphate starvation induced an approxi-

mately twofold increase in expression and ammonia

supplementation resulted in a twofold decrease in expres-

sion. These changes, however, did not affect either intra-

cellular or extracellular nodularin concentrations

(Jonasson et al., 2008).

Of interest is the observation of a putative high light-

inducible chlorophyll-binding protein (HLIP), ORF2, that

is co-transcribed with the nda cluster in N. spumigena

NSOR10 and may suggest a physiological role associated

with high light stress in nodularin-producing cyanobacte-

ria (Moffitt & Neilan, 2004). A similar open reading

frame (N9414_07721) with 98% identity to ORF2 is also

present on the newly released genome of N. spumigena

CCY9414, just downstream of the nda cluster (Voß et al.,

2013), implying a role for regulation of toxin production

under light stress. While nodularin was not shown to

bind to RuBisCo directly under high light stress, it did

bind strongly to other, as yet unidentified, proteins

(Meissner et al., 2013). Whether this is the result of

chemical binding due to increased alkalinity resulting

ccmK1-3

nodularin

HCO CO

Carboxysome: proteins Ccm K1-K4

Ccm L

RubiscoCO

HCO

CA

HCO

hv

glnB

ntc A

nif H

PS II PSI

NN

HCO HCO

P

Nif H

N

NH

Nitrogenase

rbc L

Rbc L

Rbc S

SProtein boundNodularin?

2-OG ?

nitrogen

het R

ntc A

NH

nda C nda A ORF 2

ORF 2?

NtcA

Fig. 2. Schematic figure detailing the current knowledgebase of regulation of nodularin production in nitrogen-fixing cyanobacteria forming

heterocysts. Sunlight shines onto the cell (hv) and powers photosynthesis, represented by PSI and PSII on the thylakoid membrane. The cell also

takes up CO2 and HCO�3 via various mechanisms and feeds the HCO�

3 pool in the cytoplasm and then enters the carboxysome, made of gene

products of ccmK1-4 and ccmL. Dotted lines represent expression of a gene including transcription, translation and potential processing.

Carbonic anhydrase (CA) converts HCO�3 into CO2 where it is fed to RuBisCo, composed in part of the gene product of rbcL. Nodularin is

thought to be synthesised from the nda gene cluster under the influence of NtcA in a manner similar to microcystin. Nodularin binds proteins in

cells under oxidative stress; however, it is known not to bind to RuBisCo in Nodularia spumigena CCY9414. The gene-encoding RNA polymerase

I (rpoC1) is used as a control for gene expression studies. N2 is taken up into the heterocyst cytoplasm to be converted into NHþ3 by the

nitrogenase composed in part from the gene product of nifH. Previous studies have highlighted the involvement of the ndaA, rbcL, ccmK3, ntcA,

glnB (encoding PII protein) and nif H in microcystin production. While ORF2 is known to be transcribed in N. spumigena NSOR10, the protein has

still to be identified. The concentration of 2-OG increases in vegetative cells under the conditions of increased photosynthesis/ATP levels and

reduced N availability, and is thought to signal the heterocyst to initiate/increase BNF by enhancing binding of NtcA to the nifH promoter.

Whether this results in increased nodularin production in vegetative cells and/or heterocysts is unknown.



from oxidative stress or a target-specific response remains

to be determined.

Dinitrogen fixation occurs in specialised cells, hetero-

cysts, in Nodularia and Nostoc species. Differentiation of

heterocysts is controlled in the early stages by expression

of hetR, which is indirectly regulated by the action of

NtcA. Nitrogen fixation was decreased in N. spumigena in

the presence of ammonia but not in the presence of

nitrate (Kabir & El-Shehawy, 2012). Reduction in the

transcript levels of nifH in N. spumigena grown in the

presence of ammonia was accompanied by a reduction in

ndaF transcripts, suggesting a correlation between nitro-

gen fixation and toxin regulation. The light intensity is

not provided in this study, so one cannot determine the

redox status of the cultures at the time of sampling. Pre-

vious work indicated increased nodularin production by

N. spumigena with increasing temperatures (25–28 °C)and irradiances (45–155 lmol photons m�2 s�1; Leh-

tim€aki et al., 1997).

The question arises: What would a nitrogen fixing

organism, capable of regulating its own nitrogen status,

do under nitrogen-limiting conditions with respect to

toxin production? Because of the high energy demand of

nitrogen fixation, diazotrophs often utilise other dissolved

nitrogen sources in preference to nitrogen fixation as

demonstrated for C. raciborskii (Saker et al., 1999). Given

that the nodularin-producing Nostoc spp. are terrestrial

organisms and usually found in nutrient-poor soils, the

toxin gene cluster would not easily be switched on by the

action of NtcA. Overall, it is likely that toxin production

is coupled to changes in nitrogen levels in actively grow-

ing and photosynthesising cells.

Microcystis aeruginosa is very sensitive to light with a sat-

urating light intensity of 32 lmol photons m�2 s�1 (Hesse

et al., 2001). In contrast, Nodularia spumigena is tolerant of

high light intensities in the range of 150–290 lmol pho-

tons m�2 s�1 (Jodłowska & Latała, 2010) and would there-

fore have a higher demand for nitrogen to maintain the

N/C ratio. Whether production of nodularin offers an

additional means of nitrogen storage or reduces the effects

of oxidative stress remains to be determined.

What is evident from these studies on toxin regulation

in cyanobacteria is that it is not suitable to restrict these

studies to single strains or isolates (Alexova et al.,

2011a, b). The levels of nodularin synthesis by Nostoc

species are low, in the range of 10 ng lg�1 chlorophyll a

(Gehringer et al., 2012), when compared to the approxi-

mately fourfold higher levels of toxin production

observed in the aquatic nitrogen fixing species, Nodularia

spumigena (Stolte et al., 2002). Both nda clusters

sequenced in N. spumigena have high sequence similarity

in their promoter regions and have similarly conserved

potential NtcA binding sites (Fig. S1). The difference in

toxin production between nodularin-producing Nostoc sp.

and N. spumigena spp. may be due to differences in car-

bon and nitrogen fixation rates, although errors in the

determination of cellular toxin content cannot be

excluded (Meissner et al., 2013).

Hepatotoxin production under changingclimatic conditions and the potentialimpact on CyanoHABS in the futureenvironment

A crucial element of climate change is the ongoing rise in

greenhouse gas emissions, especially carbon dioxide

(CO2), resulting largely from accelerating rates of fossil

fuel combustion and biomass burning (Houghton et al.,

2001). The consequence of this release is an expected

increase in the annual mean surface temperatures of

3–5 °C during the summer months and of 5–10 °C dur-

ing the winter time, predicted for the period 2070–2100,compared with the reference period 1960–1990 (Hough-

ton et al., 2001). Both predictions are based on a dou-

bling of CO2 emission values during the twenty-first

century or the ‘business as usual scenario’. These predic-

tions are supported by the observed average increase in

Baltic Sea surface water temperature of 0.08 °C per dec-

ade, compared with a worldwide warming of 0.05 °C per

decade, resulting in a temperature rise of c. 0.7 °C in the

last hundred years (HELCOM B, 2007). By the end of

this century, air and surface water temperatures are pre-

dicted to increase by a further 3–5 °C in the south-wes-

tern part of the Baltic Sea, particularly during summer

(Siegel et al., 2006). A greater than twofold increase in

cyanobacterial biomass, as well as prolonged growth,

averaged over a 30-year period (2069–2098) was predictedusing a coupled biological–physical model to predict

cyanobacterial growth in the Baltic Sea (Hense et al.,

2013). Their model emphasised the importance of the

biological feedback mechanism induced by increased light

absorption, suggesting a 2.3-fold increase in annual

cyanobacterial biomass and an increase in the duration of

diazotrophic cyanobacterial bloom events.

However, considering climate change from the stand-

point of water temperature alone is too simplistic. Cli-

mate change studies also need to include factors such as

the loss of ice cover, which is expected to increase: the

onset of stratification, which might occur earlier in spring

and might be prolonged in autumn, as well as hydrologi-

cal changes including salinity change and changes in pre-

cipitation and evaporation patterns (Huber et al., 2012;

Neumann et al., 2012; Paerl & Otten, 2013). Studies on

these effects and combinations thereof are even scarcer

than investigations dealing with temperature increase

alone.



Another issue gaining more importance is the loss of

alkalinity, especially in ocean habitats, also called ocean

acidification. Atmospheric CO2 concentrations already

exceed 400 lg g�1 and are predicted to increase to over

800 ppm by the end of the century (Parry, 2007). This is

predicted to lead to a lowering of pH and carbonate ion

concentrations in aquatic systems, globally lowering the

pH by c. 0.1 units compared with the beginning of the

20th century (Caldeira & Wickett, 2003). With respect to

progressively increasing CO2 emissions, a further drop in

surface water pH by up to 0.38 units is predicted by 2100

(Parry, 2007). However, it is not the magnitude of change

in pH that is alarming, but the rate of the change, which

seems to be novel in the Earth’s history (Parry, 2007; Tri-

pati et al., 2009). Dissolved inorganic carbon (DIC)

includes CO2, HCO�3 , carbonate (CO

�3 ) and carbonic acid

(H2CO3) but in the pH range of 7.5–8.1, the range

recorded in most aquatic bodies, the DIC is principally

composed of HCO�3 . In marine ecosystems, the buffering

capacity keeps the pH and speciation of inorganic DIC in

a smaller range than those typically observed in freshwa-

ters. Many lakes are supersaturated with CO2 (Cole et al.,

1994; Maberly, 1996) resulting from terrestrial carbon

inputs and sediment respiration (Cole et al., 1994). Due

to a lower buffer capacity, the pH and speciation of inor-

ganic carbon in freshwater ecosystems can alternate

widely on a daily, episodic and seasonal scale (Maberly,

1996; Qiu & Gao, 2002) with diel variations in productive

lakes as high as 2 pH units and 60 mmol DIC L�1

(Maberly, 1996). The large, diel drawdown in DIC associ-

ated with algal blooms in eutrophic lakes may cause

phytoplankton to become periodically carbon limited.

Cyanobacteria appear to be more competitive than other

eukaryotic algae under high pH and low CO2 conditions

(Qiu & Gao, 2002), as surface-dwelling taxa can directly

exploit CO2 from the atmosphere, thereby minimising

dissolved inorganic carbon (DIC) limitation of photosyn-

thetic growth and taking advantage of rising atmospheric

CO2 levels (Paerl & Ustach, 1982). Furthermore, the car-

bon-concentrating mechanisms (CCM) they possess

enable the Cyanobacteria to overcome periodical DIC lim-

itation. How alterations in temperature, light regime and

DIC availability with respect to changes in toxin produc-

tion shall be addressed in the following subsections.

Rising surface temperature and change in light

regime

The mean sea surface temperature of the brackish habitat

Baltic Sea in summer is 20–23 °C with a maximum of

around 25 °C in the Gotland Sea (HELCOM B, 2007).

Another rise in temperature by the above-mentioned

expected 3–5 °C would increase the temperature in this

region up to 30 °C. Nevertheless, studies on the impact

of global warming including temperatures above 25 °Care rare. While increased global temperatures will proba-

bly result in CyanoHAB intensification, this may not be

caused by an increase in cyanobacterial growth rates

(L€urling et al., 2013). Mean optimal growth rates for six

strains of cyanobacteria were similar at 29.2 °C at

80 lmol photons m�2; however, all grew well up to

35 °C. Increased growing temperature (12–16 °C)increased both the biovolume and photosynthetic activi-

ties of Nodularia spumigena and Aphanizomenon sp.

(Karlberg & Wulff, 2013). Changes in CO2 levels did not

affect either parameter for either organism; however,

decreased salinity levels increased the photosynthetic

activity of N. spumigena.

Cyanobacteria appear to be able to readily regulate their

pigment content and composition dependant on the light

available. Therefore, the photosynthetic capacity of cyano-

bacteria occurring in their natural environment receiving

high solar radiation shows no photo-inhibition of PS II

(Rascher et al., 2003). Acclimatisation of cyanobacteria to

high light conditions has been extensively studied in the

unicellular, nondiazotrophic and nontoxin-producing

strain of Synechocystis PCC6803 (reviewed in Muramatsu

& Hihara, 2012). Looking at studies involving whole-gen-

ome DNA analysis, they identified five acclimatory

responses: (1) increased photosystem II turnover, (2)

reduction in the capacity of the light harvesting mecha-

nism, (3) protection of photosystems from ROS/increased

light energy, (4) increase in general survival protection

mechanisms and importantly (5) modulation of C and N

fixation/assimilation. Dissipation of the excess energy gen-

erated by photosynthesis during high light periods by the

energy-demanding process of biological nitrogen fixation

(BNF) was proposed by (Wannicke et al., 2009). This

theory further illustrates the close association of carbon

and nitrogen metabolism within cyanobacteria and offers

a means for involvement in the modulation of protein

synthesis, including the regulation of expression of non-

ribosomal proteins such as microcystin and nodularin.

Expansion of these studies to encompass toxin-producing

cyanobacterial species, including diazotrophs, will offer

valuable insights into the role of microcystin and nodula-

rin in cellular metabolism, particularly under light stress.

A summary of key research published in elucidating

the regulation of hepatotoxin production in cyanobacteria

(Table 1) clearly illustrates several problems relating to

climate change research. By far the majority of studies

have focussed on unicellular, non-nitrogen-fixing Micro-

cystis aeruginosa strains, specifically M. aeruginosa

PCC7806 and mcy deletion mutants under low, nonstress-

ful light conditions and average temperatures of

20–25 °C. Comparative toxin production under varying



temperatures has seldomly been investigated, and only a

few studies have looked at toxin production at tempera-

tures over 25 °C (Long et al., 2001; Alexova et al.,

2011a, b; Dziallas & Grossart, 2011; J€ahnichen et al.,

2011; Kuniyoshi et al., 2013). A study comparing the pro-

duction of the lesser toxic form of microcystin, MC-YR,

and the acute toxic form, MC-LR, by Microcystis aerugin-

osa at three different temperatures 20, 26 and 32 °Cshowed an overall increase in the prevalence and levels of

the more toxic form MC-LR with increasing temperature

(Dziallas & Grossart, 2011). Moreover, the toxin-produc-

ing strain of M. aeruginosa was more competitive at tem-

peratures above 20 °C.The number of studies investigating toxin production

in nitrogen fixing strains, for example Nodularia spumige-

na, at temperatures over 25 °C is more restricted (Lehti-

maki et al., 1994; Jodłowska & Latała, 2010). Given the

great variety in experimental conditions in the published

literature pertaining to growth media, culture conditions,

especially regarding light and temperature, it is not easy

to draw conclusions concerning the effects of changing

culture conditions on toxin production. In some cases, it

appears that increased temperatures may result in

decreased toxin production in M. aeruginosa (J€ahnichen

et al., 2011) and in polar cyanobacterial mats incubated

at 23 °C, (Kleinteich et al., 2012) in contrast to the previ-

ously mentioned study (Dziallas & Grossart, 2011). Nod-

ularin production was enhanced at higher temperatures

accompanied by higher nitrogen fixation rates (Lehtimaki

et al., 1994; Lehtim€aki et al., 1997). The nontoxic, nitro-

gen-fixing marine cyanobacterium, Trichodesmium, signif-

icantly increased its N2 and CO2 fixation rates with

increased levels of CO2 alone (Hutchins et al., 2007) or

combined with increased light intensities (Garcia et al.,

2011). Its growth was not inhibited by nitrate and ammo-

nia or urea (10 lM) in the first generation and continued

with similar rates as determined in DIN-free media (Fu &

Bell, 2003). Whether this implies a greater level of toxin

production in hepatotoxic diazotrophs remains to be elu-

cidated.

Most culture experiments use batch cultures of single

strains to obtain a clear picture of the direct effects of

changing culture conditions on toxin production. Only

recently have competition studies involving toxin- and

nontoxin-producing strains been undertaken, again with

varying results. Toxic strains are advantaged under N

excess in batch culture (V�ezie et al., 2002) while nontoxic

strains outcompete toxic strains under continuous culture

conditions (Kardinaal & Visser, 2005; Kardinaal et al.,

2007). The involvement of light effects on toxin regula-

tion is complicated by different levels of light intensity

provided by a wide range of light sources under greatly

differing light regimes. The general consensus appears

that at a light intensity, over 40 lmol photons m�2 s�1

result in increased hepatotoxin production in toxic

M. aeruginosa strains (Zilliges et al., 2011; Meissner et al.,

2013). Considering the light intensity of a normal sunlit

day of c. 1000 lmol photons m�2 s�1 and the fact that,

in the cause of climate change, increased stratification

and changes in light regimes are possible, elevation in

toxin production is likely to occur in the future in both

marine and limnic habitats, but this correlation still has

to be proven.

What is alarmingly obvious in Table 1 is that most

studies have only calculated the soluble toxin content

(indicated by a † in Table 1) and not toxin bound to

proteins. This protein-bound fraction rapidly increases

under oxidative light stress conditions (Meissner et al.,

2013), and its detection is crucial to determining actual

toxin production within the cyanobacteria under investi-

gation. Very few studies have focussed on toxin gene

expression under conditions representing those predicted

for climate change. Combining these expression studies

with actual carbon and nitrogen fixation rates and a

re-evaluation of actual toxin production levels (Meissner

et al., 2013) may clarify some of the discrepancies in the

majority of previous toxin synthesis studies (Table 1),

which relied on the detection of soluble toxin. This

should offer a clear timeline of the metabolic processes

and their relationships during toxin production. The

release of 54 diverse cyanobacterial genome sequences this

year (Shih et al., 2013) went a long way to providing a

more reliable platform for sequence comparison of essen-

tial genes within the Cyanobacteria. While 70% of the

strains sequenced carried NRPS and PKS genes, indicative

of secondary metabolite production, some of them for

microcystin and nodularin synthesis, these gene clusters

need to be further analysed as to their potential regula-

tion by nitrogen and carbon-fixing processes. The expres-

sion of the clusters mcy and nda with respect to that of

key genes involved in photosynthesis, specifically the car-

bon concentration genes ccmK3 and ccmL (Alexova et al.,

2011a, b) and nitrogen regulation genes ntcA, glnB and

nrtA (Ginn et al., 2010; Alexova et al., 2011a, b), and

nifH (Severin & Stal, 2010a, b) is crucial to the under-

standing of the coupling of toxin synthesis to environ-

mental availability of carbon and nitrogen (Van de Waal

et al., 2009).

The increased occurrence of Nostocales blooms was

largely attributed to global warming effects (Sinha et al.,

2012; Sukenik et al., 2012). Given that cyanobacteria have

not changed their dispersal routes over the last few dec-

ades, one can assume that those cyanobacterial species

most able to adapt to changing conditions will continue

to predominate current niches, as well as potentially

expanding into new areas. The proliferation of



cyanobacterial blooms and the self-sustaining vicious

cycle of CyanoHAB development shall be addressed in

CyanoHAB proliferation in the future environment.

Rising carbon dioxide concentration

While the effects of increasing temperatures, changes in

light regimes and media nitrogen and phosphorous con-

centrations on toxin production have been extensively

investigated, the effect of increased inorganic carbon

availability has not. Only one research group has investi-

gated the potential effects of varying CO2 levels on toxin

production in non-nitrogen-fixing strains of the cyano-

bacteria, Microcystis (Van de Waal et al., 2009; Van De

Waal et al., 2011), and the results raise more questions

than they answer. Increased toxin production of a nitro-

gen-rich MC variant was observed under limiting CO2

levels at high light (50 lmol photons m�2 s�1) irradi-

ance, while nontoxic strains outcompeted toxic strains

under conditions of low light and high CO2 availability.

The effects of increased light irradiance under increased

CO2 availability in competition studies were not assessed

for toxin-producing diazotrophs; however, increased N2

fixation was observed in the nontoxin-producing strain,

Trichodesmium erythraem, under exposure to increased

CO2 levels and light intensities of 38 and 100 lmol pho-

tons m�2 s�1 (Garcia et al., 2011). Increased 2OG levels,

which indicate a higher CO2 fixation rate, resulted in

greater binding of NtcA to the mcyA promotor region

(Kuniyoshi et al., 2011).

The reason that Nostocales species appear to be domi-

nating the invasion into freshwater oligotrophic lakes as

reported in the literature (Hadas et al., 2012; Sinha et al.,

2012; Sukenik et al., 2012) may result from the fact that

these bacteria are more skilled to cope with increasing

CO2 availability and increased intracellular carbon levels

by increasing their nitrogen levels through in-house dini-

trogen fixation. Non-nitrogen-fixing organisms would be

unable to obtain enough inorganic nitrogen from internal

nitrogen cycling within the sediment to maintain a dense,

toxin or nontoxin-producing bloom (Sukenik et al.,

2012). Nitrogen fixation releases diazotrophs from the

constraints of environmental inorganic nitrogen availabil-

ity. Moreover, nitrogen fixation allows them to maintain

their N/C balance during increased carbon fixation in

contrast to Microcystis that requires the environmental

nitrogen to increase the N/C ratio (Van de Waal et al.,

2009). If the production of the hepatotoxins, microcystin

and nodularin stabilises the all-important carbon fixa-

tion-associated protein of RuBisCo or proteins of the

photosynthetic apparatus, one can expect the increase in

Nostocales-associated blooms to be increasingly toxic.

There are hints in the literature that brackish sea water

species, like Nodularia spumigena, take advantage of cli-

mate change conditions, especially increased pCO2 (Wan-

nicke et al., 2012), although no stimulation of the same

species by high pCO2 has also been shown (Czerny et al.,

2009). It is possible that increased pCO2 would provoke

an elevation in nodularin concentration per biomass and

per volume. Extending this argument into the terrestrial

sphere would suggest that there would be an increase in

the Nostocales content of biological soil crusts and the

percentage thereof that are toxic. Where nitrogen avail-

ability is not restricted, one would still expect the occur-

rence of nondiazotrophic cyanobacterial species; however,

the tendency towards increased toxicity would still be

expected (Davis et al., 2009; Wilhelm et al., 2011; Kleint-

eich et al., 2012).

CyanoHAB proliferation in the future

environment

Alterations in temperature and light regimes associated

with climate change might not only support the growth

of CyanoHAB species in general, but are also likely to

modulate the onset of CyanoHAB blooms (Fig. 3). Strati-

fication of large waterbodies could take place earlier in

spring and promote the formation CyanoHABs (Huber

et al., 2012). Destratification might be postponed to later

in autumn (Peeters et al., 2007; Elliott, 2010) and prolong

the bloom period (Huber et al., 2012). Moreover, the

structure and dynamics within food webs are likely to be

influenced. Floating cyanobacteria create thick surface

scrums, thereby increasing turbidity and shading and

hence minimising competition with other microalgae.

Buoyant species contain UV-absorbing compounds such

as mycosporine-like amino acids (MAAs) and scytonemin

which provide shelter under high irradiance conditions

(Paerl & Huisman, 2009; Paerl & Paul, 2012). Surface

blooms absorb light, thereby increasing the surface tem-

perature resulting in enhanced growth, and hence

increased biomass (Hense et al., 2013). Similarly in shal-

low waters, there is an increase in pelagic growing vegeta-

tive cells which in turn give rise to increased numbers of

benthic resting cells. The latter serve as a source for

future bloom events by rapidly increasing pelagic vegeta-

tive cell numbers under suitable conditions (Hense et al.,

2013).

Increased atmospheric CO2 levels would be expected to

positively affect cyanobacterial growth, as they would

have access to increased CO2 diffusing into the water.

This would reduce the effects of inorganic carbon limita-

tion on photosynthetic growth; however, with so few

published experiments available (Table 1), this aspect of

the cyanobacterial growth response needs to be further

investigated. On the other hand, CyanoHABs are known



to cause night-time oxygen depletion through respiration

and bacterial decomposition of dense blooms. Thus, few

studies pertaining to altered CO2 levels and toxin produc-

tion have been conducted under continuous illumination,

reducing the effects of night-time/dark phase oxygen

depletion (Van De Waal et al., 2011). Increased or pro-

longed bloom occurrences could result in periodical

hypoxia, fish kills and loss of benthic fauna and flora

(Garc�ıa & Johnstone, 2006; Paerl & Otten, 2013). Indeed,

a recent modelling study involving eight different simula-

tions suggested that cyanobacterial blooms could grow

for longer periods with a concomitant increase in anoxic

events in the Baltic Sea (Neumann et al., 2012). Because

cyanobacteria like Nodularia, but also Microcystis, are

adapted to a wide range of salinities from 0 to 20 g L�1

(Wasmund 1997; Tonk et al., 2007; M€oke et al., 2013),

increased salinisation due to increased water withdrawal

for human use and expansion of drought periods might

also increase CyanoHAB growth.

When studying Table 1, it would appear that cyano-

bacterial hepatotoxin production is positively affected by

temperature increase, light increases up to about

60 lmol photons m�2 s�1 and increased growth rates

resulting from sufficient nutrient availabilities. What the

effects of increased atmospheric CO2 in diazotrophic and

non-nitrogen-fixing cyanobacterial species under varying

conditions of nutrient availability are has not been inves-

tigated.

The envisioned earlier onset of CyanoHABs, particu-

larly hepatotoxic blooms, in combination with the

extended period of mass occurrences in freshwater and

marine habitats, will have great impact not only on the

recreational value of the ecosystem and their importance

for tourism, but also on human and animal health, drink-

ing water supply and the fishing industry.

Conclusions and future directions

Cyanobacteria are known to have highly plastic genomes

carrying large numbers of atypical genes thought to be

acquired by lateral gene transfers (Frangeul et al., 2008).

Analysis of the transcriptome of Nodularia spumigena

CCY9414 indicates a less complex transcriptional com-

plexity associated with the genes involved in dinitrogen

fixation than observed in other Nostocales. However, a

high level of complexity with regard to regulation of sec-

ondary metabolite synthesis as well as gas vesicle forma-

tion, iron and phosphorous uptake and synthesis of

N2

Greenhouse effect

Shading

Fixa

�on

Sedi

men

ta�o

n

Hypoxia

CO2

CO2

Wind

DIP

Rele

ase

DIN Uptake

Warming/ Destra�fica�on

Salinity gradient

Mar

ine

Freshwater

Fig. 3. Schematic illustration of the multiple interacting biotic and abiotic variables which control the proliferation and maintenance of

CyanoHABS and their potential hepatoxin production. Key factors of bloom development and the impact of climate change, that is increase in

CO2 concentration and temperature, are shown. The vicious self-sustaining cycle is triggered by increased atmospheric load by CO2 which

positively influences sea surface temperature. Earlier onset of stratification and lesser mixing linked to temperature elevation induces, along with

higher light availability, the onset of bloom development, provided DIP supply is sufficient. Shading of buoyant cyanobacteria negatively

influences macrophytes and microalgae. Hepatotoxin production appears to be positively influenced by increased temperature, higher light

availability and possibly higher CO2 levels accompanied by high light exposure. Decomposition of CyanoHABs and respiration provide a positive

feedback to atmospheric CO2. For a detailed description, see text (CyanoHAB proliferation in the future environment). Moreover, adaptation of

the filamentous diazotrophic species Nodularia to salinity is depicted by a grey arrow. To ensure clarity, some potential feedback mechanisms and

regulation factors for bloom development, as well as food web components, were omitted.



compatible solutes was seen (Voß et al., 2013). A project

sequencing 10 Microcystis aeruginosa genomes estimated

that about 11% of genes originated from recent horizon-

tal gene transfer events, including large gene clusters

(Humbert et al., 2013). This demonstrated the potential

of horizontal gene transfer of gene clusters responsible for

hepatotoxin synthesis, particularly if they confer an

advantage under predicted climate change conditions.

Understanding the evolutionary and regulatory processes

at work within the nonribosomal gene clusters in particu-

lar is important for regulating toxin expression in bloom

events, particularly those threatening freshwater drinking

supplies. By combining the physiological data with

sequence expression data, one would have greater insight

into the regulation of the toxin gene clusters under vary-

ing environmental conditions as proposed for the future

of climate change.

Increasing temperatures, stratification and longer

bloom-susceptible periods will affect both the composi-

tion and successional patterns of cyanobacterial and

eukaryotic algae in aquatic ecosystems. It is known that

the dominance of toxic vs. nontoxic strains of CyanoHAB

species is properly controlled by the length of the spring

and summer period and light availability (Kardinaal

et al., 2007). Understanding the fundamental genetic and

physiological regulation of hepatoxin production is a key

challenge for future studies. More importantly, the cova-

lent binding to proteins and the intracellular cycling of

hepatoxins have to be elucidated to ultimately understand

how environmental changes influence toxin production.

Moreover, hepatoxins have to be monitored in different

ecosystems on a broad level, which requires the develop-

ment and deployment of observing systems to provide

the consistent, long-term data and geographical as well as

temporal scale of monitoring.

Further elevation of temperature and atmospheric CO2

levels would promote growth and development of

CyanoHABs and might enable their further expansion

(Mehnert et al., 2010) and suggests that control of

CyanoHABs cannot be achieved by limiting the anthropo-

genic input of inorganic nutrients alone. Reduction in

dissolved inorganic nitrogen alone would appear to pro-

mote the dominance of diazotrophic cyanobacteria.

Assimilation of all the above-mentioned data would prove

invaluable to water regulatory bodies charged with ensur-

ing safe water for human consumption in the face of

changing climatic conditions.

Ultimately, the pivot element to control CyanoHABs is

the reduction of rate and extent of greenhouse gas emis-

sions which triggers global warming. Without this essen-

tial step, it is likely that future warming trends and

resultant changes in physical and chemical properties of

limnic and oceanic habitats will facilitate the vicious cycle

of CyanoHAB establishment, increase their potential tox-

icity and support their further expansion.

Acknowledgement

N.Wannicke thankfully acknowledges the financial sup-

port by the Project BIOACID of the German Federal

Ministry of Education and Research (BMBF, FKZ

03F0655 E, F).

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Supporting Information

Additional Supporting Information may be found in the

online version of this article:

Fig. S1. Sequence alignment of the promoter regions for

the nda gene clusters sequenced from Nodularia spumige-

na strains NSOR10 and CCY9414 using MUSCLE (Edgar,

2004).



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