Climate change and regulation of hepatotoxin ... - Wiley Online Library

MINIREVIEW

Climate change and regulation of hepatotoxin production in Cyanobacteria Michelle M. Gehringer1 & Nicola Wannicke2,3 1

Department 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

MICROBIOLOGY ECOLOGY

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. Understanding 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 Microcystis 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, capable of regulating their nitrogen levels by actively fixing dinitrogen. By combining data from significant studies, an overall scheme of the regulation of toxin production is presented, focussing specifically on nodularin production in diazotrophs 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 survival in a range of environments, including some of the most extreme niches. As primary producers, Cyanobacteria harvest sunlight during photosynthesis, are competent in the uptake of nutrients such as nitrogen (N), phosphorous (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

FEMS Microbiol Ecol 88 (2014) 1–25

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 suspected 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 microcystin exist and are synthesised by a wide range of cyanobacterial 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 nodularin have been identified (Mazur-Marzec et al., 2006), and production was thought to occur only in the bloom-forming Nodularia spumigena or benthic Nodularia ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

2

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 suggested 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 temperatures 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 nitrogen and non-nitrogen-fixing species (Kurmayer, 2011). Nitrogen pulses released from diazotrophs may stimulate and sustain nondiazotrophic species, including toxin producers 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 temperatures favour the growth of toxin producers, as seen for polar cyanobacterial mat samples grown at increased temperatures (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 production 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 terrestrial 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 knowledge concerning the gene regulation of hepatotoxin production in unicellular and filamentous cyanobacteria in ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

M.M. Gehringer & N. Wannicke

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 metabolites 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 presented in Table 1. We have attempted to include all seminal 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 experimental conditions that measured either toxin gene expression or synthesis, or were directly related to changing 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 summary 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 measuring toxin in methanol extracts (Meissner et al., 2013). These authors discovered that microcystin and nodularin synthesised in response to high light stress is bound rapidly and covalently to proteins, thereby making them undetectable using the standard methanol extraction techniques (Meissner et al., 2013). While this does not affect studies measuring extracellular toxin levels in water bodies, it does affect the interpretation of studies measuring 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 in context of cell metabolic processes The regulation of cyanobacterial hepatotoxin production has focussed on the synthesis of microcystin in the freshwater 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) 1–25


M. aeruginosa PCC7806 (toxic)

MASH01-A19 (toxic) *

(non-toxic)* M. aeruginosa

M. aeruginosa PCC7806 mcyB


PCC7806 mcyA & mcyB (non-toxic)

PCC7806 (toxic) M. aeruginosa

25

26

20

31 continuous 16, 31, 68

40

high 12:12 light: dark

blue, red

5, 16, 68

23

M. aeruginosa

30 continuous

38

16, 31, 68, 400 red, blue

23

20

54 lE m2 s1

0, 20,

PCC7806

M. aeruginosa

(toxic)

M. aeruginosa MASH01-A19 & 8 isolates

CYA43 (non-toxic) *

CYA 228/1 (toxic) M. aeruginosa

22

20–75 lE m2 s1

22

M. aeruginosa CYA 228/1

(toxic)* M. aeruginosa

m2 s1

Temp. °C

Strain

Light lmol photons

ND

growth rate

correlates to cellular

MC production

with ^ Fe

with ^ N ^ toxin

ND

site used under high light

-206 mcyD TSP with NtcA recognition

-254 mcyD TSP used under low light

high light

chlorosis in mutant under

mutants

in PCC7806 and not in

and red light MrpA identified

expression at high light

expression & ^ mcyB

Kaebernick et al. (2002)

Long et al. (2001)†

Hesse et al. (2001)

(2001)

Dittmann et al.

(2000)†

Kaebernick et al.

Orr and Jones (1998)†

Gjølme (1995a)

Utkilen and

Reference

m2 s1 ^ toxin

^ mcyD

Other Utkilen and Gjølme (1992a)

Toxin ^ 2.5 fold at 40 lE

levels correlate to growth rate

Limiting

Phosphate

Cellular MC

Limiting

Fe3+

mM nitrate

C/CO2 latm

0.02–0.2

0.0118–1.18 mM nitrate

Limiting

N

Table 1. Table highlighting the published research on the regulation of toxin production in Cyanobacteria

Microcystin/nodularin occurrence under climate change

3

ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved


outcompeted toxic strains decline in toxic strain in competitive experiments

M. aeruginosa V163 & CYA43 (nontoxic)*

v over 100

and isolates (toxic)

25 continuous

Non-toxic strains

20

Microcystis aeruginosa V163, CYA140

with ^ light to 60, then & v MC-RR synthesis

12:12 light: dark

competed toxic strains in competition studies

^ mcyA transcripts

Non-toxic strains out

related to C fixation

Toxin levels negatively

at max. growth rate

& protein synthesis

Max. toxin

v MC levels with time

^ toxin with ^ N

at ^ N

^ light induced increased MC-LR

nitrate

0.125, 7, 10, 15, 18 mM

mM

mM nitrate

higher light ^ toxin

under N limitation

Kardinaal et al. (2007)†

Tonk et al. (2005)†

Kardinaal & Visser (2005)†

Downing et al. (2005b)†

(2005a)†

Downing et al.

Wiedner et al. (2003)

Vezie et al. (2002)†

Non-toxic strains advantaged

Reference

Other

25, 110 6-112

25

51 continuous

0.067–0.175

1.18–21.2

light to 40. v MC with

dark

140 continuous

^ MC per cell with ^

advantaged under N excess

Toxic strains

Toxin

v in MC levels reflecting

23

20

Planktothrix arghardii (toxic)*

M. aeruginosa V163 & CYA43 (nontoxic)*

and isolates (toxic)

Microcystis aeruginosa V163, CYA140

UV027 (toxic)

M. aeruginosa PCC7806 &

23

23

M. aeruginosa

PCC7806 & UV027 (toxic)

22

M. aeruginosa PCC7806 (toxic)*

Excess

Phosphate Limit

Fe3+

Limit

C/CO2 latm

Excess

N

10–403 12:12 light:

25 continuous

20

M. aeruginosa

2 toxic strains 2 non-toxic strains

m2 s1

Temp. °C

Light lmol photons

Strain

Table 1. Continued

4 M.M. Gehringer & N. Wannicke



MASH-01A19 (toxic)

M. aeruginosa

(non-toxic)*

M. aeruginosa CYA43 & PCC7806 mcyB

CYA140 & PCC7806 (toxic)

M. aeruginosa

PCC7806 (toxic)

15, 26, 30

21.5 1000–1250 ppm

continuous

continuous

8, 40, 65

200 ppm

increased

20 mM nitrate 50, 25

16 continuous

0.2, 2,

25

M. aeruginosa

0.03,

0, 20, 200 0.1, 0.3, 1, 6 uM

CO2 levels. Non-toxic toxic strain present

light and P. v MC with v S

v MC with ^

dominant at low correlated to levels of

levels Variable S tested

and high CO2

strain dominant in low light

constant Toxic strain

with ^N. mcyB transcript: toxin

starvation ^ growth rate

MC levels

constant

Toxin/cell

expression under N limitation &

^ NtcA & mcyB

correlated to cell division rate

by a v MC/cell with increased

dark light

to Chl a content. MC inversely

–

MC content linked

between N : C and N levels

unchanged Stong correlation

production, specifically

^ total MC

MC/cell at 56 followed

Excess

272, 820 16:8 light:

Excess,

Excess

in ^ MC-RR Initial ^

24, 56, 136,

50 continuous

limit, starved

–

24

23

PCC7806 (toxic)

M. aeruginosa

UTCC299 (toxic)

M. aeruginosa

HUB 5-2-4 (toxic)*

M. aeruginosa

than non-toxic strain. TSP’s

overcame Fe deficiency better

(2011)†

Jahnichen et al.

(2011)†

Van de Waal et al.

Sevilla et al. (2010)†

Ginn et al. (2010)

(2010)†

Deblois & Juneau

(2009)†

Van de Waal et al.

with Fe limitation. Toxic strain

limitation

(non-toxic)

expression

with Fe

M. aeruginosa PCC7005

Toxin

PCC7806 (toxic)

Phosphate

Reference

Limit


Fe3+ Other

C/CO2 latm ^ mcyD

16 continuous

25

M. aeruginosa

N ^ MC levels

photons m2 s1

Temp. °C

Light lmol

Strain

Table 1. Continued


5



replete

quality of cyanobacterial toxins produced.

both MC-YR & MC-LR Overall, the

Innenalster, Germany; Lake Taihu, China)

temperature

rose with increasing

concentration of microcystins

bacteria affected the quantity and strains producing

toxic strains at higher increased with rising

32

PCC 7806 (toxic) HUB 018, P461,

3 field samples (Boulder Lake, USA;

There was a shift toward MC-LR levels

72 16:8 light: dark

20 26

M. aeruginosa HUB 524, W333,

temperatures. Heterotrophic

^ NtcA binding to mcyA promoter

25 continuous

25-30


temperatures in axenic

Dziallas & Grossart (2011)†

transcription increased in non-toxic strains.

PCC7005, HUB5.3 (non-toxic)

PCC 7806 mcyB mutant (non’toxic)

Kuniyoshi et al. (2011)

decreased in non-toxic strains. NtcA

Alexova et al. (2011b)†

(2011a)†

Alexova et al.

M. aeruginosa UWOCC CBS,

PII & NdhK proteins increased

cultures

starved cultures

10 nM 25 continuous

transcripts in 10 nM Fe starved

16 °C ^ in isiA

16 °C ^ cellular MC content in 10 nM Fe

diversity in mats at 8 &

1000, 100,

Kleinteich et al.

^ cyanobacterial

^ cyanobacterial toxin levels in mats at 8 &

(2012)†

Reference

Other

Toxin

14:10 light: dark

^ 2OG

Phosphate

10 uM,

Fe3+

90

replete

C/CO2 latm

while NrtA, CcmK3, CcmL & TrxM

28

limit

N

MRC, UWOCC MRD (toxic)

M. aeruginosa PCC7806, UWOCC

PCC7005, PCC7806 mcyH (non-toxic)

PCC7806 (toxic) M. aeruginosa

M. aeruginosa

27

23 16:8 light: dark

4, 8, 16,23

Microbial mats

(Arctic and Antarctic)

m2 s1

Temp. °C

Light lmol photons

Strain

Table 1. Continued




mM nitrate

12:12 light: dark

2 mM phosphate

0.05, 0.2,

Phosphate

PCC7806 mcyB (non-toxic)

126 (toxic) M. aeruginosa

stress.

induced oxidative

MC binds proteins under high light

with reduced phosphate with reduced phosphate ^ protein bound MC

conditions. ^ mcyD transcripts

toxin producer under favourable

strain outcompeted

and in darkness. The mutant

mcyD expression under P limitation

expression with increased light. v

^ mcyD

stress conditions.

under high light and oxidative

lower light. MC binds protein

^ MC content

^ mcyD expression

^ toxin with

under high light.

to growth rates. No strain advantage at

Toxic strain advantage

Other

MC levels correlated

Toxin

light and oxidative stress.

PII inhibit

limit

Fe3+

under high

8 continuous 250

16 continuous

0.036 & 9

5 & 39

continuous 0

28, 50, 109

70, 300, 700

16 continuous

C/CO2 latm

NIES843, Planktothrix agardhii NIVA-CYA

23

25

22

25

23

varying

N

aeruginosa

M. aeruginosa PCC7806, M.

PCC7806 (toxic)

M. aeruginosa

PCC7806 mcyB (non-toxic)

PCC7806 M. aeruginosa

M. aeruginosa

PCC7806

(non-toxic) M. aeruginosa

PCC7806 mcyB & mcyH

PCC7806 M. aeruginosa

M. aeruginosa

mcyA (non-toxic)

continuous

M. aeruginosa PCC7806

photons m2 s1 High 37 Low 8 & 17

Temp. °C

Light lmol


Strain

Table 1. Continued

Meissner et al. (2013)

(2013)†

Kuniyoshi et al.

Briand et al. (2012)†


Zilliges et al. (2011)

Phelan & Downing (2011)†

Reference


7


Gradient from 153 to 731

0.3, 0.6, 1

Phosphate

^ NOD at 20 °C

Toxin Mean optimal growth temp.

Other


20

Nodularia spumigena AV1

BA-15 (toxic)

Nodularia spumigena

15, 30

16

Nodularia spumigena CCY9414 (toxic)

(toxic)*

21

Nostoc sp. IO-102-I 7(toxic)

16:8 light: dark

10, 150, 290

16:8 light:dark

45

85 14:10 light: dark

20–27 continuous

continuous

25 2-155

20 7-28

Nodularia spumigena BY1 (toxic)

25, 50, 80 continuous

10–30

(2 isolates)

PCC6301 (non-toxic) Nodularia spumigena

addition

NH4

free

free

limit

5.4 lM

0.2–5.5

at 0.3 P

ND

in NOD detected

No variation

ND

6 MC variants detected

light at low temperature

with ^ NH4 Tolerant of high

with vP v nda expression

N2 fixation ^ nda expression

P ratio, slight decrease in

growth, elevation of C : N and N :

High CO2 caused decrease in

inorganic N

temp. & nitrogen fixation rates

low salinities and high

v NOD with high and

conditions: high light,

^ NOD at best growth

had no effect lowest toxin

light changes

at 35 °C

Fe3+

agardhii CYA116 Synechococcus elongates

0.02–42 mg L1

C/CO2 latm

of 29.5 °C. All grew well

80 16:8 light:dark

20-35

M. aeruginosa PCC7941, CYA140

N

Planktothrix agardhii CYA126 (toxic) Planktothrix

m2 s1

Temp. °C

Light lmol photons

Strain

Table 1. Continued

(2010)

Jodlowska & Latala

(2008)†

Jonasson et al.

Czerny et al. (2009)

Oksanen et al. (2004)‡

Lehtimaki et al. (1997)†

(1994)†

Lehtimaki et al.

Lurling et al. (2013)

Reference




20-35

12, 16

35 °C

of 29.5 °C. All grew well at

dark

Fv/Fm for N. spumigena

^ Fv/Fm with ^ Temp. ^ CO2 ^

induced oxidative stress

proteins under high light

BNF rate. NOD binds

Nitrates had no effect on

& expression of nifH & ndaF.

NH4 > 0.5mM caused v BNF

and N:P ratio

elevation of C:P

Mean optimal growth temp.

ND

ND

NOD produced

NOD and [L-Har2]-

High CO2 caused increase in growth, C and N2 fixation,

80 16:8 light:

dark

75 16:8 light:

398 548

12 °C ND

MC in P reduced cultures

Lurling et al. (2013)

Karlberg & Wulff (2013)

(2013)

Meissner et al.

Kabir & El-Shehawy (2012)

(2012)†

Gehringer et al.

(2012)

Wannicke et al

Reference Kurmayer (2011)†

Other Max. intracellular

Normal CO2 is approximately 380 ppm in standard air. *Continuous / semi-continuous culture experiments, otherwise results are for batch cultures. †Studies using aqueous methanol extracts for toxin determinations. ‡using acetic acid and using acetonitrile extractions for toxin determinations. ND Not determined increases are expressed relative to control values as presented in the studies listed. TSP, transcription start point; µE, micro einsteins.

gracile (non-toxic)

(neurotoxic) Anabaena sp. PCC7122 Aphanizomenon

Cylindrospermopsis raciborskii CIRF-01

Aphanizomenon KAC15 (non-toxic)

Nodularia spumigena KAC12 (toxic)

CCY9414 (toxic)

250

8 continuous

Nodularia spumigena

23

mM nitrate

dark

KAC71 (toxic)

Nodularia spumigena KAC11, KAC66,

0-1 mM NH4 or 0-100

20

16:8 light: dark

0.3 uM

free

315

detected under low

144 lM

mM nitrate light in replete medium at

Toxin

0 & 5.88

Max. MC

Fe3+ 0.14 vs.

C/CO2 latm Phosphate

N

not stated 16:8 light:

(2 isolates)

27

Nostoc strains

22

16:8 light: dark

CCY9414 (toxic)

200

23

Nodularia spumigena

1 & 50 continuous

12 & 20

Nostoc PCC9237 (originally

photons m2 s1

Light lmol

Nostoc sp. 152) (toxic)

Temp. °C

Strain

Table 1. Continued


9


10


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-containing 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 stable 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 glutamine 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 (Forchhammer, 2008) for a review. In cells of Synechococcus elongatus 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 intracellular 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 phosphorylation 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 (Martin-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

MicrocysƟn (mcy) gene cluster: MicrocysƟs 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 (MartinLuna et al., 2006). Iron limitation resulted in increased toxin production and transcription of mcyD (Sevilla et al., 2008) and isiA (iron stress-induced protein; Alexova 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 cultures while FurA transcription was reduced (Alexova et al., 2011a, b). In contrast to this, another group identified an increase in microcystin production with increasing iron availability (Utkilen & Gjølme, 1995). The discrepancies may be due to methanol extracts being used for toxin determination, with protein-bound toxin not being measured. Further work is required to determine the regulation 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 activator or as an inhibitor of nitrogen response genes by selectively binding a recognition sequence on the DNA upstream of the genes it regulates. Under conditions of high nitrogen availability, PipX, required for NtcA activation, 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 regulation. The consensus sequence is usually found c. 22 base pairs upstream of the -10 sigma hexameric signal for initiation of transcription (Luque et al., 2004). NtcA regulates its own expression and is a transcriptional activator of the genes involved in the differentiation of heterocytes (nitrogen-fixing cells) in Anabaena PCC7120 upon nitrogen 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 FEMS Microbiol Ecol 88 (2014) 1–25

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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 compared 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-limiting conditions that was 14 times greater than under nitrogen-replete conditions, suggesting strong upregulation of the mcy by NtcA binding. Additional NtcA binding 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 photons m2 s1, 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, 50 GTT 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 translation start site for mcyA (Kaebernick et al., 2002), suggesting a potential means of negative regulation by NtcA (Herrero et al., 2001). This mcyA TSP was transcribed under high light conditions (68 lmol photons m2 s1) while the TSP at 254 bp upstream was used under low (16 lmol photons m2 s1) light conditions and seemed not to be associated with any NtcA binding site (Kaebernick 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 cyanobacteria and is the most common limiting factor for growth. The effects of nitrate concentrations on microcystin production are conflicting with some studies indicating an overall increase in toxin production with increasing nitrate availability in the unicellular, non-nitrogen-fixing ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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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 assimilation 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 (Kuniyoshi et al., 2013). Nontoxin-producing M. aeruginosa strains grew better than toxin producers under low nutrient conditions. Under high nutrient load, the toxin producers increased their biomass to a larger degree than nontoxin producers (Vezie et al., 2002). Increased nitrate availability resulted in an increase in overall growth rate of M. aeruginosa PCC7806; however, the levels of microcystin 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 photons m2 s1 (Sevilla et al., 2012) or 30 lmol photons m2 s1 (Kaebernick et al., 2000). The levels fell shortly afterwards as the cells became stressed. Toxin levels showed conflicting correlations to mcyD transcription levels (Kaebernick et al., 2000; Sevilla et al., 2010, 2012). However, as methanol toxin extracts were used, additional protein-bound toxin in the cell debris may not have been detected and could account for the discrepancies (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 m2 s1 (Sevilla et al., 2010), confirming that light intensity has a greater influence than nitrogen 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 m2 s1) light exposure while no advantage was observed at lower light intensities (17 and 8 lmol photons m2 s1). 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 protection during high light stress by preventing chlorosis, as previously suggested (Hesse et al., 2001). Microcystis aeruginosa mutants unable to produce microcystin, contained less pigments in the form of chlorophyll a, b-carotene, ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved


zeaxanthin and echinenone when compared to the microcystin-producing wildtype grown under high light conditions (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 photons m2 s1 followed by a reduction in toxin levels at high photon irradiances of 136, 272 and 820 lmol photons m2 s1. 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 photosynthetic apparatus. Inhibition of the electron transfer chain of photosystem II resulted in a decrease in mcyD transcripts 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). Specifically, the large subunit of RuBisCo, RbcL, was protected from protease digestion by the binding of microcystin under conditions of light-induced oxidative stress at 70 lmol photons m2 s1 (Zilliges et al., 2011). The fact that NtcA binding regulates both the expression of the mcy gene cluster and rbcL suggests that toxin production 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 m2 s1. 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 photons m2 s1 (Zilliges et al., 2011). Of interest is the observed increase in microcystin cellular content, determined 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 production 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 FEMS Microbiol Ecol 88 (2014) 1–25

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microcystin-RR (Tonk et al., 2008). Addition of the Nrich 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 aeruginosa 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 stoichiometries, 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 microcystin-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 m2 s1). 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 FEMS Microbiol Ecol 88 (2014) 1–25

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 ribulose-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 icosahedral protein shell of repeat hexamers of the carbonconcentrating 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 (PII and 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 Synechocystis (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. Microcystin 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 characterised 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-producing 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 demonstrated 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 ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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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 identified three potential NtcA binding sequences in the nda promoter region of N. spumigena NSOR10 and N. spumigena CCY9414, upstream of ndaA (Supporting Information, 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 (Camargo et al., 2012). This raises the question of whether NtcA, and hence nitrogen levels, is involved in the regulation of nodularin production in dinitrogen-fixing cyanobacterial species? While expression of the nda cluster appears to be constitutive, phosphate starvation induced an approximately twofold increase in expression and ammonia

supplementation resulted in a twofold decrease in expression. These changes, however, did not affect either intracellular or extracellular nodularin concentrations (Jonasson et al., 2008). Of interest is the observation of a putative high lightinducible 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 cyanobacteria (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

hv CO

HCO

NH

N N

PS II

PSI

glnB

HCO

HCO P

ntc A

Carboxysome: proteins Ccm K1-K4

HCO

Ccm L

HCO C A CO

NH Rubisco 2-OG ?

Nitrogenase

ccmK1-3

nitrogen

Rbc S

ntc A

rbc L nodularin

nda C

nda A

N

S

Rbc L

Protein bound Nodularin?

NtcA

nif H Nif H

ORF 2

het R ORF 2?

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 HCO3 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 NHþ 3 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, heterocysts, 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 nitrogen 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. Previous work indicated increased nodularin production by N. spumigena with increasing temperatures (25–28 °C) and irradiances (45–155 lmol photons m2 s1; Lehtim€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 growing and photosynthesising cells. Microcystis aeruginosa is very sensitive to light with a saturating light intensity of 32 lmol photons m2 s1 (Hesse et al., 2001). In contrast, Nodularia spumigena is tolerant of high light intensities in the range of 150–290 lmol photons m2 s1 (Jodłowska & Latała, 2010) and would therefore 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 lg1 chlorophyll a (Gehringer et al., 2012), when compared to the approximately 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 FEMS Microbiol Ecol 88 (2014) 1–25

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toxin production between nodularin-producing Nostoc sp. and N. spumigena spp. may be due to differences in carbon and nitrogen fixation rates, although errors in the determination of cellular toxin content cannot be excluded (Meissner et al., 2013).

Hepatotoxin production under changing climatic conditions and the potential impact on CyanoHABS in the future environment 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 during the winter time, predicted for the period 2070–2100, compared with the reference period 1960–1990 (Houghton et al., 2001). Both predictions are based on a doubling of CO2 emission values during the twenty-first century or the ‘business as usual scenario’. These predictions are supported by the observed average increase in Baltic Sea surface water temperature of 0.08 °C per decade, 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 predicted to increase by a further 3–5 °C in the south-western 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 predicted using 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 standpoint of water temperature alone is too simplistic. Climate 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 hydrological changes including salinity change and changes in precipitation 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. ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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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 g1 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; Tripati et al., 2009). Dissolved inorganic carbon (DIC) includes CO2, HCO 3 , carbonate (CO3 ) 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 freshwaters. 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 inorganic 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 L1 (Maberly, 1996). The large, diel drawdown in DIC associated 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 photosynthetic growth and taking advantage of rising atmospheric CO2 levels (Paerl & Ustach, 1982). Furthermore, the carbon-concentrating mechanisms (CCM) they possess enable the Cyanobacteria to overcome periodical DIC limitation. How alterations in temperature, light regime and DIC availability with respect to changes in toxin production 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 ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved


region up to 30 °C. Nevertheless, studies on the impact of global warming including temperatures above 25 °C are rare. While increased global temperatures will probably 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 m2; however, all grew well up to 35 °C. Increased growing temperature (12–16 °C) increased both the biovolume and photosynthetic activities 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 cyanobacteria 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-genome DNA analysis, they identified five acclimatory responses: (1) increased photosystem II turnover, (2) reduction in the capacity of the light harvesting mechanism, (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 generated 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 nonribosomal 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 nodularin 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 Microcystis aeruginosa strains, specifically M. aeruginosa PCC7806 and mcy deletion mutants under low, nonstressful light conditions and average temperatures of 20–25 °C. Comparative toxin production under varying FEMS Microbiol Ecol 88 (2014) 1–25


temperatures has seldomly been investigated, and only a few studies have looked at toxin production at temperatures 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 production of the lesser toxic form of microcystin, MC-YR, and the acute toxic form, MC-LR, by Microcystis aeruginosa at three different temperatures 20, 26 and 32 °C showed an overall increase in the prevalence and levels of the more toxic form MC-LR with increasing temperature (Dziallas & Grossart, 2011). Moreover, the toxin-producing strain of M. aeruginosa was more competitive at temperatures above 20 °C. The number of studies investigating toxin production in nitrogen fixing strains, for example Nodularia spumigena, at temperatures over 25 °C is more restricted (Lehtimaki 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 previously mentioned study (Dziallas & Grossart, 2011). Nodularin production was enhanced at higher temperatures accompanied by higher nitrogen fixation rates (Lehtimaki et al., 1994; Lehtim€aki et al., 1997). The nontoxic, nitrogen-fixing marine cyanobacterium, Trichodesmium, significantly 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 ammonia 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 elucidated. 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 (Vezie 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 regulation 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 FEMS Microbiol Ecol 88 (2014) 1–25

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that at a light intensity, over 40 lmol photons m2 s1 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 m2 s1 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 investigation. 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 essential 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 regulation by nitrogen and carbon-fixing processes. The expression of the clusters mcy and nda with respect to that of key genes involved in photosynthesis, specifically the carbon 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 understanding of the coupling of toxin synthesis to environmental 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 decades, 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 ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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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 concentrations on toxin production have been extensively investigated, the effect of increased inorganic carbon availability has not. Only one research group has investigated the potential effects of varying CO2 levels on toxin production in non-nitrogen-fixing strains of the cyanobacteria, 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 nitrogen-rich MC variant was observed under limiting CO2 levels at high light (50 lmol photons m2 s1) irradiance, 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 photons m2 s1 (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 dominating 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 dinitrogen 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 availability. 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 fixation-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 ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved


species, like Nodularia spumigena, take advantage of climate change conditions, especially increased pCO2 (Wannicke 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 availability is not restricted, one would still expect the occurrence of nondiazotrophic cyanobacterial species; however, the tendency towards increased toxicity would still be expected (Davis et al., 2009; Wilhelm et al., 2011; Kleinteich 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). Stratification 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 temperature resulting in enhanced growth, and hence increased biomass (Hense et al., 2013). Similarly in shallow waters, there is an increase in pelagic growing vegetative 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 vegetative 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 limitation 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 FEMS Microbiol Ecol 88 (2014) 1–25

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CO2

Greenhouse eﬀect

CO2 N2 Fixaon

Wind

Shading

Release

DIP

Freshwater

Hypoxia

Salinity gradient

Marine

DIN Uptake

Sedimentaon

Warming/ Destraﬁcaon

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.

to cause night-time oxygen depletion through respiration and bacterial decomposition of dense blooms. Thus, few studies pertaining to altered CO2 levels and toxin production have been conducted under continuous illumination, reducing the effects of night-time/dark phase oxygen depletion (Van De Waal et al., 2011). Increased or prolonged bloom occurrences could result in periodical hypoxia, fish kills and loss of benthic fauna and flora (Garcıa & Johnstone, 2006; Paerl & Otten, 2013). Indeed, a recent modelling study involving eight different simulations 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 L1 (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 cyanobacterial hepatotoxin production is positively affected by temperature increase, light increases up to about 60 lmol photons m2 s1 and increased growth rates resulting from sufficient nutrient availabilities. What the FEMS Microbiol Ecol 88 (2014) 1–25

effects of increased atmospheric CO2 in diazotrophic and non-nitrogen-fixing cyanobacterial species under varying conditions of nutrient availability are has not been investigated. The envisioned earlier onset of CyanoHABs, particularly 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, drinking 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 complexity associated with the genes involved in dinitrogen fixation than observed in other Nostocales. However, a high level of complexity with regard to regulation of secondary metabolite synthesis as well as gas vesicle formation, iron and phosphorous uptake and synthesis of ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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compatible solutes was seen (Voß et al., 2013). A project sequencing 10 Microcystis aeruginosa genomes estimated that about 11% of genes originated from recent horizontal 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 particular 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 varying environmental conditions as proposed for the future of climate change. Increasing temperatures, stratification and longer bloom-susceptible periods will affect both the composition 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 covalent 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 development 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 anthropogenic input of inorganic nutrients alone. Reduction in dissolved inorganic nitrogen alone would appear to promote the dominance of diazotrophic cyanobacteria. Assimilation of all the above-mentioned data would prove invaluable to water regulatory bodies charged with ensuring 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 emissions which triggers global warming. Without this essential 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 ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved


of CyanoHAB establishment, increase their potential toxicity and support their further expansion.

Acknowledgement N.Wannicke thankfully acknowledges the financial support by the Project BIOACID of the German Federal Ministry of Education and Research (BMBF, FKZ 03F0655 E, F).

References Alexova R, Haynes PA, Ferrari BC & Neilan BA (2011a) Comparative protein expression in different strains of the bloom-forming cyanobacterium Microcystis aeruginosa. Mol Cell Proteomics 10: 1–16. Alexova R, Fujii M, Birch D, Cheng J, Waite TD, Ferrari BC & Neilan BA (2011b) Iron uptake and toxin synthesis in the bloom-forming Microcystis aeruginosa under iron limitation. Environ Microbiol 13: 1064–1077. Badger MR & Price GD (2003) CO2 concentrating mechanisms in cyanobacteria: molecular components, their diversity and evolution. J Exp Bot 54: 609–622. Badger MR, Pfanz H, B€ udel B, Heber U & Lange OL (1993) Evidence for the functioning of photosynthetic CO2-concentrating mechanisms in lichens containing green algal and cyanobacterial photobionts. Planta 191: 57–70. Beversdorf LJ, Miller TR & McMahon KD (2013) The role of nitrogen fixation in cyanobacterial bloom toxicity in a temperate, eutrophic lake. PLoS One 8: E56103. Bothe H, Schmitz O, Yates MG & Newton WE (2010) Nitrogen fixation and hydrogen metabolism in cyanobacteria. Microbiol Mol Biol Rev 74: 529–551. Briand E, Bormans M, Quiblier C, Salencßon M-J & Humbert J-F (2012) Evidence of the cost of the production of Microcystins by Microcystis aeruginosa under differing light and nitrate environmental conditions. PLoS One 7: e29981. Caldeira K & Wickett ME (2003) Oceanography: anthropogenic carbon and ocean pH. Nature 425: 365. Camargo S, Valladares A, Flores E & Herrero A (2012) Transcription activation by NtcA in the absence of consensus NtcA-binding sites in an anabaena heterocyst differentiation gene promoter. J Bacteriol 194: 2939–2948. Cole JJ, Caraco NF, Kling GW & Kratz TK (1994) Carbon dioxide supersaturation in the surface waters of lakes. Science 265: 1568. Czerny J, Barcelos e Ramos J & Riebesell U (2009) Influence of elevated CO2 concentrations on cell division and nitrogen fixation rates in the bloom-forming cyanobacterium Nodularia spumigena. Biogeosciences (BG) 6: 1865–1875. Davis TW, Berry DL, Boyer GL & Gobler CJ (2009) The effects of temperature and nutrients on the growth and dynamics of toxic and non-toxic strains of Microcystis during cyanobacteria blooms. Harmful Algae 8: 715–725.



Deblois CP & Juneau P (2010) Relationship between photosynthetic processes and microcystin in Microcystis aeruginosa grown under different photon irradiances. Harmful Algae 9: 18–24. Dittmann E, Erhard M, Kaebernick M, Scheler C, Neilan BA, Von D€ ohren H & B€ orner T (2001) Altered expression of two light-dependent genes in a microcystin-lacking mutant of Microcystis aeruginosa PCC 7806. Microbiology 147: 3113– 3119. Downing TG, Sember CS, Gehringer MM & Leukes W (2005a) Medium N:P ratios and specific growth rate comodulatemicrocystin and protein content in Microcystis aeruginosa PCC7806 and M. aeruginosa UV027. Microb Ecol 49: 468–473. Downing TG, Meyer C, Gehringer MM & Van De Venter M (2005b) Microcystin content of Microcystis aeruginosa is modulated by nitrogen uptake rate relative to specific growth rate or carbon fixation rate. Environ Toxicol 20: 257–262. Dziallas C & Grossart H-P (2011) Increasing oxygen radicals and water temperature select for toxic Microcystis sp. PLoS One 6: e25569. Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32: 1792–1797. Elliott J (2010) The seasonal sensitivity of cyanobacteria and other phytoplankton to changes in flushing rate and water temperature. Glob Change Biol 16: 864–876. Elliott JA (2012) Is the future blue-green? A review of the current model predictions of how climate change could affect pelagic freshwater cyanobacteria. Water Res 46: 1364– 1371. El-Shehawy R, Gorokhova E, Fernandez-Pi~ nas F & del Campo FF (2012) Global warming and hepatotoxin production by cyanobacteria: What can we learn from experiments? Water Res 46: 1420–1429. Ermilova EV & Forchhammer K (2013) PII signaling proteins of cyanobacteria and green algae. New features of conserved proteins. Russ J Plant Physiol 60: 483–490. Forchhammer K (2004) Global carbon/nitrogen control by P II signal transduction in cyanobacteria: from signals to targets. FEMS Microbiol Rev 28: 319–333. Forchhammer K (2008) PII signal transducers: novel functional and structural insights. Trends Microbiol 16: 65– 72. Frangeul L, Quillardet P, Castets AM et al. (2008) Highly plastic genome of Microcystis aeruginosa PCC 7806, a ubiquitous toxic freshwater cyanobacterium. BMC Genomics 9: 274. Fu F-X & Bell P (2003) Effect of salinity on growth, pigmentation, N2 fixation and alkaline phosphatase activity of cultured Trichodesmium sp. Mar Ecol-Prog Ser 257: 69– 76. Garcıa R & Johnstone RW (2006) Effects of Lyngbya majuscula (Cyanophycea) blooms on sediment nutrients and


21

meiofaunal assemblages in seagrass beds in Moreton Bay, Australia. Mar Freshw Res 57: 155–165. Garcia NS, Fu FX, Breene CL, Bernhardt PW, Mulholland MR, Sohm JA & Hutchins DA (2011) Interactive effects of irradiance and CO2 on CO2 fixation and N2 fixation in the diazotroph Trichodesmium erythraeum (cyanobacteria). J Phycol 47: 1292–1303. Gehringer MM, Adler L, Roberts AA, Moffitt MC, Mihali TK, Mills TJT, Fieker C & Neilan BA (2012) Nodularin, a cyanobacterial toxin, is synthesized in planta by symbiotic Nostoc sp. ISME J 6: 1834–1847. Ginn HP, Pearson LA & Neilan BA (2010) NtcA from Microcystis aeruginosa PCC 7806 is autoregulatory and binds to the microcystin promoter. Appl Environ Microbiol 76: 4362–4368. Hadas O, Pinkas R, Malinsky-Rushansky N, Nishri A, Kaplan A, Rimmer A & Sukenik A (2012) Appearance and establishment of diazotrophic cyanobacteria in Lake Kinneret, Israel. Freshw Biol 57: 1214–1227. HELCOM B (2007) The HELCOM Baltic Sea Action Plan. Helsinki Commission for the Protection of the Baltic Marine Environment Helsinki, Finland. Baltic Sea environment proceedings no. 111. Hense I, Meier HEM & Sonntag S (2013) Projected climate change impact on Baltic Sea cyanobacteria – climate change impact on cyanobacteria. Clim Change 119: 391–406. Herrero A, Muro-Pastor AM & Flores E (2001) Nitrogen control in cyanobacteria. J Bacteriol 183: 411–425. Hesse K, Dittmann E & B€ orner T (2001) Consequences of impaired microcystin production for light-dependent growth and pigmentation of Microcystis aeruginosa PCC 7806. FEMS Microbiol Ecol 37: 39–43. Hisbergues M, Jeanjean R, Joset F, Tandeau de Marsac N & Bedu S (1999) Protein PII regulates both inorganic carbon and nitrate uptake and is modified by a redox signal in Synechocystis PCC 6803. FEBS Lett 463: 216–220. Honkanen RE, Zwiller J, Moore RE, Daily SL, Khatra BS, Dukelow M & Boynton AL (1990) Characterization of microcystin-LR, a potent inhibitor of type 1 and type 2A protein phosphatases. J Biol Chem 265: 19401–19404. Honkanen RE, Dukelow M, Zwiller J, Moore RE, Khatra BS & Boynton AL (1991) Cyanobacterial nodularin is a potent inhibitor of type 1 and type 2A protein phosphatases. Mol Pharmacol 40: 577–583. Houghton J, Ding Y, Griggs D, Noguer M, Van der Linden P, Dai X, Maskell K & Johnson C (2001) IPCC, 2001: Climate Change 2001: The Scientific Basis. Contribution of Working Group I to the Third Assessment Report of the Intergovernmental Panel on Climate Change, Vol. 881. Cambridge University Press, Cambridge, UK, New York, NY, p. 9. Huber V, Wagner C, Gerten D & Adrian R (2012) To bloom or not to bloom: contrasting responses of cyanobacteria to recent heat waves explained by critical thresholds of abiotic drivers. Oecologia 169: 245–256.


22

Humbert JF, Barbe V, Latifi A et al. (2013) A tribute to disorder in the genome of the bloom-forming freshwater cyanobacterium Microcystis aeruginosa. PLoS One 8: e70747. Hutchins D, Fu F, Zhang Y, Warner M, Feng Y, Portune K, Bernhardt P & Mulholland M (2007) CO2 control of Trichodesmium N2 fixation, photosynthesis, growth rates, and elemental ratios: implications for past, present, and future ocean biogeochemistry. Limnol Oceanogr 52: 1293. J€ahnichen S, Long BM & Petzoldt T (2011) Microcystin production by Microcystis aeruginosa: direct regulation by multiple environmental factors. Harmful Algae 12: 95–104. Jiang F, Hellman U, Sroga GE, Bergman B & Mannervik B (1995) Cloning, sequencing, and regulation of the glutathione reductase gene from the cyanobacterium Anabaena PCC 7120. J Biol Chem 270: 22882–22889. Jiang F, Wisen S, Widersten M, Bergman B & Mannervik B (2000) Examination of the transcription factor NtcA-binding motif by in vitro selection of DNA sequences from a random library. J Mol Biol 301: 783–793. Jochimsen EM, Carmichael WW, An J et al. (1998) Liver failure and death after exposure to microcystins at a hemodialysis center in Brazil. N Engl J Med 338: 873–878. Jodłowska S & Latała A (2010) Photoacclimation strategies in the toxic cyanobacterium Nodularia spumigena (Nostocales, Cyanobacteria). Phycologia 49: 203–211. Jonasson S, Vintila S, Sivonen K & El-Shehawy R (2008) Expression of the nodularin synthetase genes in the Baltic Sea bloom-former cyanobacterium Nodularia spumigena strain AV1. FEMS Microbiol Ecol 65: 31–39. Kaasalainen U, Fewer DP, Jokela J, Wahlsten M, Sivonen K & Rikkinen J (2012) Cyanobacteria produce a high variety of hepatotoxic peptides in lichen symbiosis. P Natl Acad Sci USA 109: 5886–5891. Kaasalainen U, Fewer DP, Jokela J, Wahlsten M, Sivonen K & Rikkinen J (2013) Lichen species identity and diversity of cyanobacterial toxins in symbiosis. New Phytol 198: 647– 651. Kabir A & El-Shehawy R (2012) Expression of nifH, hetR, and ndaF genes in Nodularia spumigena under nitrogen sources. Plant Biosyst 146: 992–1000. Kaebernick M, Neilan BA, Borner T & Dittmann E (2000) Light and the transcriptional response of the microcystin biosynthesis gene cluster. Appl Environ Microbiol 66: 3387– 3392. Kaebernick M, Dittmann E, B€ orner T & Neilan BA (2002) Multiple alternate transcripts direct the biosynthesis of microcystin, a cyanobacterial nonribosomal peptide. Appl Environ Microbiol 68: 449–455. Kaplan A, Harel M, Kaplan-Levy RN, Hadas O, Sukenik A & Dittmann E (2012) The languages spoken in the water body (or the biological role of cyanobacterial toxins). Front Microbiol 3: article 138. Kardinaal WEA & Visser PM (2005) Dynamics of cyanobacterial toxins: sources of variability in microcystin concentrations. (Huisman J, Matthijs HCP & Visser PM,



eds), Harmful Cyanobacteria, Aquatic Ecology Series 3: pp. 41–63. Springer, the Netherlands. Kardinaal WEA, Tonk L, Janse I, Hol S, Slot P, Huisman J & Visser PM (2007) Competition for light between toxic and nontoxic strains of the harmful cyanobacterium Microcystis. Appl Environ Microbiol 73: 2939–2946. Karlberg M & Wulff A (2013) Impact of temperature and species interaction on filamentous cyanobacteria may be more important than salinity and increased pCO2 levels. Mar Biol 160: 2063–2072. Kleinteich J, Wood SA, K€ upper FC, Camacho A, Quesada A, Frickey T & Dietrich DR (2012) Temperature-related changes in polar cyanobacterial mat diversity and toxin production. Nat Clim Chang 2: 356–360. Kuniyoshi TM, Gonzalez A, Lopez-Gomollon S, Valladares A, Bes MT, Fillat MF & Peleato ML (2011) 2-oxoglutarate enhances NtcA binding activity to promoter regions of the microcystin synthesis gene cluster. FEBS Lett 585: 3921–3926. Kuniyoshi TM, Sevilla E, Bes MT, Fillat MF & Peleato ML (2013) Phosphate deficiency (N/P 40:1) induces mcyD transcription and microcystin synthesis in Microcystis aeruginosa PCC7806. Plant Physiol Biochem 65: 120–124. Kurmayer R (2011) The toxic cyanobacterium Nostoc sp. strain 152 produces highest amounts of microcystin and nostophycin under stress conditions. J Phycol 47: 200–207. Lehtim€aki J, Sivonen K, Luukkainen R & Niemela S (1994) The effects of incubation time, temperature, light, salinity, and phosphorus on growth and hepatotoxin production by Nodularia strains. Archiv f€ ur Hydrobiologie 130: 269–282. Lehtim€aki J, Moisander P, Sivonen K & Kononen K (1997) Growth, nitrogen fixation, and nodularin production by two Baltic Sea cyanobacteria. Appl Environ Microbiol 63: 1647– 1656. Long BM, Jones GJ & Orr PT (2001) Cellular microcystin content in N-limited Microcystis aeruginosa can be predicted from growth rate. Appl Environ Microbiol 67: 278–283. Luque I, Vazquez-Berm udez MF, Paz-Yepes J, Flores E & Herrero A (2004) In vivo activity of the nitrogen control transcription factor NtcA is subjected to metabolic regulation in Synechococcus sp. strain PCC 7942. FEMS Microbiol Lett 236: 47–52. L€ urling M, Eshetu F, Faassen EJ, Kosten S & Huszar VLM (2013) Comparison of cyanobacterial and green algal growth rates at different temperatures. Freshw Biol 58: 552–559. Maberly S (1996) Diel, episodic and seasonal changes in pH and concentrations of inorganic carbon in a productive lake. Freshw Biol 35: 579–598. Marcozzi C, Cumino AC & Salerno GL (2009) Role of NtcA, a cyanobacterial global nitrogen regulator, in the regulation of sucrose metabolism gene expression in Anabaena sp. PCC 7120. Arch Microbiol 191: 255–263. Martin-Luna B, Hernandez JA, Bes MT, Fillat MF & Peleato ML (2006) Identification of a ferric uptake regulator from Microcystis aeruginosa PCC7806. FEMS Microbiol Lett 254: 63–70.



Mazur-Marzec H, Krezel A, Kobos J & Pli nski M (2006) Toxic Nodularia spumigena blooms in the coastal waters of the Gulf of Gda nsk: a ten-year survey. Oceanologia 48: 255–273. Mehnert G, Leunert F, Cires S, J€ ohnk KD, R€ ucker J, Nixdorf B & Wiedner C (2010) Competitiveness of invasive and native cyanobacteria from temperate freshwaters under various light and temperature conditions. J Plankton Res 32: 1009– 1021. Meissner S, Fastner J & Dittmann E (2013) Microcystin production revisited: conjugate formation makes a major contribution. Environ Microbiol 15: 1810–1820. Metcalf JS, Richer R, Cox PA & Codd GA (2012) Cyanotoxins in desert environments may present a risk to human health. Sci Total Environ 421–422: 118–123. M€ oke F, Wasmund N, Bauwe H & Hagemann M (2013) Salt acclimation of Nodularia spumigena CCY9414—a cyanobacterium adapted to brackish water. Aquat Microb Ecol 70: 207–214. Moffitt MC & Neilan BA (2004) Characterization of the nodularin synthetase gene cluster and proposed theory of the evolution of cyanobacterial hepatotoxins. Appl Environ Microbiol 70: 6353–6362. Mollenhauer D, Bengtsson R & Lindstrøm EA (1999) Macroscopic cyanobacteria of the genus Nostoc: a neglected and endangered constituent of European inland aquatic biodiversity. Eur J Phycol 34: 349–360. Muramatsu M & Hihara Y (2012) Acclimation to high-light conditions in cyanobacteria: from gene expression to physiological responses. J Plant Res 125: 11–39. Muro-Pastor AM, Herrero A & Flores E (2001) Nitrogen-regulated group 2 sigma factor from Synechocystis sp. strain PCC 6803 involved in survival under nitrogen stress. J Bacteriol 183: 1090–1095. Neilan BA, Pearson LA, Moffitt MC, Mihali KT, Kaebernick M, Kellmann R & Pomati F (2008) The genetics and genomics of cyanobacterial toxicity. Adv Exp Med Biol 619: 417–452. Neumann T, Eilola K, Gustafsson B, M€ uller-Karulis B, Kuznetsov I, Meier HEM & Savchuk OP (2012) Extremes of temperature, oxygen and blooms in the baltic sea in a changing climate. Ambio 41: 574–585. Ohlendieck U, Gundersen K, Meyerh€ ofer M, Fritsche P, Nachtigall K & Bergmann B (2007) The significance of nitrogen fixation to new production during early summer in the Baltic Sea. Biogeosciences 4: 63–73. Oksanen I, Jokela J, Fewer DP, Wahlsten M, Rikkinen J & Sivonen K (2004) Discovery of rare and highly toxic microcystins from lichen-associated cyanobacterium Nostoc sp. strain IO-102-I. Appl Environ Microbiol 70: 5756–5763. O’Neil JM, Davis TW, Burford MA & Gobler CJ (2012) The rise of harmful cyanobacteria blooms: the potential roles of eutrophication and climate change. Harmful Algae 14: 313– 334. Orr PT & Jones GJ (1998) Relationship between microcystin production and cell division rates in nitrogen-limited


23

Microcystis aeruginosa cultures. Limnol Oceanogr 43: 1604– 1614. Paerl HW & Huisman J (2009) Climate change: a catalyst for global expansion of harmful cyanobacterial blooms. Environ Microbiol Rep 1: 27–37. Paerl HW & Otten TG (2013) Harmful cyanobacterial blooms: causes, consequences, and controls. Microb Ecol 65: 995– 1010. Paerl HW & Paul VJ (2012) Climate change: links to global expansion of harmful cyanobacteria. Water Res 46: 1349– 1363. Paerl HW & Ustach J (1982) Blue green algal scums: an explanation for their occurrence during freshwater blooms. Limnol Oceanogr 27: 212–217. Parry ML (2007) Climate Change 2007: Impacts, Adaptation and Vulnerability: Working Group II Contribution to the Fourth Assessment Report of the IPCC Intergovernmental Panel on Climate Change. Cambridge University Press, Cambridge. Peeters F, Straile D, Lorke A & Livingstone DM (2007) Earlier onset of the spring phytoplankton bloom in lakes of the temperate zone in a warmer climate. Glob Change Biol 13: 1898–1909. Phelan RR & Downing TG (2011) A growth advantage for microcystin production by Microcystis PCC7806 under high light. J Phycol 47: 1241–1246. Qiu B & Gao K (2002) Effects of CO2 enrichment on the bloom-forming cyanobacterium Microcystis aeruginosa (Cyanophyceae): physiological responses and relationships with the acailibility of dissolved inorganic carbon. J Phycol 38: 721–729. Ramasubramanian TS, Wei TF, Oldham AK & Golden JW (1996) Transcription of the Anabaena sp. strain PCC 7120 ntcA gene: multiple transcripts and NtcA binding. J Bacteriol 178: 922–926. Rascher U, Lakatos M, B€ udel B & L€ uttge U (2003) Photosynthetic field capacity of cyanobacteria of a tropical inselberg of the Guiana Highlands. Eur J Phycol 38: 247– 256. Rouhiainen L, Vakkilainen T, Siemer BL, Buikema W, Haselkorn R & Sivonen K (2004) Genes coding for hepatotoxic heptapeptides (microcystins) in the cyanobacterium Anabaena strain 90. Appl Environ Microbiol 70: 686–692. Runnegar MT, Kong S & Berndt N (1993) Protein phosphatase inhibition and in vivo hepatotoxicity of microcystins. Am J Physiol Gastrointest Liver Physiol 265: G224–G230. Saker ML, Neilan BA & Griffiths DJ (1999) Two morphological forms of Cylindrospermopsis raciborskii (Cyanobacteria) isolated from Solomon Dam, Palm Island, Queensland. J Phycol 35: 599–606. Schopf JW & Packer BM (1987) Early archean (3.3-billion to 3.5-billion-year-old) microfossils from Warrawoona Group, Australia. Science 237: 70–73.


24

Severin I & Stal LJ (2010a) Spatial and temporal variability in nitrogenase activity and diazotrophic community composition in coastal microbial mats. Mar Ecol Prog Ser 417: 13–25. Severin I & Stal LJ (2010b) NifH expression by five groups of phototrophs compared with nitrogenase activity in coastal microbial mats. FEMS Microbiol Ecol 73: 55–67. Severin I, Confurius-Guns V & Stal LJ (2012) Effect of salinity on nitrogenase activity and composition of the active diazotrophic community in intertidal microbial mats. Arch Microbiol 194: 483–491. Sevilla E, Martin-Luna B, Vela L, Bes MT, Fillat MF & Peleato ML (2008) Iron availability affects mcyD expression and microcystin-LR synthesis in Microcystis aeruginosa PCC7806. Environ Microbiol 10: 2476–2483. Sevilla E, Martin-Luna B, Vela L, Teresa Bes M, Luisa Peleato M & Fillat MF (2010) Microcystin-LR synthesis as response to nitrogen: transcriptional analysis of the mcyD gene in Microcystis aeruginosa PCC7806. Ecotoxicology 19: 1167– 1173. Sevilla E, Martin-Luna B, Bes MT, Fillat MF & Peleato ML (2012) An active photosynthetic electron transfer chain required for mcyD transcription and microcystin synthesis in Microcystis aeruginosa PCC7806. Ecotoxicology 21: 811– 819. Shih PM, Wu D, Latifi A, Axen SD, Fewer DP, Talla E, Calteau A, Cai F, de Marsac NT & Rippka R (2013) Improving the coverage of the cyanobacterial phylum using diversity-driven genome sequencing. P Natl Acad Sci USA 110: 1053–1058. Siegel H, Gerth M & Tschersich G (2006) Sea surface temperature development of the Baltic Sea in the period 1990–2004. Oceanologia 48(S): 119–131. Sinha R, Pearson LA, Davis TW, Burford MA, Orr PT & Neilan BA (2012) Increased incidence of Cylindrospermopsis raciborskii in temperate zones - Is climate change responsible? Water Res 46: 1408–1419. Sivonen K & Jones G (1999) Cyanobacterial toxins. Toxic Cyanobacteria in Water — A Guide to Their Public Health Consequences and Management, (Chorus I, Bartram J, eds), E & F Spon, London, pp. 41–111. Stolte W, Karlsson C, Carlsson P & Graneli E (2002) Modeling the increase of nodularin content in Baltic Sea Nodularia spumigena during stationary phase in phosphorus-limited batch cultures. FEMS Microbiol Ecol 41: 211–220. Su Z, Olman V, Mao F & Xu Y (2005) Comparative genomics analysis of NtcA regulons in cyanobacteria: regulation of nitrogen assimilation and its coupling to photosynthesis. Nucleic Acids Res 33: 5156–5171. € J, Lehtiniemi M, Suikkanen S, Pulina S, Engstr€ om-Ost Lehtinen S & Brutemark A (2013) Climate change and eutrophication induced shifts in northern summer plankton communities. PLoS One 8: e66475. Sukenik A, Hadas O, Kaplan A & Quesada A (2012) Invasion of Nostocales (cyanobacteria) to subtropical and temperate



freshwater lakes – physiological, regional, and global driving forces. Front Microbiol 3: article 86. Tanaka S, Kerfeld CA, Sawaya MR, Cai F, Heinhorst S, Cannon GC & Yeates TO (2008) Atomic-level models of the bacterial carboxysome shell. Science 319: 1083–1086. Tonk L, Visser PM, Christiansen G, Dittmann E, Snelder EOFM, Wiedner C, Mur LR & Huisman J (2005) The microcystin composition of the cyanobacterium Planktothrix agardhii changes toward a more toxic variant with increasing light intensity. Appl Environ Microbiol 71: 5177– 5181. Tonk L, Bosch K, Visser PM & Huisman J (2007) Salt tolerance of the harmful cyanobacterium Microcystis aeruginosa. Aquat Microb Ecol 46: 117–123. Tonk L, van de Waal DB, Slot P, Huisman J, Matthijs HCP & Visser PM (2008) Amino acid variability determines the ratio of microcystin variants in the cyanobacterium Planktothrix agardhii. FEMS Microbiol Ecol 65: 383–390. Tripati AK, Roberts CD & Eagle RA (2009) Coupling of CO2 and ice sheet stability over major climate transitions of the last 20 million years. Science 326: 1394–1397. Utkilen H & Gjølme N (1992) Toxin production by Microcystis aeruginosa as a function of light in continuous cultures and its ecological significance. Appl Environ Microbiol 58: 1321– 1325. Utkilen H & Gjølme N (1995) Iron-stimulated toxin production in Microcystis aeruginosa. Appl Environ Microbiol 61: 797–800. Van de Waal DB, Verspagen JM, L€ urling M, Van Donk E, Visser PM & Huisman J (2009) The ecological stoichiometry of toxins produced by harmful cyanobacteria: an experimental test of the carbon-nutrient balance hypothesis. Ecol Lett 12: 1326–1335. Van De Waal DB, Verspagen JMH, Finke JF et al. (2011) Reversal in competitive dominance of a toxic versus non-toxic cyanobacterium in response to rising CO2. ISME J 5: 1438–1450. Vezie C, Rapala J, Vaitomaa J, Seitsonen J & Sivonen K (2002) Effect of nitrogen and phosphorus on growth of toxic and nontoxic Microcystis strains and on intracellular microcystin concentrations. Microb Ecol 43: 443–454. Voß B, Bolhuis H, Fewer DP et al. (2013) Insights into the physiology and ecology of the brackish-water-adapted cyanobacterium Nodularia spumigena CCY9414 Based on a Genome-Transcriptome Analysis. PLoS One 8: e60224. Wannicke N, Koch BP & Voss M (2009) Release of fixed N2 and C as dissolved compounds by Trichodesmium erythreum and Nodularia spumigena under the influence of high light and high nutrient (P). Aquat Microb Ecol 57: 175–189. Wannicke N, Endres S, Engel A, Grossart HP, Nausch M, Unger J & Voss M (2012) Response of Nodularia spumigena to pCO2 - Part 1: growth, production and nitrogen cycling. Biogeosciences 9: 2973–2988. Wannicke N, Korth F, Liskow I & Voss M (2013) Incorporation of diazotrophic fixed N2 by mesozooplankton


25


- Case studies in the southern Baltic Sea. J Mar Syst 117– 118: 1–13. Wasmund N. (1997) Occurrence of Cyanobacterial blooms in the Baltic Sea in relation to environmental conditions. Int Revue ges Hydrobiol 82: 169–184. Wiedner C, Visser PM, Fastner J, Metcalf JS, Codd GA & Mur LR (2003) Effects of light on the microcystin content of Microcystis strain PCC 7806. Appl Environ Microbiol 69: 1475–1481. Wilhelm SW, Farnsley SE, LeCleir GR, Layton AC, Satchwell MF, DeBruyn JM, Boyer GL, Zhu G & Paerl HW (2011) The relationships between nutrients, cyanobacterial toxins and the microbial community in Taihu (Lake Tai), China. Harmful Algae 10: 207–215. Wilhelm L, Singer GA, Fasching C, Battin TJ & Besemer K (2013) Microbial biodiversity in glacier-fed streams. ISME J 7: 1651–1660. Wood SA, Kuhajek JM, de Winton M & Phillips NR (2012) Species composition and cyanotoxin production in periphyton mats from three lakes of varying trophic status. FEMS Microbiol Ecol 79: 312–326.


Zhao MX, Jiang YL, He YX, Chen YF, Teng YB, Chen Y, Zhang CC & Zhou CZ (2010) Structural basis for the allosteric control of the global transcription factor NtcA by the nitrogen starvation signal 2-oxoglutarate. P Natl Acad Sci USA 107: 12487–12492. Zilliges Y, Kehr JC, Meissner S, Ishida K, Mikkat S, Hagemann M, Kaplan A, B€ orner T & Dittmann E (2011) The cyanobacterial hepatotoxin microcystin binds to proteins and increases the fitness of Microcystis under oxidative stress conditions. PLoS One 6: e17615.

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 spumigena strains NSOR10 and CCY9414 using MUSCLE (Edgar, 2004).


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