1. RESEARCH LETTER
Quorum sensing N-acylhomoserine lactone signals a¡ect nitrogen
¢xation in the cyanobacterium Anabaena sp. PCC7120
Manuel Romero1, Alicia M. Muro-Pastor2 & Ana Otero1
1
Departamento de Microbiolog´a y Parasitolog´a, Facultad de Biolog´a-CIBUS, Universidad de Santiago de Compostela, Santiago de Compostela, Spain;
ı ı ı
and 2Instituto de Bioqu´mica Vegetal y Fotos´ntesis, Consejo Superior de Investigaciones Cient´ficas and Universidad de Sevilla, Seville, Spain
ı ı ı
Correspondence: Ana Otero, Departamento Abstract
de Microbiolog´a y Parasitolog´a, Facultad de
ı ı
Biolog´a-CIBUS, Universidad de Santiago de
ı
Bacteria secrete small signal molecules into the environment that induce self and
Compostela, 15782 Santiago de Compostela, neighbour gene expression. This phenomenon, termed quorum sensing, allows
Spain. Tel.: 134 981 563 100, ext. 16913; cooperative behaviours that increase the fitness of the group. The best-studied
fax: 134 981 528 006; e-mail: signal molecules are the N-acylhomoserine lactones (AHLs), secreted by a growing
anamaria.otero@usc.es number of bacterial species including important pathogen species such as
Pseudomonas aeruginosa. These molecules have recently been proposed to have
Received 4 October 2010; revised 23 November properties other than those of signalling, functioning as iron quelants or
2010; accepted 24 November 2010.
antibiotics. As the presence of an acylase capable of inactivating long-chain AHLs
Final version published online January 2011.
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
DOI:10.1111/j.1574-6968.2010.02175.x and substitutions on the growth and nitrogen metabolism of the cyanobacterium
Anabaena sp. PCC7120. All the AHLs tested strongly inhibited nitrogen fixation.
Editor: Karl Forchhammer The inhibition seems to take place at post-transcriptional level, as no effect on
heterocyst differentiation or on the expression of nitrogenase was observed.
Keywords Moreover, N-(3-oxodecanoyl)-L-homoserine lactone (OC10-HSL) showed a spe-
cyanobacteria; N-acylhomoserine lactones; cific cytotoxic effect on this cyanobacterium in the presence of a combined
tetramic acid; nitrogen fixation.
MICROBIOLOGY LETTERS
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.
have now been described, but the most studied QS signalling
Introduction system involves N-acylhomoserine lactones (AHLs) em-
The term ‘quorum sensing’ (QS) (Fuqua et al., 1994) ployed by diverse Gram-negative bacteria. AHLs differ in
describes a phenomenon of bacterial communication that the acyl side chain, which is usually 4–18 carbons in length,
confers on these organisms the ability to perceive and with or without saturation or C3 hydroxy- or oxo-substitu-
respond to the community density through coordinated tions (Whitehead et al., 2001). AHLs have been initially
regulation of gene expression, thus being able to adopt an described as being exclusively produced by a relatively small
advantageous social behaviour. Bacteria communicate their number of Alpha-, Beta- and Gammaproteobacteria (Wil-
presence to others by secreting small chemical signals called liams et al., 2007), but recently the production of these
autoinducers, allowing the individuals to distinguish be- signals has also been reported for the colonial cyanobacter-
tween high and low population densities. ium Gloeothece (Sharif et al., 2008) and different marine
By means of QS, bacterial populations can coordinate Bacteroidetes (Huang et al., 2008; Romero et al., 2010),
important biological functions including motility, swarm- which might indicate a significant role for QS systems in
ing, aggregation, plasmid conjugal transfer, luminescence, natural populations/environment.
antibiotic biosynthesis, virulence, symbiosis and biofilm Besides acting as quorum signals, some AHLs have been
maintenance and differentiation (Williams et al., 2007). proposed to have other possible biological functions, for
Several chemically distinct families of QS signal molecules example acting as iron quelants and antibiotics (Kaufmann
FEMS Microbiol Lett 315 (2011) 101–108
c 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved

2. 102 M. Romero et al.
et al., 2005; Schertzer et al., 2009). A naturally occurring In this work, we study the effects of exogenous AHL
degradation product of N-(3-oxododecanoyl)-L-homoserine addition to cultures of the filamentous heterocyst-forming
lactone (OC12-HSL), one of the AHL signals produced by cyanobacterium Anabaena sp. PCC7120 to assess the possi-
Pseudomonas aeruginosa, is the tetramic acid 3-(1-hydroxy- ble physiological role of the AHL-acylase present in this
decylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione, which cyanobacterium.
exhibits iron-binding ability. This AHL derivative is able
to bind iron in a 3 : 1 complex with an affinity comparable Materials and methods
to that exhibited by standard quelators and siderophores
(Schertzer et al., 2009). In addition, antibiotic properties of Growth conditions
the tetramic acid derivative of OC12-HSL have been de-
Stock cultures of Anabaena sp. PCC7120 were maintained
scribed, through the disruption of membrane potential and
photoautotrophically at 30 1C with a continuous irradiance
proton gradient of bacteria, thus eliminating the proton-
of 75 mE mÀ2 sÀ1. Cultures were aerated by connecting each
motive force and leading to bacterial death (Lowery et al.,
culture unit to an aeration system with a continuous filtered
2009).
(0.45 mm) air flow or carbon dioxide (CO2)-enriched air
The existence of QS blockage systems adopted by compe-
(1% v/v).
titors to destroy or inhibit the functions of AHLs also
Diazotrophic cultures were carried out in BG110C med-
indicates the ecological importance of these molecules. The
ium [BG11 medium (Rippka et al., 1979) without NaNO3
different mechanisms of interference with QS communica-
and supplemented with 0.84 g LÀ1 of NaHCO3 (C)].
tion systems have been generally termed ‘quorum quench-
Nondiazotrophic cultures of Anabaena sp. PCC7120 were
ing’ (QQ) (Dong et al., 2001). An example of QQ is the
established in BG110C supplemented with either 17 mM
enzymatic inactivation of AHLs, with two groups of AHL-
NaNO3 (BG11C) or 6 mM NH4Cl and 12 mM of N-Tris(hy-
degrading enzymes identified so far. The lactonases hydro-
droxymethyl)methyl-2-aminoethanesulphonic acid-NaOH
lyse the homoserine lactone (HSL) ring of the AHL molecule
buffer pH 7.5 (BG110C1NH1). To study the effect of AHL
4
to produce acyl homoserines (Dong et al., 2007), whereas
addition on the process of heterocyst differentiation, the
the acylases cleave the AHL amide bond, generating the
biomass of nondiazotrophic cultures was collected by filtra-
corresponding fatty acid and HSL ring (Dong et al., 2007).
tion (0.45 mm), washed and resuspended in fresh BG110C
Enzymatic QQ activity has been described in Gram-positive
(nitrogen step-down procedure).
and -negative bacteria and more recently in the cyanobac-
Solid media plates were prepared mixing equal volumes
terium Anabaena sp. PCC7120 (Romero et al., 2008).
of double-concentrated sterilized BG110 or BG1101NH1 4
Anabaena sp. PCC7120 is a filamentous cyanobacterium
and agar 10 g LÀ1. Plates inoculated with Anabaena sp.
simultaneously able to perform photosynthesis and dinitro-
PCC7120 were incubated at 30 1C with light.
gen fixation under aerobic conditions. In the presence of a
source of combined nitrogen, filaments grow as undiffer-
Addition of synthetic AHLs to cultures
entiated chains of vegetative cells. In contrast, when Ana-
baena sp. PCC7120 is deprived of combined nitrogen, AHLs were first assayed in solid media to check a possible
approximately 10% of the cells differentiate into morpholo- antibiotic effect (Lowery et al., 2009). Cells from a liquid
gically distinct heterocysts that supply the rest of the exponentially growing culture of Anabaena sp. PCC7120 in
filament with fixed nitrogen and in return receive carbohy- BG110C1NH1 were harvested by filtration, washed and
4
drate from vegetative cells (Wolk et al., 1994). In the absence resuspended in BG110C at a concentration of 5 mg chloro-
of combined nitrogen the heterocysts are spaced along the phyll a (Chl a) mLÀ1 and 100 mL of the suspension was
filament in a semi-regular pattern that is controlled by a spread on top of BG1101NH1 or BG110 plates. Small holes
4
regulatory loop established between two master regulators, were made in the centre of each plate and filled with 100 mL
NtcA and HetR (Muro-Pastor et al., 2002). of 100 mM AHL or acetonitrile (as control). Growth was
Because AHLs have been described in natural environ- checked after 7 days of incubation at 30 1C with light.
ments where cyanobacteria are prevalent, such as microbial Synthetic AHLs were also added to liquid cultures of
mats and algal blooms (McLean et al., 1997; Bachofen Anabaena sp. PCC7120 both under nondiazotrophic condi-
Schenk, 1998), the acylase-type QQ activity found in tions (BG110C1NH1 medium) and during nitrogen step-
4
Anabaena sp. PCC7120 (Romero et al., 2008) could serve down. Anabaena sp. PCC7120 was grown to exponential
either to mitigate possible negative effects of AHLs them- phase in BG110C1NH1 [cultures with about 5 mg Chl
4
selves and/or their tetramic acid derivatives (Kaufmann a mLÀ1; Chl a levels were determined in methanolic extracts
et al., 2005; Schertzer et al., 2009) or to confer a competitive (Mackinney, 1941)]. The cells were filtered, washed with
advantage against AHL-producing competitors through the BG110C, inoculated in fresh BG110C1NH11AHL 4
disruption of their communication system. (100 mM) or BG110C1AHL (100 mM) and bubbled with air
c 2011 Federation of European Microbiological Societies FEMS Microbiol Lett 315 (2011) 101–108
Published by Blackwell Publishing Ltd. All rights reserved

3. AHLs inhibit nitrogen fixation in cyanobacteria 103
or CO2-enriched air with a final Chl a concentration of after acetylene injection to determine the concentration of
4 mg mLÀ1. The AHLs used were: N-butyryl-homoserine the ethylene produced using a GC-MS (HP 5890 series II)
lactone (C4-HSL), N-(3-oxobutyryl)-L-homoserine (OC4- equipped with injector, column (Porapak Q) and flame
HSL), N-(3-hydroxybutyryl)-L-homoserine (OHC4-HSL), ionization detector (kept at 100, 80 and 150 1C, respec-
N-decanoyl-L-homoserine (C10-HSL) N-(3-oxodecanoyl)-L- tively). The detected signals were processed with the com-
homoserine lactone (OC10-HSL), N-(3-hydroxydecanoyl)- puting integrator PYE Unicam DP88. The equipment was
L-homoserine (OHC10-HSL), N-dodecanoyl-L-homoserine calibrated with known concentrations of ethylene.
(C12-HSL) OC12-HSL and N-(3-hydroxydodecanoyl)- To determine the nitrogenase activity of the cultures per
L-homoserine (OHC12-HSL) (unsubstituted AHLs were pur- unit Chl a, the following formula was used: nitrogenase
chased from Sigma-Aldrich, all other AHLs were kindly activity = nmol ethylene in sample  14 mL/2  mg Chl a
provided by Prof. Miguel C´ mara from the University of
a mLÀ1; where 14 was the atmosphere volume in 17-mL flasks
Nottingham). AHL stock solutions of 1 mg mLÀ1 were pre- and 2 the volume of culture in the flask.
pared in acetonitrile. Parallel control assays were carried out C10-HSL was also added to BG110C cultures of Anabaena
using equal amounts of acetonitrile (AHL solvent). In nitrogen sp. PCC7120 with mature heterocysts (24 h after nitrogen
step-down cultures, the differentiation of heterocysts was step-down) and the nitrogenase activity then measured as
monitored by Alcian blue staining of polysaccharides in the described before.
heterocyst envelope (Olmedo-Verd et al., 2006).
To further evaluate the lethal effect observed for OC10-
RNA isolation and analysis
HSL in ammonium-grown nondiazotrophic cultures of
Anabaena sp. PCC7120 (BG110C1NH1), different concen-
4 To assess a possible effect of AHLs on the expression of genes
trations of this signal (0.01, 0.1, 1, 10, 25, 50, 75 and involved in nitrogen fixation, Northern hybridization was
100 mM) as well as OC12-tetramic acid (100 mM) were also carried out with probes for the nifH and fdxH genes.
assayed. The effect of OC10-HSL (100 mM) was also tested in Samples of 50 mL were taken at 0, 3, 6, 20 and 24 h after
cultures with nitrate as combined nitrogen source (BG11C). nitrogen step-down. Cells were filtered, washed and resus-
OD600 nm of the cultures was measured at different time pended in 1 mL of Tris 50 mM/EDTA 100 mM, centrifuged
points after treatment (Kuznetsova et al., 2008). 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
Nitrogenase activity measurement
previously (Muro-Pastor et al., 2002).
Biomass (200 mL, 2–3 mg mLÀ1 Chl a) from BG110C1NH1 4 For Northern analysis, 30 mg of RNA was loaded per lane
aerated cultures of Anabaena sp. PCC7120 was harvested, and electrophoresed in 1% agarose denaturing formaldehyde
washed and resuspended in fresh BG110C at a Chl a gels. Transfer and fixation to Hybond-N1 membranes (Amer-
concentration of 2 mg mLÀ1 to induce the differentiation of sham Biosciences) were carried out using 0.1 M NaOH.
heterocysts. Cultures of 20 mL were established in flasks Hybridization was performed at 65 1C according to the
supplemented with AHLs (100 mM) or acetonitrile as con- recommendations of the manufacturer of the membranes.
trol. After 20 h of incubation at 30 1C, 120 r.p.m. and light, The nifH and fdxH probes were fragments of these genes
the nitrogenase activity was measured as follows: cells were amplified by PCR. The nifH probe was amplified using
concentrated to 4 mL by removing part of the supernatant plasmid pCSAV60 (containing the nifH gene cloned in
after centrifugation, and they were then divided in two 17- pGEM-T vector) as a template and oligonucleotides NH-1
mL flasks sealed with silicon caps (2 mL each, 10 mg Chl a). (corresponding to positions À 334 to À 314 with respect to
For each AHL, one flask was incubated under standard the translation start of nifH) and NH-4 (complementary to
aerobic conditions. Another flask was incubated with an nucleotides 1884 to 1863 with respect to the translation start
anaerobic atmosphere by injecting argon for 3 min and of nifH) (Valladares et al., 2007). The fdxH probe was
adding 10 mM 3-(3,4dichlorophenyl)-1,1-dimethylurea amplified using plasmid pCSAV164 (containing the fdxH gene
(DCMU) to inhibit photosynthesis and therefore oxygen cloned in pGEM-T vector) as a template and oligonucleotides
(O2) production (Rippka Stanier, 1978) to avoid a FH-1 (corresponding to nucleotides13 to 120 with respect to
possible inhibition of nitrogenase activity derived from the the translation start of fdxH) and FH-2 (complementary to
formation of abnormal heterocyst cell walls during matura- nucleotides 1297 to 1269 with respect to the translation start
tion or the damage from other mechanisms responsible for of fdxH) (Valladares et al., 2007). rnpB, encoding the RNA
maintaining low O2 concentration within the heterocysts. subunit of RNase P (Vioque, 1997), was used as a loading and
After 1-h incubation at 30 1C, 2 mL of acetylene was transfer control. All probes were 32P-labeled with a Ready-to-
injected. Samples of 1 mL from the air in the sealed flask Go DNA labeling kit (Amersham Biosciences) using
were taken at different times during 20 h starting 15 min [a-32P]dCTP. Images of radioactive filters and gels were
FEMS Microbiol Lett 315 (2011) 101–108
c 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved

4. 104 M. Romero et al.
obtained and quantified with a Cyclone storage phosphor nitrogen assimilation, which also controls the early phases of
system and OPTIQUANT image analysis software (Packard). heterocyst differentiation (Herrero et al., 2004). Expression
of gfp in this strain is induced in specific cells upon nitrogen
Results and discussion step-down, indicating the induction of ntcA during hetero-
cyst differentiation (Olmedo-Verd et al., 2006). To test for
Effect of synthetic AHLs addition possible effects of AHLs, cells of strain CSEL4a grown in the
presence of BG110C1NH1 were transferred to BG110C in
4
AHLs were added to Anabaena sp. PCC7120 cultures to the presence of AHLs (100 mM). Induction of the expression
evaluate possible effects on growth and nitrogen metabolism of gfp from the ntcA promoter proceeded in the same way
of the cyanobacterial filaments both in solid and liquid both in the presence and in the absence of AHLs, indicating
media. We selected saturated and substituted representatives that the AHLs were not affecting the process of heterocyst
of short- (C4, OC4 and OHC4-HSL), middle- (C10, OC10 differentiation (data not shown).
and OHC10-HSL) and long-chain AHLs (C12, OC12 and In contrast, and consistent with the results obtained in
OHC12-HSL). A first experiment was carried out in solid solid plates, a strong cytotoxic effect was observed after only
media, as described in Materials and methods. Growth 5 h for OC10-HSL (100 mM) in BG110C1NH1 in liquid 4
inhibition halos surrounding the wells were observed after media (Fig. 2a). The same effect could also be observed in
7 days for OC10-HSL and OC12-HSL in cultures subjected cultures with nitrate as nitrogen source (BG11C) supple-
to nitrogen step-down (transferred to nitrogen-free BG110 mented with OC10-HSL at the same concentration (data
medium) (Fig. 1). OC10-HSL also inhibited growth in the not shown). This effect could not be observed for any of the
presence of combined nitrogen (BG1101NH1, data not
4 other AHLs tested. To determine the OC10-HSL minimal
shown). These observations suggested that at least these lethal concentration, the assay was repeated using: 0.01, 0.1,
two AHLs could have an effect on heterocyst differentiation 1, 10, 25, 50, 75 and 100 mM of OC10-HSL in BG110C1
or maturation, which was further investigated. NH1 cultures. Concentrations 4 25 mM were lethal (Fig. 2a
4
AHLs were also added to liquid cultures under nondiazo- and b) and the filaments appeared completely lysed under
trophic conditions (BG110C1NH1) and to cultures sub-
4 the microscope after 5 h of culture. Cells incubated in the
jected to nitrogen step-down to study the effect on growth presence of 25 mM of OC10-HSL showed black dots, resem-
and heterocyst differentiation. None of the tested AHLs bling cyanophycin granules, in the inner side of the cell walls
showed cytotoxic effects in liquid cultures subjected to step- (data not shown). No lethal effect on Anabaena sp. PCC7120
down after 20 h of exposure. Moreover, no effect on hetero- was observed in cultures supplemented with 100 mM OC12-
cyst differentiation and distribution pattern was found in HSL or OC12-tetramic acid (data not shown). The half
step-down cultures for any of the tested AHLs after Alcian maximal effective concentration (EC50) observed for other
blue staining and microscope observation (data not shown). bacteria is between 8 and 55 mM for the OC12-HSL-derived
The discrepancy between the inhibitory effects obtained for tetramic acid and between 22.1 and 100 mM for OC12-HSL
OC10 and OC12-HSL in solid plates (Fig. 1) and in liquid itself, depending on the bacterial strain (Kaufmann et al.,
cultures could be derived from the longest period of 2005). These ranges match the lethal concentration observed
incubation of solid plates or could also be due to the higher for OC10-HSL in BG110C1NH1 cultures of Anabaena sp.
4
initial cell concentration in the liquid cultures compared PCC7120, but it should be noted that this activity was
with plates resulting in a higher AHL-acylase activity described only for Gram-positive bacteria, as the outer
(Romero et al., 2008) that would diminish the effect of Gram-negative membrane seems represent a permeability
initial AHL concentration. barrier for tetramic acids (Lowery et al., 2009). Nevertheless,
Possible effects of AHLs on heterocyst differentiation the antibiotic effect observed for OC10-HSL under non-
were also tested with Anabaena sp. PCC7120 strain CSEL4a diazotrophic conditions seems to be highly specific and
(Olmedo-Verd et al., 2006). This strain expresses gfp gene different from the antibiotic effect described so far for
under the control of ntcA promoter, the master regulator of tetramic acids, as no cytotoxic effect of OC12-HSL or its
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
Control OC10-HSL OC12-HSL
at 30 1C.
c 2011 Federation of European Microbiological Societies FEMS Microbiol Lett 315 (2011) 101–108
Published by Blackwell Publishing Ltd. All rights reserved

5. AHLs inhibit nitrogen fixation in cyanobacteria 105
tetramic acid derivative could be observed. It has been rophores (Kaufmann et al., 2005; Schertzer et al., 2009),
reported that a degradation product of oxo-substituted therefore the cytotoxic effect of OC10-HSL could be related
AHLs such as OC12-HSL is a tetramic acid with a high to iron quelant properties, but this could not explain the
affinity for iron, comparable to standard quelants and side- 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
(a) that OC10-HSL is acting through the disruption of mem-
brane potential, as already described for OC12-HSL or its
C 100 µM 75 µM 50 µM 25 µM
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
(b) 2.5
nitrogen in liquid media could be the result of a specific
inhibitory effect of this molecule on the metabolism of
2 combined nitrogen. Alternatively, OC10-HSL signal might
lead to the activation of the wrong pathways. For instance,
1.5
overactivation of arginine biosynthesis in the presence of
OD600 nm
combined nitrogen could lead to cyanophycin accumulation
(dense, presumptive cyanophycin granules are observed in
1 the damaged filaments), blocking the entire nitrogen meta-
bolism and resulting in cell death.
0.5
Nitrogenase activity
0 Although no macroscopic effect of AHLs on survival and
0 5 10 15 20 25
heterocyst differentiation was recorded in diazotrophic
Time (h)
cultures in short-time experiments, the effect of the signals
Fig. 2. (a) Antibiotic effect of different concentrations of OC10-HSL on the nitrogenase activity was evaluated in BG110C1NH1 4
(25–100 mM) on Anabaena sp. PCC7120 cultures in BG110C1NH1. The 4 cultures transferred to BG110C for the induction of hetero-
photo was taken 7 h after AHL addition. C, control culture containing
cyst formation and nitrogen fixation in the presence of the
acetonitrile. (b) Evolution of OD600 nm of Anabaena sp. PCC7120 cultures
in BG110C1NH1 with different concentrations of OC10-HSL (, 25 mM;
AHLs. Nitrogenase measurements were carried out 20 h
4
m, 50 mM; , 75 mM; and ^, 100 mM) and acetonitrile (B) as control. after the nitrogen step-down treatment to allow formation
Time 0, addition of OC10-HSL. of mature heterocysts. A strong inhibition of the nitrogenase
100
80
Nitrogenase activity (%)
60
Fig. 3. Anabaena sp. PCC7120 nitrogenase
activities under aerobic (black bars) and
anaerobic (grey bars) conditions in BG110C
40
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 20
expressed as percentages of the value for
control culture, which corresponds to 2.04
(aerobic) and 6.5 (anaerobic) nmol ethylene per 0
mg ChlÀ1hÀ1. C C4 OC4 OHC4 C10 OC10 OHC10 C12 OC12 OHC12
FEMS Microbiol Lett 315 (2011) 101–108
c2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved

6. 106 M. Romero et al.
activity was recorded for all AHLs tested (Fig. 3). The lower Effect on the expression of nitrogen fixation-
ethylene production in AHL-treated cultures was already related genes
evident 5 min after acetylene addition. The inhibition was
Because all tested AHLs showed inhibitory activity on
specially marked in cultures treated with OC10 and OC12-
nitrogen fixation mostly in newly formed heterocysts, to
HSL, in which none or residual nitrogenase activity could be
study possible effects at the level of expression of nitrogen
detected (Fig. 3). This result is consistent with the inhibition
metabolism genes, Northern blots were carried out to detect
of growth observed in the cyanobacterium, with these two
changes in expression of the dinitrogenase reductase subunit
AHLs in solid BG110 media (Fig. 1).
gene (nifH) and fdxH, encoding a heterocyst-specific ferro-
To evaluate whether the inhibition of nitrogenase activity
doxin that is a likely electron donor to dinitrogenase
was due to defects in heterocyst wall formation or defects in
reductase (Razquin et al., 1995).
any of the other mechanisms driving the creation of a
No significant differences in the expression of either gene
microoxic environment inside the heterocysts, nitrogenase
could be detected at 20 and 24 h after nitrogen step-down
activity was also measured under anaerobic atmosphere (Fig.
(no expression of nifH and fdxH was detected at 0, 3 or 6 h)
3). Air inside the flasks was substituted by argon and DCMU
in total RNA extracted from C10-HSL-treated cultures when
was added to the cultures to inhibit PSII-dependent O2
compared with control samples (Fig. 4). This indicates that
production. As expected, slightly higher nitrogenase activity
the process of heterocyst differentiation proceeds normally
was observed in anaerobic conditions than in aerobic ones
in the presence of AHLs and therefore AHL inhibition could
(Valladares et al., 2007), but the effect of AHL addition was
be affecting either the expression of other genes related to
still observed (Fig. 3). This indicates that the lower nitrogen-
nitrogen fixation or be acting on nitrogenase-related genes
ase activity observed in the presence of AHLs was not due to
at a post-transcriptional level.
alterations in the microoxic environment of the heterocysts
and confirms that they have no effect on heterocyst differ-
Control C10-HSL treated
entiation as observed in AHL-supplemented cultures de- 0 3 6 20 24 3 6 20 24 h
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 hesAB-fdxH
on nitrogen fixation took place only in developing hetero-
cysts, nitrogenase activity was also measured in diazotrophic fdxH
cultures in which C10-HSL (100 mM) 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
nifHDK
30% decrease of nitrogenase activity (data not shown). The
nifHD
progressive increase of the inhibitory effect of AHLs in
acclimated cultures could perhaps be caused by the entry of nifH
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
rnpB
periplasmic space (Flores et al., 2006; Mariscal et al., 2007),
entering in both mature and forming heterocysts, these
Fig. 4. Effect of C10-HSL addition on heterocyst differentiation upon
molecules could only act at the molecular level in newly
nitrogen step-down. RNA (30 mg) was isolated from samples taken at 0
formed heterocysts. In that case the results observed would (NH1), 3, 6, 20 and 24 h in the presence or absence of C10-HSL. Cultures
4
suggest a nonreversible inhibition of nitrogenase in very contained 100 mM C10-HSL or acetonitrile as control. Hybridizations
early stages at the level of either gene expression or its were carried out with a probe for the nifH or fdxH gene or for the rnpB
enzymatic activity. gene (Vioque, 1997), which was used as a loading and transfer control.
c 2011 Federation of European Microbiological Societies FEMS Microbiol Lett 315 (2011) 101–108
Published by Blackwell Publishing Ltd. All rights reserved

7. AHLs inhibit nitrogen fixation in cyanobacteria 107
Finally, the strong inhibition of nitrogenase demonstrated References
for all the AHLs tested and the cytotoxic effect of OC10-HSL Bachofen R Schenk A (1998) Quorum sensing autoinducers –
in the presence of combined nitrogen represent novel do they play a role in natural microbial habitats? Microbiol Res
biological activities of these signal molecules. The observa- 153: 61–63.
tion that antibiotics cannot easily reach the lethal concen- Burton EO, Read HW, Pellitteri MC Hickey WJ (2005)
trations in natural environments has led to a questioning of Identification of acyl-homoserine lactone signal molecules
whether these molecules could act, in subinhibitory con- produced by Nitrosomonas europaea strain Schmidt. Appl
centrations, as signal molecules (Davies, 2006; Linares et al., Environ Microb 71: 4906–4909.
2006). Low concentrations of several antibiotics can alter Davies J (2006) Are antibiotics naturally antibiotics? J Ind
expression patterns of bacteria without any effect on growth Microbiol Biot 33: 496–499.
rate (Davies et al., 2006), which resembles the mode of Davies J, Spiegelman GB Yim G (2006) The world of
action of QS signals. Thus one possibility is that the AHL subinhibitory antibiotic concentrations. Curr Opin Microbiol
signals have inhibitory effects when added at higher con- 9: 445–453.
centrations than those found in natural environments. In Dong YH, Wang LH, Xu JL, Zhang HB, Zhang XF Zhang LH
fact, the concentrations reported in the literature for AHLs (2001) Quenching quorum-sensing-dependent bacterial
in the culture media of the model microorganism Vibrio infection by an N-acyl homoserine lactonase. Nature 411:
fischeri usually range between 0.4 and 400 nM (Kaplan 813–817.
Greenberg, 1985; Schaefer et al., 2002; Burton et al., 2005), Dong YH, Wang LH Zhang LH (2007) Quorum-quenching
significantly lower than the concentrations exhibiting in- microbial infections: mechanisms and implications. Philos T
hibitory activity against Anabaena sp. PCC7120. Roy Soc B 362: 1201–1211.
In conclusion, AHLs strongly inhibit nitrogen fixation in Flores E, Herrero A, Wolk CP Maldener I (2006) Is the
Anabaena sp. PCC7120, although they do not affect the periplasm continuous in filamentous multicellular
process of heterocyst differentiation because no changes cyanobacteria? Trends Microbiol 14: 439–443.
were observed in the frequency, pattern of differentiation, Fuqua WC, Winans SC Greenberg EP (1994) Quorum sensing
permeability of the heterocyst cell wall or expression of in bacteria: the LuxR-LuxI family of cell density-responsive
regulatory genes whose products are involved in differentia- transcriptional regulators. J Bacteriol 176: 269–275.
tion (ntcA). The strong inhibition of nitrogenase activity Herrero A, Muro-Pastor AM, Valladares A Flores E (2004)
observed could be related to nitrogen fixation blockage at a Cellular differentiation and the NtcA transcription factor in
post-transcriptional level, mainly on newly formed hetero- filamentous cyanobacteria. FEMS Microbiol Rev 28: 469–487.
Huang YL, Ki JS, Case RJ Quian PY (2008) Diversity and acyl-
cysts. Moreover, a possible new activity of AHL signals was
homoserine lactone production among subtidal biofilm-
found for OC10-HSL in the presence of combined nitrogen,
forming bacteria. Aquat Microb Ecol 52: 185–193.
differing from those activities described for oxo-substituted
Kaplan HB Greenberg EP (1985) Diffusion of autoinducer is
and AHL tetramic acid derivatives. The presence of acylase
involved in regulation of the Vibrio fischeri luminescence
activity against long-chain AHLs described in the biomass of
system. J Bacteriol 163: 1210–1214.
Anabaena sp. PCC7120 (Romero et al., 2008) could be
Kaufmann GF, Sartorio R, Lee SH, Rogers CJ, Meijler MM, Moss
related to the negative effects of AHLs in this cyanobacter-
JA, Clapham B, Brogan AP, Dickerson TJ Janda KD (2005)
ium. This AHL-degradation mechanism would protect the
Revisiting quorum sensing: discovery of additional chemical
filaments, at normal environmental concentrations, from and biological functions for 3-oxo-N-acyl-homoserine
exogenous signals with potential cytotoxic and inhibitory lactones. P Natl Acad Sci USA 102: 309–314.
activities on the cyanobacterium. Kuznetsova NI, Azizbekyan RR, Konyukhov IV, Pogosyan SI
Rubin AB (2008) Inhibition of photosynthesis in
cyanobacteria and plankton algae by the bacterium
Acknowledgements Brevibacillus laterosporus metabolites. Dokl Biochem Biophys
421: 181–184.
´
This work was financed by a grant from Consellerıa de Linares JF, Gustafsson I, Baquero F Mart´nez JL (2006)
ı
´
Innovacion e Industria, Xunta de Galicia PGIDIT06P- Antibiotics as intermicrobial signaling agents instead of
XIB200045PR. M.R. was supported by an FPU fellowship weapons. P Natl Acad Sci USA 103: 19484–19489.
from the Spanish Ministry of Education and Science and a Lowery CA, Park J, Gloeckner C et al. (2009) Defining the mode
´
predoctoral fellowship from Diputacion de A Coru˜ a. We
n of action of tetramic acid antibacterials derived from
would like to thank Prof. Kim D. Janda and Dr Gunnar F. Pseudomonas aeruginosa quorum sensing signals. J Am Chem
Kaufmann for kindly providing us with OC12-tetramic acid. Soc 131: 14473–14479.
´
We also would like to thank Prof. Miguel Camara for Mackinney G (1941) Absorption of light by chlorophyll solutions.
providing us with synthetic AHLs. J Biol Chem 140: 315–322.
FEMS Microbiol Lett 315 (2011) 101–108
c2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved

8. 108 M. Romero et al.
Mariscal V, Herrero A Flores E (2007) Continuous periplasm in degradation by the fish pathogen Tenacibaculum maritimum, a
a filamentous, heterocyst-forming cyanobacterium. Mol member of the Cytophaga–Flavobacterium–Bacteroides (CFB)
Microbiol 65: 1139–1145. division. FEMS Microbiol Lett 304: 131–139.
McLean RJC, Whiteley M, Stickler DJ Fuqua WC (1997) Schaefer AL, Taylor TA, Beatty JT Greenberg EP (2002) Long-
Evidence of autoinducer activity in naturally-occurring chain acyl-homoserine lactone quorum-sensing regulation of
biofilms. FEMS Microbiol Lett 154: 259–263. Rhodobacter capsulatus gene transfer agent production. J
Muro-Pastor AM, Valladares A, Flores E Herrero A (2002) Bacteriol 184: 6515–6521.
Mutual dependence of the expression of the cell differentiation Schertzer JW, Boulette ML Whiteley M (2009) More than a
regulatory protein HetR and the global nitrogen regulator signal: non-signaling properties of quorum sensing molecules.
NtcA during heterocyst development. Mol Microbiol 44: Trends Microbiol 17: 189–195.
1377–1385. Sharif DI, Gallon J, Smith CJ Dudley E (2008) Quorum sensing
Olmedo-Verd E, Muro-Pastor AM, Flores E Herrero A (2006) in Cyanobacteria: N-octanoyl-homoserine lactone release and
Localized induction of the ntcA regulatory gene in developing response, by the epilithic colonial cyanobacterium Gloeothece
heterocysts of Anabaena sp. strain PCC 7120. J Bacteriol 188: PCC6909. ISME J 2: 1171–1182.
6694–6699. Valladares A, Maldener I, Muro-Pastor AM, Flores E Herrero A
´
Razquin P, Schmitz S, Peleato ML, Fillat MF, Gomez-Moreno C (2007) Heterocyst development and diazotrophic metabolism
B¨ hme H (1995) Differential activities of heterocyst
o in terminal respiratory oxidase mutants of the cyanobacterium
ferredoxin, vegetative cell ferredoxin, and flavodoxin as Anabaena sp. strain PCC7120. J Bacteriol 189: 4425–4430.
electron carriers in nitrogen fixation and photosynthesis in Vioque A (1997) The RNase P RNA from cyanobacteria: short
Anabaena sp. Photosynth Res 43: 35–40. tandemly repeated repetitive (STRR) sequences are present
Rippka R Stanier RY (1978) The effects of anaerobiosis on within the RNase P RNA gene in heterocyst-forming
nitrogenase synthesis and heterocyst development by cyanobacteria. Nucleic Acids Res 25: 3471–3477.
nostocacean cyanobacteria. J Gen Microbiol 105: 83–94. Whitehead NA, Barnard AM, Slater H, Simpson NJ Salmond
Rippka R, Deruelles J, Waterbury JB, Herdman M Stanier RY GP (2001) Quorum-sensing in Gram-negative bacteria. FEMS
(1979) Generic assignments, strain histories and properties of Microbiol Rev 25: 365–404.
pure cultures of cyanobacteria. J Gen Microbiol 111: 1–61. Williams P, Winzer K, Chan WC C´ mara M (2007) Look who’s
a
Romero M, Diggle SP, Heeb S, C´ mara M Otero A (2008)
a talking: communication and quorum sensing in the bacterial
Quorum quenching activity in Anabaena sp. PCC 7120: world. Philos T Roy Soc B 362: 1119–1134.
identification of AiiC, a novel AHL-acylase. FEMS Microbiol Wolk CP, Ernst A Elhai J (1994) Heterocyst metabolism and
Lett 280: 73–80. development. The Molecular Biology of Cyanobacteria (Bryant
Romero M, Avenda˜ o-Herrera R, Magari˜ os B, C´ mara M
n n a DA, ed), pp. 769–823. Kluwer Academic Publishers,
Otero A (2010) Acyl homoserine lactone production and Dordrecht.
c 2011 Federation of European Microbiological Societies FEMS Microbiol Lett 315 (2011) 101–108
Published by Blackwell Publishing Ltd. All rights reserved