RESEARCH LETTER                       Quorum sensing N-acylhomoserine lactone signals a¡ect nitrogen                      ...
102                                                                                                                  M. Ro...
AHLs inhibit nitrogen fixation in cyanobacteria                                                                            ...
104                                                                                                                   M. R...
AHLs inhibit nitrogen fixation in cyanobacteria                                                                            ...
106                                                                                                                       ...
AHLs inhibit nitrogen fixation in cyanobacteria                                                                            ...
108                                                                                                                     M....
Upcoming SlideShare
Loading in …5
×

1

405 views

Published on

0 Comments
0 Likes
Statistics
Notes
  • Be the first to comment

  • Be the first to like this

No Downloads
Views
Total views
405
On SlideShare
0
From Embeds
0
Number of Embeds
2
Actions
Shares
0
Downloads
3
Comments
0
Likes
0
Embeds 0
No embeds

No notes for slide

1

  1. 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. 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 AHLdegradation product of N-(3-oxododecanoyl)-L-homoserine addition to cultures of the filamentous heterocyst-forminglactone (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 thisdecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione, which cyanobacterium.exhibits iron-binding ability. This AHL derivative is ableto bind iron in a 3 : 1 complex with an affinity comparable Materials and methodsto that exhibited by standard quelators and siderophores(Schertzer et al., 2009). In addition, antibiotic properties of Growth conditionsthe tetramic acid derivative of OC12-HSL have been de- Stock cultures of Anabaena sp. PCC7120 were maintainedscribed, through the disruption of membrane potential and photoautotrophically at 30 1C with a continuous irradianceproton gradient of bacteria, thus eliminating the proton- of 75 mE mÀ2 sÀ1. Cultures were aerated by connecting eachmotive force and leading to bacterial death (Lowery et al., culture unit to an aeration system with a continuous filtered2009). (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 NaNO3different 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 wereing’ (QQ) (Dong et al., 2001). An example of QQ is the established in BG110C supplemented with either 17 mMenzymatic 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-NaOHlyse the homoserine lactone (HSL) ring of the AHL molecule buffer pH 7.5 (BG110C1NH1). To study the effect of AHL 4to produce acyl homoserines (Dong et al., 2007), whereas addition on the process of heterocyst differentiation, thethe 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 BG110CEnzymatic 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 volumesterium 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 asource of combined nitrogen, filaments grow as undiffer- Addition of synthetic AHLs to culturesentiated 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 possibleapproximately 10% of the cells differentiate into morpholo- antibiotic effect (Lowery et al., 2009). Cells from a liquidgically distinct heterocysts that supply the rest of the exponentially growing culture of Anabaena sp. PCC7120 infilament with fixed nitrogen and in return receive carbohy- BG110C1NH1 were harvested by filtration, washed and 4drate 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 wasfilament in a semi-regular pattern that is controlled by a spread on top of BG1101NH1 or BG110 plates. Small holes 4regulatory loop established between two master regulators, were made in the centre of each plate and filled with 100 mLNtcA 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 ofmats 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- 4Anabaena sp. PCC7120 (Romero et al., 2008) could serve down. Anabaena sp. PCC7120 was grown to exponentialeither to mitigate possible negative effects of AHLs them- phase in BG110C1NH1 [cultures with about 5 mg Chl 4selves and/or their tetramic acid derivatives (Kaufmann a mLÀ1; Chl a levels were determined in methanolic extractset al., 2005; Schertzer et al., 2009) or to confer a competitive (Mackinney, 1941)]. The cells were filtered, washed withadvantage against AHL-producing competitors through the BG110C, inoculated in fresh BG110C1NH11AHL 4disruption of their communication system. (100 mM) or BG110C1AHL (100 mM) and bubbled with airc 2011 Federation of European Microbiological Societies FEMS Microbiol Lett 315 (2011) 101–108Published by Blackwell Publishing Ltd. All rights reserved
  3. 3. AHLs inhibit nitrogen fixation in cyanobacteria 103or CO2-enriched air with a final Chl a concentration of after acetylene injection to determine the concentration of4 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 flameHSL), 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 wasL-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 perL-homoserine (OHC12-HSL) (unsubstituted AHLs were pur- unit Chl a, the following formula was used: nitrogenasechased from Sigma-Aldrich, all other AHLs were kindly activity = nmol ethylene in sample  14 mL/2  mg Chl aprovided by Prof. Miguel C´ mara from the University of a mLÀ1; where 14 was the atmosphere volume in 17-mL flasksNottingham). 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 Anabaenausing equal amounts of acetonitrile (AHL solvent). In nitrogen sp. PCC7120 with mature heterocysts (24 h after nitrogenstep-down cultures, the differentiation of heterocysts was step-down) and the nitrogenase activity then measured asmonitored 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 analysisHSL in ammonium-grown nondiazotrophic cultures ofAnabaena sp. PCC7120 (BG110C1NH1), different concen- 4 To assess a possible effect of AHLs on the expression of genestrations of this signal (0.01, 0.1, 1, 10, 25, 50, 75 and involved in nitrogen fixation, Northern hybridization was100 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 aftercultures 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, centrifugedpoints 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 describedNitrogenase 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 laneaerated cultures of Anabaena sp. PCC7120 was harvested, and electrophoresed in 1% agarose denaturing formaldehydewashed 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 thesupplemented 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 genesthe nitrogenase activity was measured as follows: cells were amplified by PCR. The nifH probe was amplified usingconcentrated to 4 mL by removing part of the supernatant plasmid pCSAV60 (containing the nifH gene cloned inafter centrifugation, and they were then divided in two 17- pGEM-T vector) as a template and oligonucleotides NH-1mL flasks sealed with silicon caps (2 mL each, 10 mg Chl a). (corresponding to positions À 334 to À 314 with respect toFor each AHL, one flask was incubated under standard the translation start of nifH) and NH-4 (complementary toaerobic conditions. Another flask was incubated with an nucleotides 1884 to 1863 with respect to the translation startanaerobic atmosphere by injecting argon for 3 min and of nifH) (Valladares et al., 2007). The fdxH probe wasadding 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 topossible inhibition of nitrogenase activity derived from the the translation start of fdxH) and FH-2 (complementary toformation of abnormal heterocyst cell walls during matura- nucleotides 1297 to 1269 with respect to the translation starttion or the damage from other mechanisms responsible for of fdxH) (Valladares et al., 2007). rnpB, encoding the RNAmaintaining 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) usingwere taken at different times during 20 h starting 15 min [a-32P]dCTP. Images of radioactive filters and gels wereFEMS Microbiol Lett 315 (2011) 101–108 c 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
  4. 4. 104 M. Romero et al.obtained and quantified with a Cyclone storage phosphor nitrogen assimilation, which also controls the early phases ofsystem and OPTIQUANT image analysis software (Packard). heterocyst differentiation (Herrero et al., 2004). Expression of gfp in this strain is induced in specific cells upon nitrogenResults and discussion step-down, indicating the induction of ntcA during hetero- cyst differentiation (Olmedo-Verd et al., 2006). To test forEffect of synthetic AHLs addition possible effects of AHLs, cells of strain CSEL4a grown in the presence of BG110C1NH1 were transferred to BG110C in 4AHLs were added to Anabaena sp. PCC7120 cultures to the presence of AHLs (100 mM). Induction of the expressionevaluate possible effects on growth and nitrogen metabolism of gfp from the ntcA promoter proceeded in the same wayof the cyanobacterial filaments both in solid and liquid both in the presence and in the absence of AHLs, indicatingmedia. We selected saturated and substituted representatives that the AHLs were not affecting the process of heterocystof 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 inOHC12-HSL). A first experiment was carried out in solid solid plates, a strong cytotoxic effect was observed after onlymedia, as described in Materials and methods. Growth 5 h for OC10-HSL (100 mM) in BG110C1NH1 in liquid 4inhibition halos surrounding the wells were observed after media (Fig. 2a). The same effect could also be observed in7 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 (datamedium) (Fig. 1). OC10-HSL also inhibited growth in the not shown). This effect could not be observed for any of thepresence of combined nitrogen (BG1101NH1, data not 4 other AHLs tested. To determine the OC10-HSL minimalshown). 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 BG110C1or 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 undertrophic conditions (BG110C1NH1) and to cultures sub- 4 the microscope after 5 h of culture. Cells incubated in thejected 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 wallsshowed cytotoxic effects in liquid cultures subjected to step- (data not shown). No lethal effect on Anabaena sp. PCC7120down 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 halfstep-down cultures for any of the tested AHLs after Alcian maximal effective concentration (EC50) observed for otherblue staining and microscope observation (data not shown). bacteria is between 8 and 55 mM for the OC12-HSL-derivedThe discrepancy between the inhibitory effects obtained for tetramic acid and between 22.1 and 100 mM for OC12-HSLOC10 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 observedincubation of solid plates or could also be due to the higher for OC10-HSL in BG110C1NH1 cultures of Anabaena sp. 4initial cell concentration in the liquid cultures compared PCC7120, but it should be noted that this activity waswith 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 permeabilityinitial 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 forunder 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) 101–108Published by Blackwell Publishing Ltd. All rights reserved
  5. 5. AHLs inhibit nitrogen fixation in cyanobacteria 105tetramic 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 relatedAHLs such as OC12-HSL is a tetramic acid with a high to iron quelant properties, but this could not explain theaffinity 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 signalsFig. 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 theacetonitrile. (b) Evolution of OD600 nm of Anabaena sp. PCC7120 culturesin 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 formationTime 0, addition of OC10-HSL. of mature heterocysts. A strong inhibition of the nitrogenase 100 80 Nitrogenase activity (%) 60Fig. 3. Anabaena sp. PCC7120 nitrogenaseactivities under aerobic (black bars) andanaerobic (grey bars) conditions in BG110C 40supplemented with the AHLs: C4, OC4, OHC4,C10, OC10, OHC10, C12, OC12 andOHC12-HSL (100 mM). Control culture was setwith acetonitrile (C). Nitrogenase activities are 20expressed as percentages of the value forcontrol culture, which corresponds to 2.04(aerobic) and 6.5 (anaerobic) nmol ethylene per 0mg ChlÀ1hÀ1. C C4 OC4 OHC4 C10 OC10 OHC10 C12 OC12 OHC12FEMS Microbiol Lett 315 (2011) 101–108 c2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
  6. 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 genesevident 5 min after acetylene addition. The inhibition was Because all tested AHLs showed inhibitory activity onspecially marked in cultures treated with OC10 and OC12- nitrogen fixation mostly in newly formed heterocysts, toHSL, in which none or residual nitrogenase activity could be study possible effects at the level of expression of nitrogendetected (Fig. 3). This result is consistent with the inhibition metabolism genes, Northern blots were carried out to detectof growth observed in the cyanobacterium, with these two changes in expression of the dinitrogenase reductase subunitAHLs 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 dinitrogenasewas 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 genemicrooxic environment inside the heterocysts, nitrogenase could be detected at 20 and 24 h after nitrogen step-downactivity 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 whenwas added to the cultures to inhibit PSII-dependent O2 compared with control samples (Fig. 4). This indicates thatproduction. As expected, slightly higher nitrogenase activity the process of heterocyst differentiation proceeds normallywas 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 tostill observed (Fig. 3). This indicates that the lower nitrogen- nitrogen fixation or be acting on nitrogenase-related genesase activity observed in the presence of AHLs was not due to at a post-transcriptional level.alterations in the microoxic environment of the heterocystsand confirms that they have no effect on heterocyst differ- Control C10-HSL treatedentiation as observed in AHL-supplemented cultures de- 0 3 6 20 24 3 6 20 24 hscribed before. As observed under aerobic conditions, theOC10 and OC12-HSL signals had the strongest inhibitoryeffect on nitrogenase activity (Fig. 3). Twenty hours after theaddition of acetylene still no recovery of normal levels ofnitrogenase activity of the cultures was observed either inaerobic or anaerobic conditions (data not shown). To determine whether the inhibitory effect of the AHLs hesAB-fdxHon nitrogen fixation took place only in developing hetero-cysts, nitrogenase activity was also measured in diazotrophic fdxHcultures in which C10-HSL (100 mM) was added 24 h afternitrogen step-down, when mature heterocysts are alreadypresent. The amount of ethylene produced in early sampleswas similar in cultures with or without C10-HSL but,interestingly, a progressively decreased ethylene productionwas observed in the C10-HSL-treated culture, resulting in a nifHDK30% decrease of nitrogenase activity (data not shown). The nifHDprogressive increase of the inhibitory effect of AHLs inacclimated cultures could perhaps be caused by the entry of nifHthe AHLs in the new generations of heterocysts, as theimpermeability of the wall of mature heterocysts couldprevent the penetration of the AHLs. Nonetheless it cannotbe excluded that although AHLs could enter throughvegetative walls and spread along the filaments by the rnpBperiplasmic 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 uponmolecules could only act at the molecular level in newly nitrogen step-down. RNA (30 mg) was isolated from samples taken at 0formed heterocysts. In that case the results observed would (NH1), 3, 6, 20 and 24 h in the presence or absence of C10-HSL. Cultures 4suggest a nonreversible inhibition of nitrogenase in very contained 100 mM C10-HSL or acetonitrile as control. Hybridizationsearly 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 rnpBenzymatic 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) 101–108Published by Blackwell Publishing Ltd. All rights reserved
  7. 7. AHLs inhibit nitrogen fixation in cyanobacteria 107 Finally, the strong inhibition of nitrogenase demonstrated Referencesfor 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 Resbiological 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 moleculeswhether these molecules could act, in subinhibitory con- produced by Nitrosomonas europaea strain Schmidt. Applcentrations, 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 Indexpression 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 ofaction of QS signals. Thus one possibility is that the AHL subinhibitory antibiotic concentrations. Curr Opin Microbiolsignals 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 LHfact, the concentrations reported in the literature for AHLs (2001) Quenching quorum-sensing-dependent bacterialin 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-quenchingsignificantly lower than the concentrations exhibiting in- microbial infections: mechanisms and implications. Philos Thibitory 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 theAnabaena sp. PCC7120, although they do not affect the periplasm continuous in filamentous multicellularprocess 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 sensingpermeability of the heterocyst cell wall or expression of in bacteria: the LuxR-LuxI family of cell density-responsiveregulatory 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 inpost-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 isand AHL tetramic acid derivatives. The presence of acylase involved in regulation of the Vibrio fischeri luminescenceactivity 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, Mossrelated 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 chemicalfilaments, at normal environmental concentrations, from and biological functions for 3-oxo-N-acyl-homoserineexogenous 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 bacteriumAcknowledgements 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 ofXIB200045PR. 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 fromwould like to thank Prof. Kim D. Janda and Dr Gunnar F. Pseudomonas aeruginosa quorum sensing signals. J Am ChemKaufmann 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. 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. JMuro-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 sensingOlmedo-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 presentRippka 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 SalmondRippka 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 aRomero 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 (BryantRomero 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) 101–108Published by Blackwell Publishing Ltd. All rights reserved

×