quorum sensing n-acylhomoserine lactone signals affect nitrogen fixation in the cyanobacterium...

R E S E A R C H L E T T E R

Quorum sensingN-acylhomoserine lactone signals a¡ect nitrogen¢xation in the cyanobacteriumAnabaena sp.PCC7120Manuel Romero1, Alicia M. Muro-Pastor2 & Ana Otero1

1Departamento de Microbiologıa y Parasitologıa, Facultad de Biologıa-CIBUS, Universidad de Santiago de Compostela, Santiago de Compostela, Spain;

and 2Instituto de Bioquımica Vegetal y Fotosıntesis, Consejo Superior de Investigaciones Cientıficas and Universidad de Sevilla, Seville, Spain

Correspondence: Ana Otero, Departamento

de Microbiologıa y Parasitologıa, Facultad de

Biologıa-CIBUS, Universidad de Santiago de

Compostela, 15782 Santiago de Compostela,

Spain. Tel.: 134 981 563 100, ext. 16913;

fax: 134 981 528 006; e-mail:

[email protected]

Received 4 October 2010; revised 23 November

2010; accepted 24 November 2010.

Final version published online January 2011.

DOI:10.1111/j.1574-6968.2010.02175.x

Editor: Karl Forchhammer

Keywords

cyanobacteria; N-acylhomoserine lactones;

tetramic acid; nitrogen fixation.

Abstract

Bacteria secrete small signal molecules into the environment that induce self and

neighbour gene expression. This phenomenon, termed quorum sensing, allows

cooperative behaviours that increase the fitness of the group. The best-studied

signal molecules are the N-acylhomoserine lactones (AHLs), secreted by a growing

number of bacterial species including important pathogen species such as

Pseudomonas aeruginosa. These molecules have recently been proposed to have

properties other than those of signalling, functioning as iron quelants or

antibiotics. As the presence of an acylase capable of inactivating long-chain AHLs

in Anabaena sp. PCC7120 could constitute a defence mechanism against these

molecules, in this work we analyse the effects of different AHLs varying in length

and substitutions on the growth and nitrogen metabolism of the cyanobacterium

Anabaena sp. PCC7120. All the AHLs tested strongly inhibited nitrogen fixation.

The inhibition seems to take place at post-transcriptional level, as no effect on

heterocyst differentiation or on the expression of nitrogenase was observed.

Moreover, N-(3-oxodecanoyl)-L-homoserine lactone (OC10-HSL) showed a spe-

cific cytotoxic effect on this cyanobacterium in the presence of a combined

nitrogen source, but the mechanism involved seems to be different from that

described so far for tetramic acid derivatives of oxo-substituted AHLs. These

results suggest a variety of new unexpected activities for AHLs, at least on

cyanobacterial populations.

Introduction

The term ‘quorum sensing’ (QS) (Fuqua et al., 1994)

describes a phenomenon of bacterial communication that

confers on these organisms the ability to perceive and

respond to the community density through coordinated

regulation of gene expression, thus being able to adopt an

advantageous social behaviour. Bacteria communicate their

presence to others by secreting small chemical signals called

autoinducers, allowing the individuals to distinguish be-

tween high and low population densities.

By means of QS, bacterial populations can coordinate

important biological functions including motility, swarm-

ing, aggregation, plasmid conjugal transfer, luminescence,

antibiotic biosynthesis, virulence, symbiosis and biofilm

maintenance and differentiation (Williams et al., 2007).

Several chemically distinct families of QS signal molecules

have now been described, but the most studied QS signalling

system involves N-acylhomoserine lactones (AHLs) em-

ployed by diverse Gram-negative bacteria. AHLs differ in

the acyl side chain, which is usually 4–18 carbons in length,

with or without saturation or C3 hydroxy- or oxo-substitu-

tions (Whitehead et al., 2001). AHLs have been initially

described as being exclusively produced by a relatively small

number of Alpha-, Beta- and Gammaproteobacteria (Wil-

liams et al., 2007), but recently the production of these

signals has also been reported for the colonial cyanobacter-

ium Gloeothece (Sharif et al., 2008) and different marine

Bacteroidetes (Huang et al., 2008; Romero et al., 2010),

which might indicate a significant role for QS systems in

natural populations/environment.

Besides acting as quorum signals, some AHLs have been

proposed to have other possible biological functions, for

example acting as iron quelants and antibiotics (Kaufmann

FEMS Microbiol Lett 315 (2011) 101–108 c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

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et al., 2005; Schertzer et al., 2009). A naturally occurring

degradation product of N-(3-oxododecanoyl)-L-homoserine

lactone (OC12-HSL), one of the AHL signals produced by

Pseudomonas aeruginosa, is the tetramic acid 3-(1-hydroxy-

decylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione, which

exhibits iron-binding ability. This AHL derivative is able

to bind iron in a 3 : 1 complex with an affinity comparable

to that exhibited by standard quelators and siderophores

(Schertzer et al., 2009). In addition, antibiotic properties of

the tetramic acid derivative of OC12-HSL have been de-

scribed, through the disruption of membrane potential and

proton gradient of bacteria, thus eliminating the proton-

motive force and leading to bacterial death (Lowery et al.,

2009).

The existence of QS blockage systems adopted by compe-

titors to destroy or inhibit the functions of AHLs also

indicates the ecological importance of these molecules. The

different mechanisms of interference with QS communica-

tion systems have been generally termed ‘quorum quench-

ing’ (QQ) (Dong et al., 2001). An example of QQ is the

enzymatic inactivation of AHLs, with two groups of AHL-

degrading enzymes identified so far. The lactonases hydro-

lyse the homoserine lactone (HSL) ring of the AHL molecule

to produce acyl homoserines (Dong et al., 2007), whereas

the acylases cleave the AHL amide bond, generating the

corresponding fatty acid and HSL ring (Dong et al., 2007).

Enzymatic QQ activity has been described in Gram-positive

and -negative bacteria and more recently in the cyanobac-

terium Anabaena sp. PCC7120 (Romero et al., 2008).

Anabaena sp. PCC7120 is a filamentous cyanobacterium

simultaneously able to perform photosynthesis and dinitro-

gen fixation under aerobic conditions. In the presence of a

source of combined nitrogen, filaments grow as undiffer-

entiated chains of vegetative cells. In contrast, when Ana-

baena sp. PCC7120 is deprived of combined nitrogen,

approximately 10% of the cells differentiate into morpholo-

gically distinct heterocysts that supply the rest of the

filament with fixed nitrogen and in return receive carbohy-

drate from vegetative cells (Wolk et al., 1994). In the absence

of combined nitrogen the heterocysts are spaced along the

filament in a semi-regular pattern that is controlled by a

regulatory loop established between two master regulators,

NtcA and HetR (Muro-Pastor et al., 2002).

Because AHLs have been described in natural environ-

ments where cyanobacteria are prevalent, such as microbial

mats and algal blooms (McLean et al., 1997; Bachofen &

Schenk, 1998), the acylase-type QQ activity found in

Anabaena sp. PCC7120 (Romero et al., 2008) could serve

either to mitigate possible negative effects of AHLs them-

selves and/or their tetramic acid derivatives (Kaufmann

et al., 2005; Schertzer et al., 2009) or to confer a competitive

advantage against AHL-producing competitors through the

disruption of their communication system.

In this work, we study the effects of exogenous AHL

addition to cultures of the filamentous heterocyst-forming

cyanobacterium Anabaena sp. PCC7120 to assess the possi-

ble physiological role of the AHL-acylase present in this

cyanobacterium.

Materials and methods

Growth conditions

Stock cultures of Anabaena sp. PCC7120 were maintained

photoautotrophically at 30 1C with a continuous irradiance

of 75 mE m�2 s�1. Cultures were aerated by connecting each

culture unit to an aeration system with a continuous filtered

(0.45 mm) air flow or carbon dioxide (CO2)-enriched air

(1% v/v).

Diazotrophic cultures were carried out in BG110C med-

ium [BG11 medium (Rippka et al., 1979) without NaNO3

and supplemented with 0.84 g L�1 of NaHCO3 (C)].

Nondiazotrophic cultures of Anabaena sp. PCC7120 were

established in BG110C supplemented with either 17 mM

NaNO3 (BG11C) or 6 mM NH4Cl and 12 mM of N-Tris(hy-

droxymethyl)methyl-2-aminoethanesulphonic acid-NaOH

buffer pH 7.5 (BG110C1NH41). To study the effect of AHL

addition on the process of heterocyst differentiation, the

biomass of nondiazotrophic cultures was collected by filtra-

tion (0.45 mm), washed and resuspended in fresh BG110C

(nitrogen step-down procedure).

Solid media plates were prepared mixing equal volumes

of double-concentrated sterilized BG110 or BG1101NH41

and agar 10 g L�1. Plates inoculated with Anabaena sp.

PCC7120 were incubated at 30 1C with light.

Addition of synthetic AHLs to cultures

AHLs were first assayed in solid media to check a possible

antibiotic effect (Lowery et al., 2009). Cells from a liquid

exponentially growing culture of Anabaena sp. PCC7120 in

BG110C1NH41 were harvested by filtration, washed and

resuspended in BG110C at a concentration of 5 mg chloro-

phyll a (Chl a) mL�1 and 100mL of the suspension was

spread on top of BG1101NH41 or BG110 plates. Small holes

were made in the centre of each plate and filled with 100mL

of 100 mM AHL or acetonitrile (as control). Growth was

checked after 7 days of incubation at 30 1C with light.

Synthetic AHLs were also added to liquid cultures of

Anabaena sp. PCC7120 both under nondiazotrophic condi-

tions (BG110C1NH41 medium) and during nitrogen step-

down. Anabaena sp. PCC7120 was grown to exponential

phase in BG110C1NH41 [cultures with about 5 mg Chl

a mL�1; Chl a levels were determined in methanolic extracts

(Mackinney, 1941)]. The cells were filtered, washed with

BG110C, inoculated in fresh BG110C1NH411AHL

(100 mM) or BG110C1AHL (100mM) and bubbled with air

FEMS Microbiol Lett 315 (2011) 101–108c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

102 M. Romero et al.

or CO2-enriched air with a final Chl a concentration of

4mg mL�1. The AHLs used were: N-butyryl-homoserine

lactone (C4-HSL), N-(3-oxobutyryl)-L-homoserine (OC4-

HSL), N-(3-hydroxybutyryl)-L-homoserine (OHC4-HSL),

N-decanoyl-L-homoserine (C10-HSL) N-(3-oxodecanoyl)-L-

homoserine lactone (OC10-HSL), N-(3-hydroxydecanoyl)-

L-homoserine (OHC10-HSL), N-dodecanoyl-L-homoserine

(C12-HSL) OC12-HSL and N-(3-hydroxydodecanoyl)-

L-homoserine (OHC12-HSL) (unsubstituted AHLs were pur-

chased from Sigma-Aldrich, all other AHLs were kindly

provided by Prof. Miguel Camara from the University of

Nottingham). AHL stock solutions of 1 mg mL�1 were pre-

pared in acetonitrile. Parallel control assays were carried out

using equal amounts of acetonitrile (AHL solvent). In nitrogen

step-down cultures, the differentiation of heterocysts was

monitored by Alcian blue staining of polysaccharides in the

heterocyst envelope (Olmedo-Verd et al., 2006).

To further evaluate the lethal effect observed for OC10-

HSL in ammonium-grown nondiazotrophic cultures of

Anabaena sp. PCC7120 (BG110C1NH41), different concen-

trations of this signal (0.01, 0.1, 1, 10, 25, 50, 75 and

100 mM) as well as OC12-tetramic acid (100mM) were also

assayed. The effect of OC10-HSL (100mM) was also tested in

cultures with nitrate as combined nitrogen source (BG11C).

OD600 nm of the cultures was measured at different time

points after treatment (Kuznetsova et al., 2008).

Nitrogenase activity measurement

Biomass (200 mL, 2–3mg mL�1 Chl a) from BG110C1NH41

aerated cultures of Anabaena sp. PCC7120 was harvested,

washed and resuspended in fresh BG110C at a Chl a

concentration of 2 mg mL�1 to induce the differentiation of

heterocysts. Cultures of 20 mL were established in flasks

supplemented with AHLs (100 mM) or acetonitrile as con-

trol. After 20 h of incubation at 30 1C, 120 r.p.m. and light,

the nitrogenase activity was measured as follows: cells were

concentrated to 4 mL by removing part of the supernatant

after centrifugation, and they were then divided in two 17-

mL flasks sealed with silicon caps (2 mL each, 10 mg Chl a).

For each AHL, one flask was incubated under standard

aerobic conditions. Another flask was incubated with an

anaerobic atmosphere by injecting argon for 3 min and

adding 10 mM 3-(3,4dichlorophenyl)-1,1-dimethylurea

(DCMU) to inhibit photosynthesis and therefore oxygen

(O2) production (Rippka & Stanier, 1978) to avoid a

possible inhibition of nitrogenase activity derived from the

formation of abnormal heterocyst cell walls during matura-

tion or the damage from other mechanisms responsible for

maintaining low O2 concentration within the heterocysts.

After 1-h incubation at 30 1C, 2 mL of acetylene was

injected. Samples of 1 mL from the air in the sealed flask

were taken at different times during 20 h starting 15 min

after acetylene injection to determine the concentration of

the ethylene produced using a GC-MS (HP 5890 series II)

equipped with injector, column (Porapak Q) and flame

ionization detector (kept at 100, 80 and 150 1C, respec-

tively). The detected signals were processed with the com-

puting integrator PYE Unicam DP88. The equipment was

calibrated with known concentrations of ethylene.

To determine the nitrogenase activity of the cultures per

unit Chl a, the following formula was used: nitrogenase

activity = nmol ethylene in sample� 14 mL/2� mg Chl a

mL�1; where 14 was the atmosphere volume in 17-mL flasks

and 2 the volume of culture in the flask.

C10-HSL was also added to BG110C cultures of Anabaena

sp. PCC7120 with mature heterocysts (24 h after nitrogen

step-down) and the nitrogenase activity then measured as

described before.

RNA isolation and analysis

To assess a possible effect of AHLs on the expression of genes

involved in nitrogen fixation, Northern hybridization was

carried out with probes for the nifH and fdxH genes.

Samples of 50 mL were taken at 0, 3, 6, 20 and 24 h after

nitrogen step-down. Cells were filtered, washed and resus-

pended in 1 mL of Tris 50 mM/EDTA 100 mM, centrifuged

and the pellet was frozen in liquid nitrogen before RNA

extraction. RNA from whole filaments was extracted in the

presence of ribonucleoside–vanadyl complex as described

previously (Muro-Pastor et al., 2002).

For Northern analysis, 30mg of RNA was loaded per lane

and electrophoresed in 1% agarose denaturing formaldehyde

gels. Transfer and fixation to Hybond-N1 membranes (Amer-

sham Biosciences) were carried out using 0.1 M NaOH.

Hybridization was performed at 65 1C according to the

recommendations of the manufacturer of the membranes.

The nifH and fdxH probes were fragments of these genes

amplified by PCR. The nifH probe was amplified using

plasmid pCSAV60 (containing the nifH gene cloned in

pGEM-T vector) as a template and oligonucleotides NH-1

(corresponding to positions � 334 to � 314 with respect to

the translation start of nifH) and NH-4 (complementary to

nucleotides 1884 to 1863 with respect to the translation start

of nifH) (Valladares et al., 2007). The fdxH probe was

amplified using plasmid pCSAV164 (containing the fdxH gene

cloned in pGEM-T vector) as a template and oligonucleotides

FH-1 (corresponding to nucleotides13 to 120 with respect to

the translation start of fdxH) and FH-2 (complementary to

nucleotides 1297 to 1269 with respect to the translation start

of fdxH) (Valladares et al., 2007). rnpB, encoding the RNA

subunit of RNase P (Vioque, 1997), was used as a loading and

transfer control. All probes were 32P-labeled with a Ready-to-

Go DNA labeling kit (Amersham Biosciences) using

[a-32P]dCTP. Images of radioactive filters and gels were


103AHLs inhibit nitrogen fixation in cyanobacteria

obtained and quantified with a Cyclone storage phosphor

system and OPTIQUANT image analysis software (Packard).

Results and discussion

Effect of synthetic AHLs addition

AHLs were added to Anabaena sp. PCC7120 cultures to

evaluate possible effects on growth and nitrogen metabolism

of the cyanobacterial filaments both in solid and liquid

media. We selected saturated and substituted representatives

of short- (C4, OC4 and OHC4-HSL), middle- (C10, OC10

and OHC10-HSL) and long-chain AHLs (C12, OC12 and

OHC12-HSL). A first experiment was carried out in solid

media, as described in Materials and methods. Growth

inhibition halos surrounding the wells were observed after

7 days for OC10-HSL and OC12-HSL in cultures subjected

to nitrogen step-down (transferred to nitrogen-free BG110

medium) (Fig. 1). OC10-HSL also inhibited growth in the

presence of combined nitrogen (BG1101NH41, data not

shown). These observations suggested that at least these

two AHLs could have an effect on heterocyst differentiation

or maturation, which was further investigated.

AHLs were also added to liquid cultures under nondiazo-

trophic conditions (BG110C1NH41) and to cultures sub-

jected to nitrogen step-down to study the effect on growth

and heterocyst differentiation. None of the tested AHLs

showed cytotoxic effects in liquid cultures subjected to step-

down after 20 h of exposure. Moreover, no effect on hetero-

cyst differentiation and distribution pattern was found in

step-down cultures for any of the tested AHLs after Alcian

blue staining and microscope observation (data not shown).

The discrepancy between the inhibitory effects obtained for

OC10 and OC12-HSL in solid plates (Fig. 1) and in liquid

cultures could be derived from the longest period of

incubation of solid plates or could also be due to the higher

initial cell concentration in the liquid cultures compared

with plates resulting in a higher AHL-acylase activity

(Romero et al., 2008) that would diminish the effect of

initial AHL concentration.

Possible effects of AHLs on heterocyst differentiation

were also tested with Anabaena sp. PCC7120 strain CSEL4a

(Olmedo-Verd et al., 2006). This strain expresses gfp gene

under the control of ntcA promoter, the master regulator of

nitrogen assimilation, which also controls the early phases of

heterocyst differentiation (Herrero et al., 2004). Expression

of gfp in this strain is induced in specific cells upon nitrogen

step-down, indicating the induction of ntcA during hetero-

cyst differentiation (Olmedo-Verd et al., 2006). To test for

possible effects of AHLs, cells of strain CSEL4a grown in the

presence of BG110C1NH41 were transferred to BG110C in

the presence of AHLs (100mM). Induction of the expression

of gfp from the ntcA promoter proceeded in the same way

both in the presence and in the absence of AHLs, indicating

that the AHLs were not affecting the process of heterocyst

differentiation (data not shown).

In contrast, and consistent with the results obtained in

solid plates, a strong cytotoxic effect was observed after only

5 h for OC10-HSL (100mM) in BG110C1NH41 in liquid

media (Fig. 2a). The same effect could also be observed in

cultures with nitrate as nitrogen source (BG11C) supple-

mented with OC10-HSL at the same concentration (data

not shown). This effect could not be observed for any of the

other AHLs tested. To determine the OC10-HSL minimal

lethal concentration, the assay was repeated using: 0.01, 0.1,

1, 10, 25, 50, 75 and 100mM of OC10-HSL in BG110C1

NH41 cultures. Concentrations 4 25mM were lethal (Fig. 2a

and b) and the filaments appeared completely lysed under

the microscope after 5 h of culture. Cells incubated in the

presence of 25 mM of OC10-HSL showed black dots, resem-

bling cyanophycin granules, in the inner side of the cell walls

(data not shown). No lethal effect on Anabaena sp. PCC7120

was observed in cultures supplemented with 100mM OC12-

HSL or OC12-tetramic acid (data not shown). The half

maximal effective concentration (EC50) observed for other

bacteria is between 8 and 55mM for the OC12-HSL-derived

tetramic acid and between 22.1 and 100 mM for OC12-HSL

itself, depending on the bacterial strain (Kaufmann et al.,

2005). These ranges match the lethal concentration observed

for OC10-HSL in BG110C1NH41 cultures of Anabaena sp.

PCC7120, but it should be noted that this activity was

described only for Gram-positive bacteria, as the outer

Gram-negative membrane seems represent a permeability

barrier for tetramic acids (Lowery et al., 2009). Nevertheless,

the antibiotic effect observed for OC10-HSL under non-

diazotrophic conditions seems to be highly specific and

different from the antibiotic effect described so far for

tetramic acids, as no cytotoxic effect of OC12-HSL or its

Control OC10-HSL OC12-HSL

Fig. 1. Anabaena sp. PCC7120 growth

inhibition halos surrounding wells filled with

100 mL of OC10-HSL and OC12-HSL (100 mM) in

comparison with normal growth (control with

acetonitrile) in BG110 plates. Plates were

incubated for 7 days with continuous light

at 30 1C.



tetramic acid derivative could be observed. It has been

reported that a degradation product of oxo-substituted

AHLs such as OC12-HSL is a tetramic acid with a high

affinity for iron, comparable to standard quelants and side-

rophores (Kaufmann et al., 2005; Schertzer et al., 2009),

therefore the cytotoxic effect of OC10-HSL could be related

to iron quelant properties, but this could not explain the

dramatic lethal effect observed, with total lysis of the

filaments already after 5 h of the addition of OC10-HSL to

nondiazotrophic cultures. Moreover, it is highly improbable

that OC10-HSL is acting through the disruption of mem-

brane potential, as already described for OC12-HSL or its

tetramic acid derivative (Lowery et al., 2009), because no

effect was recorded for these two compounds, which are

expected to be more active than OC10-HSL in this respect

(Schertzer et al., 2009). Therefore, the observation that

OC10-HSL is lethal only in the presence of combined

nitrogen in liquid media could be the result of a specific

inhibitory effect of this molecule on the metabolism of

combined nitrogen. Alternatively, OC10-HSL signal might

lead to the activation of the wrong pathways. For instance,

overactivation of arginine biosynthesis in the presence of

combined nitrogen could lead to cyanophycin accumulation

(dense, presumptive cyanophycin granules are observed in

the damaged filaments), blocking the entire nitrogen meta-

bolism and resulting in cell death.

Nitrogenase activity

Although no macroscopic effect of AHLs on survival and

heterocyst differentiation was recorded in diazotrophic

cultures in short-time experiments, the effect of the signals

on the nitrogenase activity was evaluated in BG110C1NH41

cultures transferred to BG110C for the induction of hetero-

cyst formation and nitrogen fixation in the presence of the

AHLs. Nitrogenase measurements were carried out 20 h

after the nitrogen step-down treatment to allow formation

of mature heterocysts. A strong inhibition of the nitrogenase

0

20

40

60

80

100

C C4 OC4 OHC4 C10 OC10 OHC10 C12 OC12 OHC12

Nit

rog

enas

e ac

tivi

ty (

%)

Fig. 3. Anabaena sp. PCC7120 nitrogenase

activities under aerobic (black bars) and

anaerobic (grey bars) conditions in BG110C

supplemented with the AHLs: C4, OC4, OHC4,

C10, OC10, OHC10, C12, OC12 and

OHC12-HSL (100 mM). Control culture was set

with acetonitrile (C). Nitrogenase activities are

expressed as percentages of the value for

control culture, which corresponds to 2.04

(aerobic) and 6.5 (anaerobic) nmol ethylene per

mg Chl�1h�1.

0

0.5

1

1.5

2

2.5

0 5 10 15 20 25Time (h)

OD

600

nm

(a)

(b)

C 100 µM 75 µM 50 µM 25 µM

Fig. 2. (a) Antibiotic effect of different concentrations of OC10-HSL

(25–100 mM) on Anabaena sp. PCC7120 cultures in BG110C1NH41. The

photo was taken 7 h after AHL addition. C, control culture containing

acetonitrile. (b) Evolution of OD600 nm of Anabaena sp. PCC7120 cultures

in BG110C1NH41 with different concentrations of OC10-HSL (&, 25 mM;

m, 50mM; �, 75 mM; and ^, 100 mM) and acetonitrile (B) as control.

Time 0, addition of OC10-HSL.



activity was recorded for all AHLs tested (Fig. 3). The lower

ethylene production in AHL-treated cultures was already

evident 5 min after acetylene addition. The inhibition was

specially marked in cultures treated with OC10 and OC12-

HSL, in which none or residual nitrogenase activity could be

detected (Fig. 3). This result is consistent with the inhibition

of growth observed in the cyanobacterium, with these two

AHLs in solid BG110 media (Fig. 1).

To evaluate whether the inhibition of nitrogenase activity

was due to defects in heterocyst wall formation or defects in

any of the other mechanisms driving the creation of a

microoxic environment inside the heterocysts, nitrogenase

activity was also measured under anaerobic atmosphere (Fig.

3). Air inside the flasks was substituted by argon and DCMU

was added to the cultures to inhibit PSII-dependent O2

production. As expected, slightly higher nitrogenase activity

was observed in anaerobic conditions than in aerobic ones

(Valladares et al., 2007), but the effect of AHL addition was

still observed (Fig. 3). This indicates that the lower nitrogen-

ase activity observed in the presence of AHLs was not due to

alterations in the microoxic environment of the heterocysts

and confirms that they have no effect on heterocyst differ-

entiation as observed in AHL-supplemented cultures de-

scribed before. As observed under aerobic conditions, the

OC10 and OC12-HSL signals had the strongest inhibitory

effect on nitrogenase activity (Fig. 3). Twenty hours after the

addition of acetylene still no recovery of normal levels of

nitrogenase activity of the cultures was observed either in

aerobic or anaerobic conditions (data not shown).

To determine whether the inhibitory effect of the AHLs

on nitrogen fixation took place only in developing hetero-

cysts, nitrogenase activity was also measured in diazotrophic

cultures in which C10-HSL (100mM) was added 24 h after

nitrogen step-down, when mature heterocysts are already

present. The amount of ethylene produced in early samples

was similar in cultures with or without C10-HSL but,

interestingly, a progressively decreased ethylene production

was observed in the C10-HSL-treated culture, resulting in a

30% decrease of nitrogenase activity (data not shown). The

progressive increase of the inhibitory effect of AHLs in

acclimated cultures could perhaps be caused by the entry of

the AHLs in the new generations of heterocysts, as the

impermeability of the wall of mature heterocysts could

prevent the penetration of the AHLs. Nonetheless it cannot

be excluded that although AHLs could enter through

vegetative walls and spread along the filaments by the

periplasmic space (Flores et al., 2006; Mariscal et al., 2007),

entering in both mature and forming heterocysts, these

molecules could only act at the molecular level in newly

formed heterocysts. In that case the results observed would

suggest a nonreversible inhibition of nitrogenase in very

early stages at the level of either gene expression or its

enzymatic activity.

Effect on the expression of nitrogen fixation-related genes

Because all tested AHLs showed inhibitory activity on

nitrogen fixation mostly in newly formed heterocysts, to

study possible effects at the level of expression of nitrogen

metabolism genes, Northern blots were carried out to detect

changes in expression of the dinitrogenase reductase subunit

gene (nifH) and fdxH, encoding a heterocyst-specific ferro-

doxin that is a likely electron donor to dinitrogenase

reductase (Razquin et al., 1995).

No significant differences in the expression of either gene

could be detected at 20 and 24 h after nitrogen step-down

(no expression of nifH and fdxH was detected at 0, 3 or 6 h)

in total RNA extracted from C10-HSL-treated cultures when

compared with control samples (Fig. 4). This indicates that

the process of heterocyst differentiation proceeds normally

in the presence of AHLs and therefore AHL inhibition could

be affecting either the expression of other genes related to

nitrogen fixation or be acting on nitrogenase-related genes

at a post-transcriptional level.

rnpB

fdxH

nifH

hesAB-fdxH

nifHD

nifHDK

Control C10-HSL treated0 3 6 20 24 3 6 20 24 h

Fig. 4. Effect of C10-HSL addition on heterocyst differentiation upon

nitrogen step-down. RNA (30 mg) was isolated from samples taken at 0

(NH41), 3, 6, 20 and 24 h in the presence or absence of C10-HSL. Cultures

contained 100mM C10-HSL or acetonitrile as control. Hybridizations

were carried out with a probe for the nifH or fdxH gene or for the rnpB

gene (Vioque, 1997), which was used as a loading and transfer control.



Finally, the strong inhibition of nitrogenase demonstrated

for all the AHLs tested and the cytotoxic effect of OC10-HSL

in the presence of combined nitrogen represent novel

biological activities of these signal molecules. The observa-

tion that antibiotics cannot easily reach the lethal concen-

trations in natural environments has led to a questioning of

whether these molecules could act, in subinhibitory con-

centrations, as signal molecules (Davies, 2006; Linares et al.,

2006). Low concentrations of several antibiotics can alter

expression patterns of bacteria without any effect on growth

rate (Davies et al., 2006), which resembles the mode of

action of QS signals. Thus one possibility is that the AHL

signals have inhibitory effects when added at higher con-

centrations than those found in natural environments. In

fact, the concentrations reported in the literature for AHLs

in the culture media of the model microorganism Vibrio

fischeri usually range between 0.4 and 400 nM (Kaplan &

Greenberg, 1985; Schaefer et al., 2002; Burton et al., 2005),

significantly lower than the concentrations exhibiting in-

hibitory activity against Anabaena sp. PCC7120.

In conclusion, AHLs strongly inhibit nitrogen fixation in

Anabaena sp. PCC7120, although they do not affect the

process of heterocyst differentiation because no changes

were observed in the frequency, pattern of differentiation,

permeability of the heterocyst cell wall or expression of

regulatory genes whose products are involved in differentia-

tion (ntcA). The strong inhibition of nitrogenase activity

observed could be related to nitrogen fixation blockage at a

post-transcriptional level, mainly on newly formed hetero-

cysts. Moreover, a possible new activity of AHL signals was

found for OC10-HSL in the presence of combined nitrogen,

differing from those activities described for oxo-substituted

and AHL tetramic acid derivatives. The presence of acylase

activity against long-chain AHLs described in the biomass of

Anabaena sp. PCC7120 (Romero et al., 2008) could be

related to the negative effects of AHLs in this cyanobacter-

ium. This AHL-degradation mechanism would protect the

filaments, at normal environmental concentrations, from

exogenous signals with potential cytotoxic and inhibitory

activities on the cyanobacterium.

Acknowledgements

This work was financed by a grant from Consellerıa de

Innovacion e Industria, Xunta de Galicia PGIDIT06P-

XIB200045PR. M.R. was supported by an FPU fellowship

from the Spanish Ministry of Education and Science and a

predoctoral fellowship from Diputacion de A Coruna. We

would like to thank Prof. Kim D. Janda and Dr Gunnar F.

Kaufmann for kindly providing us with OC12-tetramic acid.

We also would like to thank Prof. Miguel Camara for

providing us with synthetic AHLs.

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