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Applied and Environmental Microbiology, June 2004, p. 3715-3723, Vol. 70, No. 6
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.6.3715-3723.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Use of Sinorhizobium meliloti as an Indicator for Specific Detection of Long-Chain N-Acyl Homoserine Lactones

Inmaculada Llamas, Neela Keshavan, and Juan E. González*

Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, Texas 75083-0688

Received 5 December 2003/ Accepted 20 February 2004


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ABSTRACT
 
Population-density-dependent gene expression in gram-negative bacteria involves the production of signal molecules characterized as N-acyl homoserine lactones (AHLs). The synthesis of AHLs by numerous microorganisms has been identified by using biosensor strains based on the Agrobacterium tumefaciens and Chromobacterium violaceum quorum-sensing systems. The symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti is rapidly becoming a model organism for the study of quorum sensing. This organism harbors at least three different quorum-sensing systems (Sin, Mel, and Tra), which play a role in its symbiotic relationship with its host plant, alfalfa. The Sin system is distinguished among them for the production of long-chain AHLs, including C18-HL, the longest AHL reported so far. In this work, we show that construction of a sinI::lacZ transcriptional fusion results in a strain that detects long-chain AHLs with exquisite sensitivity. Overexpression of the SinR regulator protein from a vector promoter increases its sensitivity without loss of specificity. We also show that the resulting indicator strain can recognize long-chain AHLs produced by unrelated bacteria such as Paracoccus denitrificans and Rhodobacter capsulatus. This S. meliloti indicator strain should serve as a tool for the specific detection of long-chain AHLs in new systems.


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INTRODUCTION
 
Over the past few years, it has become increasingly evident that numerous bacteria can establish a sophisticated cell-cell communication system within a bacterial community by producing diffusible signal molecules. These signals, called autoinducers, control gene expression in response to bacterial cell density in a process generally known as quorum sensing. The best-characterized gram-negative bacterial autoinducers are N-acyl homoserine lactones (AHLs), which are usually synthesized by a member of the LuxI protein family. As the density of the population increases, AHL molecules accumulate in the growth medium, and upon reaching a critical threshold concentration, they bind and activate an AHL receptor protein belonging to the LuxR family of transcriptional regulators. The LuxR homolog-AHL complex then activates or represses the expression of target genes, including in many instances, the activation of the autoinducer synthase, which results in the production of more AHLs (positive feedback loop) (for reviews, see references 4, 5, 8 and 35).

Most of the quorum-sensing systems characterized so far occur in bacteria that are able to establish relationships, either pathogenic or symbiotic, with plant or animal hosts. In these cases, quorum-sensing mechanisms have been demonstrated to regulate genes involved in biofilm formation in Pseudomonas aeruginosa (3, 4, 24), production of exoenzymes and antibiotics in Chromobacterium violaceum (19), exopolysaccharide production in Pantoea stewartii and Sinorhizobium meliloti (16, 34), and plasmid transfer in Agrobacterium tumefaciens and S. meliloti (10, 28; M. M. Marketon, I. Llamas, M. R. Gronquist, A. Eberhard, and J. E. González, submitted for publication).

S. meliloti is a gram-negative soil bacterium characterized by its ability to establish a nitrogen-fixing symbiosis with the host plant, Medicago sativa (alfalfa). S. meliloti Rm1021 and Rm41 have been recently shown to harbor at least two and three quorum-sensing systems, respectively (17, 18; Marketon et al., submitted). One of these, the Sin system, is located on the chromosome of both strains and is responsible for making several long-chain AHLs ranging in size from 12 to 18 carbons, the longest reported so far. One of these AHLs (N-2-hexadecenoyl-DL-homoserine lactone or C16:1-HL) plays a role in the synthesis of low-molecular-weight EPS II, the biologically active fraction of this exopolysaccharide. Moreover, plants inoculated with sin mutants show a defect in nodulation efficiency (16, 18). A second system, referred to as the Mel system, is also encoded in the chromosome of both strains and is involved in the synthesis of short-chain AHLs including C8-HL (18; Marketon et al., submitted). In addition to the Sin and Mel systems, Rm41 carries the Tra system, which is located on the 200-kb plasmid pRme41a. The Tra quorum-sensing system is also responsible for the production of short-chain AHLs and regulates conjugal plasmid transfer in S. meliloti Rm41 (Marketon et al., submitted).

AHL molecules consist of a variable acyl chain (length from 4 to 18 carbons) attached to a conserved homoserine lactone head group. The differences in the length of the acyl chain, the nature of the substituent on the third carbon, and the presence of a double bond confer the signal specificity and determine their permeability through the cell membrane (6, 12). For instance, long-chain AHLs, containing 12 or more carbons, cannot diffuse freely and may need to be exported through the cell membrane by efflux pumps (27).

The discovery that numerous unrelated bacteria have the ability to produce autoinducer molecules in response to a high cell density has been achieved by using biosensors. Most of the biosensors or indicator strains developed so far (1) are composed of a quorum-sensing-controlled promoter fused to the lux operon (2, 33, 36) or reporter genes such as lacZ (26, 32). These biosensor strains have a functional regulator protein but lack the AHL synthase enzyme; therefore, promoter activity can be induced by the presence of exogenous AHLs. However, some microorganisms may produce signals that are not detected by one of the indicator strains or they produce molecules at levels below the threshold of sensitivity of the reporter (32). For example, the detection of the long-chain AHL C16-HL produced by Rhodobacter capsulatus and Paracoccus denitrificans required the use of a radioactive incorporation assay (31).

A recently described indicator strain developed by Zhu et al. can detect long-chain AHLs with high sensitivity (39). Unfortunately, AHLs with more than 14 carbons cannot be specifically detected by any of the known biosensors. This leads us to propose the use of S. meliloti as a new indicator for long-chain AHLs based on its ability to make a broad range of AHLs with long acyl chains. We present here the development of an S. meliloti strain for the specific detection of long-chain AHLs, including those with more than 14 carbons. This indicator strain was constructed by the disruption of the Sin quorum-sensing system and the construction of a sinI::lacZ transcriptional fusion by single recombination using a suicide plasmid. We also show that an Rm41 sinI::lacZ fusion strain that overexpresses the SinR regulator from a vector promoter (referred to as pJNSinR) exhibits twofold more ß-galactosidase activity than the same reporter fusion in the strain that expresses wild-type levels of SinR. This strain, Rm41 sinI::lacZ, in the presence or absence of high-level expression of SinR, detects specifically AHLs such as 3-oxo-C14-HL, C16-HL, C16:1-HL, and 3-oxo-C16:1. In addition, this biosensor can also detect the long-chain AHLs produced by unrelated bacteria such as P. denitrificans and R. capsulatus. Based on these findings, we propose that Rm41 sinI::lacZ(pJNSinR) could act as a specific indicator for long-acyl chain AHLs, providing a new and valuable tool for the analysis of quorum sensing in other bacterial species.


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MATERIALS AND METHODS
 
Bacterial strains and media.
The bacterial strains used in this study are listed in Table 1. S. meliloti strains were cultured in Luria-Bertani broth supplemented with 2.5 mM CaCl2 and 2.5 mM MgSO4 (LB/MC), MGM minimal medium (11 g of Na2HPO4, 3 g of KH2PO4, 0.5 g of NaCl, 1 g of glutamate, 10 g of mannitol, 1 mg of biotin, 27.8 mg of CaCl2, and 246 mg of MgSO4 per liter), or MMGly medium (11 g of Na2HPO4, 3 g of KH2PO4, 0.5 g of NaCl, 1 g of NH4Cl, 5 ml of glycerol, 1 mg of biotin, 27.8 mg of CaCl2, and 246 mg of MgSO4 per liter). Antibiotics were added, as appropriate, at the following final concentrations: streptomycin, 500 µg/ml; neomycin, 200 µg/ml; gentamicin, 50 µg/ml; and rifampin, 50 µg/ml. A. tumefaciens NTL4(pZLR4) was cultured in LB broth (30) and in MGM minimal medium containing 50 µg of gentamicin per ml. Escherichia coli strains were grown on LB medium containing 25 µg of kanamycin/ml and 10 µg of streptomycin/ml. S. meliloti and A. tumefaciens were incubated at 30°C, while E. coli was grown at 37°C.


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  		TABLE 1. Bacterial strains and plasmids used in this work

Constructions of sinI::lacZ transcriptional fusions.
The complete sinI gene and an internal piece of sinI were cloned separately into the suicide plasmid pVIK112, containing a promoterless lacZ gene (11). The sinI gene, including its promoter, was amplified from S. meliloti chromosomal DNA by using the following primers, which contain EcoRI and XbaI restriction sites (underlined) at their respective 5' ends: 5'-GAATTCCGGGTTGCCGGCCCGGGACAGG-3' and 5'-TCTAGAGTGCCGTTTCAAGCGACATCGGG-3'. PCR was performed by using 30 cycles of 30 s at 95°C, 30 s at 70°C, and 1.30 min at 72°C. The PCR fragment was purified, digested with EcoRI and XbaI, and cloned into the suicide plasmid pVIK112, creating pVIKSinI.

A 350-bp internal piece from the sinI gene was amplified from S. meliloti chromosomal DNA by using the following primers, which contain EcoRI and XbaI restriction sites (underlined) at their respective 5' ends: 5'-GAATTCGCCAGCACCCCCAGGCCATCGAC-3' and 5'-TCTAGAGCGATGGTGACCTGGTTCGATGC-3'. PCR was performed by using 30 cycles of 30 s at 95°C, 30 s at 75°C, and 30 s at 72°C. The PCR fragment was purified, digested with EcoRI and XbaI, and cloned into the suicide plasmid pVIK112, creating pVIKSinIsub. The resulting plasmids, pVIKSin and pVIKSinIsub, containing sinI::lacZ transcriptional fusions, were then transformed into S17 {lambda}pir and transferred into a rifampin-resistant Rm1021 derivative (Rm11554) by biparental mating, where the gene was replaced by single recombination. The fusion strain contains the lacZ gene inserted after bp 369 (codon 124) of the sinI gene. S. meliloti clones carrying the chromosomal lacZ transcriptional fusions were selected by plating them on minimal medium (MGM) containing the appropriate antibiotics and 40 µg of 5-bromo-4-chloro-3-indolyl-ß-D-galactosidase (X-Gal)/ml.

Cloning of the sinR gene in a multicopy plasmid.
The sinR gene was amplified from S. meliloti chromosomal DNA by using the following primers, which contain EcoRI and XbaI restriction sites (underlined) at their respective 5' ends: 5'-GAATTCATGTTTATTATAGGGGC-3' and 5'-TCTAGAGATGGTGGGGATCAGAGCATGTCG-3'. PCR was performed by using 30 cycles of 30 s at 95°C, 30 s at 75°C, and 1 min at 72°C. The PCR fragment was purified, digested with EcoRI and XbaI, and cloned into plasmid pJN105. This broad-host-range expression vector is a pBBR-1 MCS5 derivative (13) that carries the L-arabinose-inducible E. coli araBAD promoter and the araC regulator (araC-PBAD) (23). This plasmid construction, pJNSinR, was transferred by triparental mating into an Rm41 strain containing the sinI::lacZ fusion described above (Rm11558).

Genetic manipulations.
The sinI::lacZ fusion mutation was transduced from Rm8501 into Rm8530 (an Rm1021 expR+ derivative) and Rm41, by using phages {phi}M12 and {phi}M12h1, respectively.

Preparation of crude AHL extracts.
AHL extraction was done following the technique previously described by Marketon and González (17). Briefly, 5-ml cultures were grown to early stationary phase (optical density at 600 nm [OD600] of ~2.4 for Rm1021 derivatives and OD600 of ~2.2 for Rm41 derivatives). Whole cultures were extracted twice with equal volumes of dichloromethane, and the extracts were dried and resuspended in 5 µl (Rm1021 and its derivatives) or 500 µl (Rm41 and its derivatives) of 70% (vol/vol) methanol.

TLC analysis of AHLs.
Five-microliter portions of AHL crude extracts were analyzed by reverse-phase C18-thin layer chromatography (RP-C18 TLC) using 70% (vol/vol) methanol-30% (vol/vol) water as the solvent. The detection of the different synthetic and commercial long-chain AHLs was achieved by spotting 5 nmol of each AHL and 5 µl of AHL crude extracts onto RP-C18 TLC. Once dried, the plates were overlaid with top agar containing the indicator organism and incubated overnight at 30°C.

Autoinducer bioassays.
An overnight culture of the indicator strains, NTL4(pZLR4), Rm41 sinI::lacZ (Rm11558), Rm41 sinI::lacZ(pJNSinR) (Rm11559), and Rm8530 sinI::lacZ (Rm11557), grown in LB/MC broth and the appropriate drugs was subcultured (1:100) in MGM minimal medium. After incubation for 6 h at 30°C with shaking, an overlay was prepared by mixing each culture with equal volumes of fresh medium and 1.5% (wt/vol) agar (Difco) and 80 µg of X-Gal per ml.

ß-Galactosidase assays.
Strains Rm41 sinI::lacZ (Rm11558) and Rm41 sinI::lacZ(pJNSinR) (Rm11559) were grown to log phase (OD600 of ~0.5) in 2 ml of minimal medium or LB/MC broth with or without 1% (wt/vol) L-arabinose at 30°C. The cultures were diluted 1:10 in Z buffer (0.06 M Na2HPO4, 0.04 M KCl, 0.001 M MgSO4, 0.05 M ß-mercaptoethanol) and assayed as previously described (21) to determine Miller units of activity, using o-nitrophenyl-ß-D-galactopyranoside as the substrate. Each sample was assayed in triplicate, and each experiment was repeated at least three times. AHL crude extracts from Rm1021 or Rm41 or the different synthetic and commercial long-chain AHLs were added to the medium at the time of inoculation.

AHL standards were purchased from Fluka [N-butyryl-DL-homoserine lactone (C4-HL), N-hexanoyl-DL-homoserine lactone (C6-HL), N-octanoyl-DL-homoserine lactone (C8-HL), N-dodecanoyl-DL-homoserine lactone (C12-HL), and N-tetradecanoyl-DL-homoserine lactone (C14-HL)] or synthesized [N-(3-oxo-octanoyl)-DL-homoserine lactone (oxo-C8-HL), N-(3-hydroxy-octanoyl)-DL-homoserine lactone (oxo-C8-HL), N-hexadecanoyl-DL-homoserine lactone (C16-HL), N-(3-oxo-tetradecanoyl)-DL-homoserine lactone (oxo-C14-HL), N-2-hexadecenoyl-DL-homoserine lactone (C16:1-HL), N-3-oxo-2-hexadecenoyl-DL-homoserine lactone (oxo-C16:1), and N-octadecanoyl-L-homoserine lactone (C18-HL)] as previously described (16). L-Arabinose was obtained from Sigma Chemical Co.


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RESULTS
 
The sinI::lacZ transcriptional fusion inactivates the production of sinI-encoded long-chain AHLs in S. meliloti.
Our laboratory has previously characterized the different quorum-sensing systems present in S. meliloti strains Rm1021 and Rm41 (17, 18; Marketon et al., submitted). One of them, the Sin system, is specifically responsible for the production of long-chain AHLs, including C12-HL, oxo-C14-HL, oxo-C16:1-HL, C16:1-HL, and the longest AHL molecule identified to date, C18-HL. Aware of the fact that no bioassay has been reported for the specific detection of long-chain AHLs with more than 14 carbons, we decided to construct an indicator based on the ability of S. meliloti to synthesize long-chain AHLs.

To accomplish this, we introduced two different constructions into the suicide plasmid pVIK112 (11). We cloned an intact sinI gene (including its promoter) (Fig. 1A) and an internal sinI fragment of 350 bp (Fig. 1B) upstream of a promoterless lacZ gene in pVIK112. These two resulting constructs, pVIKSinI and pVIKSinIsub, were transferred by biparental mating into Rm8501 (a lac mutant Rm1021 derivative) and integrated in the chromosome by single recombination. In the first construction, the resulting strain contains a sinI::lacZ fusion and a complete copy of the sinI gene in cis (Fig. 1A). We refer to this construction as sinI+::lacZ. In the second construction, the resulting strain has a fusion followed by a truncated allele of the sinI gene (sinI::lacZ) (Fig. 1B). The Rm8501 sinI::lacZ transcriptional fusion candidates were checked by PCR using primers from the beginning of the gene and the end of the lacZ gene. The fusions were analyzed on plates containing the appropriate drugs and X-Gal to determine the differences in lacZ gene expression. The sinI+::lacZ fusion constructs containing a copy of the complete sinI gene showed colonies with an intense blue color. However, the fusion constructs that have an internal piece of the sinI gene recombined were slightly blue, suggesting that the sinI gene was disrupted in this case. To confirm and characterize both of the sinI::lacZ transcriptional fusion constructs that we described above, we analyzed their AHL patterns. AHL extractions were performed as indicated in Material and Methods, and the extracts were separated on a reverse-phase C18-TLC plate and then overlaid with the A. tumefaciens NTL4(pZLR4) indicator strain. Figure 2, lane 2, shows that the sinI::lacZ fusion construct did not synthesize the sinI-encoded AHLs that normally migrate close to the origin of the TLC plate. However, they could still be produced by the sinI+::lacZ fusion construct, which contains the intact sinI gene (Fig. 2, lane 1).



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  		FIG. 1. Schematic representation of the mechanism used to create sinI::lacZ fusions by single recombination. (A) Integration using a fragment that contains the complete 5' end of the gene. (B) Integration using an internal fragment of the sinI gene.



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  		FIG. 2. TLC analysis of AHLs produced by sinI::lacZ fusion mutants. The TLC plates were overlaid with the A. tumefaciens indicator strain. Lane 1, Rm8501 sinI+::lacZ containing an intact sinI gene; lane 2, Rm8501 sinI::lacZ containing a disrupted sinI gene; lane 3, Rm41 wild type; lane 4, Rm41 sinI::lacZ containing a disrupted sinI gene. Each lane contains 5 µl of extract.

In addition, we transduced the sinI::lacZ transcriptional fusion into Rm8530, an Rm1021 derivative that has an intact expR gene. The expR gene encodes the ExpR regulator, a LuxR homolog. The colonies from the Rm8530 sinI::lacZ strain became flat and nonmucoid in contrast to the very mucoid colonies present in the derivative containing an intact sinI gene (Fig. 3). This phenotype has been reported previously for other Rm8530 sinI mutants and results from the fact that the Sin quorum-sensing system controls the production of the symbiotically active exopolysaccharide EPS II in S. meliloti. The control of EPS II production by the Sin system depends on the presence of a functional ExpR regulator.



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  		FIG. 3. Effect of sinI::lacZ disruption on production of the symbiotically important exopolysaccharide EPS II in an Rm8530 background (Rm1021 expR+ derivative). (A) Mucoid colonies produced by Rm8530 containing an intact sinI gene. (B) Nonmucoid colonies produced by the Rm8530 sinI::lacZ fusion strain containing a disrupted sinI gene.

In order to improve the expression of the sinI::lacZ fusions, the construction was transduced into the Rm41 strain by using phage {phi}M12h1. The Rm41 strain has been shown to produce at least 10-fold-more AHLs than Rm1021 derivatives (Fig. 2, lane 3) (19). The Rm41 sinI::lacZ fusion was tested for AHL patterns. Figure 2, lane 4, shows, as expected, that the sinI-encoded AHLs were not produced by the derivatives that contain the sinI::lacZ fusion.

sinI expression is specifically induced by long-chain AHLs.
To determine the range of AHLs that can activate the sinI::lacZ transcriptional fusion, we tested the putative indicator strains Rm41 sinI::lacZ and Rm8530 sinI::lacZ and compared their activations with that of the known A. tumefaciens NTL4(pZLR4) indicator strain. We spotted 10 µl of AHL crude extracts from both Rm41 (containing long- and short-chain autoinducer molecules) (Fig. 4A and B, spots i) and Rm41 sinI (producing only short-chain AHLs) (Fig. 4A and B, spots ii) on a TLC plate and then overlaid the plates with the indicator organisms. We confirmed that NTL4(pZLR4) is a broad-range AHL detector (Fig. 4A). However, the S. meliloti sinI::lacZ fusion strains were highly specific for the detection of long-chain AHLs (Fig. 4B, spot i), while no induction was seen in the presence of short-chain AHLs (Fig. 4B, spot ii). We also noticed that the Rm41 sinI::lacZ strain produced a greater response than Rm8530 sinI::lacZ (data not shown). This result suggests that the Rm41 sinI::lacZ strain could be used as a specific indicator for the detection of long-chain AHLs (Fig. 4B).



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  		FIG. 4. Specific detection of long-chain AHLs by the Rm41 sinI::lacZ indicator strain. Rm41 crude extract (i), which contains short- and long-chain AHLs, and Rm41 sinI crude extract (ii), which contains only short-chain AHLs, were spotted on TLC plates (10 µl of each extract). (A) Plate overlaid with A. tumefaciens NTL4(pZLR4); (B) plate overlaid with Rm41 sinI::lacZ.

Recognition of specific long-chain AHLs by the Rm41 sinI::lacZ fusion strain expressing wild-type levels of sinR.
Since sinI gene expression was induced specifically by long-chain AHLs, we sought to determine which AHLs were responsible for the activation of the fusion by loading 5 nmol of individual synthetic and commercial long-chain AHLs onto a TLC plate. Among them were AHLs whose acyl chains ranged in size from 12 to 18 carbons, including unsubstituted AHLs (C12-HL, C14-HL, C16-HL, and C18-HL), 3-oxo derivatives (3-oxo-C14-HL and 3-oxo-C16:1-HL), and molecules with double bonds (C16:1-HL) in their acyl chain. The plate was then overlaid with an Rm41 sinI::lacZ fusion culture in MGM medium containing X-Gal. We found that among the unsubstituted molecules tested, only C16-HL activated sinI::lacZ expression, while both of the 3-oxo-derivatives and the ones with double bonds were recognized by the Sin quorum-sensing system (Fig. 5).



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  		FIG. 5. Specific detection of different long-chain AHLs using the Rm41 sinI::lacZ fusion strain as an indicator organism. Ten-microliter portions of Rm41 and Rm1021 AHL crude extracts and 5 nmol of each synthetic AHL (C12-HL, C14-HL, oxo-C14-HL, C16-HL, C16:1-HL, oxo-C16:1-HL, and C18-HL) were spotted.

Activation of the sinI::lacZ fusion by specific AHLs was confirmed by directly measuring ß-galactosidase activity (Fig. 6). Ten microliters of Rm41 AHL crude extract and 5 nmol of each synthetic and commercial long-chain AHL were added individually to LB/MC cultures of Rm41 sinI::lacZ. We found that maximal induction was achieved with 3-oxo-C14-HL. We also confirmed that high levels of expression were seen in the presence of AHLs such as 3-oxo-C16:1-HL, C16:1-HL, and C16-HL. However, no induction of the sinI::lacZ fusion was observed when C12-HL, C14-HL, or C18-HL was added. Addition of up to 50 nmol of synthetic short-chain AHLs (C4, C6, C8, and OH-C8-HL) failed to activate the sinI::lacZ fusion. We did observe a slight increase (<1.5-fold) in expression of the sinI::lacZ fusion when we added 5 nmol of oxo-C8-HL. Addition of 50 nmol of this AHL did not result in further increases in fusion expression (data not shown).



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  		FIG. 6. ß-Galactosidase activity of Rm41 sinI::lacZ and Rm41 sinI::lacZ(pJNSinR) measured after addition of different long-chain AHLs. Ten-microliter portions of Rm41, R. capsulatus, and P. denitrificans crude extracts and 5 nmol of each long-chain AHL were added to 2-ml cultures of Rm41 sinI::lacZ and Rm41 sinI::lacZ(pJNSinR) in LB/MC.

Recognition of autoinducer molecules using a strain that overexpresses SinR.
Zhu et al. (38, 39) showed that a reporter fusion in A. tumefaciens WCF47, containing TraR overexpressed from a vector promoter, produces more ß-galactosidase activity than the same reporter fusion in the strain that express wild-type levels of TraR. Aware of this fact, we decided to overexpress SinR in the Rm41 sinI::lacZ background and determine whether this overexpression affects the detection of AHLs. To address this possibility, we constructed an Rm41 sinI::lacZ derivative that contains the SinR regulator cloned in plasmid pJN105 (referred to as pJNSinR) (13). This vector carries the L-arabinose-inducible E. coli araBAD promoter and the araC regulator (araC-PBAD) and has been used successfully in A. tumefaciens A136 (23). We found that in S. meliloti this vector provides control of gene expression and was regulated by the presence of L-arabinose. The highest level of induction of pJNSinR expression was reached at 1% (wt/vol) L-arabinose (data not shown).

To determine the ability of the reporter strain Rm41 sinI::lacZ(pJNSinR) to respond to long-chain AHLs, we measured the ß-galactosidase activity (Fig. 6). Ten microliters of Rm41 AHL crude extract and 5 nmol of each synthetic and commercial long-chain AHL were added individually to LB/MC cultures of Rm41 sinI::lacZ(pJNSinR) containing 1% L-arabinose. Figure 6 shows that the highest induction of this strain was achieved with 3-oxo-C14-HL, 3-oxo-C16:1-HL, C16:1-HL, and C16-HL and that no response was found in the presence of C12-HL, C14-HL, and C18-HL. This AHL induction profile is the same as that found in the reporter strain Rm41 sinI::lacZ. The sinI::lacZ fusion in strain Rm41 sinI::lacZ(pJNSinR) expressed about twofold-more ß-galactosidase than the same reporter fusion in the strain that expresses wild-type levels of SinR. The higher basal-level expression seen in Rm41 sinI::lacZ(pJNSinR) has also been reported for A. tumefaciens harboring pBBR PBAD::lacZ. Newman and Fuqua (23) proposed that it is likely due to a limit on AraC-dependent repression of PBAD activity. We do not know the reason for the elevated basal expression of the sinI::lacZ fusion in the presence of pJNSinR. It could be due to a weak response by SinR to the AHLs that remain in the sinI mutant strain or to a possible inducer-independent SinR activation.

Based on the knowledge that the induction of the PBAD promoter is affected in the presence of different nutritional supplements (23), we analyzed the expression of the sinI::lacZ fusion in minimal medium. Ten microliters of Rm41 AHL crude extract and 5 nmol of each synthetic and commercial long-chain AHL were added individually to cultures of Rm41 sinI::lacZ(pJNSinR) containing 1% (wt/vol) L-arabinose. We found that expression levels of this strain in minimal medium supplemented with mannitol (MGM) is the same as in LB/MC medium but the basal-level expression is higher (data not shown). We also tested sinI::lacZ expression in minimal medium with 0.5% (vol/vol) glycerol (MM Gly) added instead of mannitol. Figure 7 shows that the ß-galactosidase activity values are higher in this case. However, surprisingly, the difference in the expression levels in the cultures with Rm41 AHL crude extract added was only about twofold more than the basal level, in contrast to the threefold-more activity seen in LB/MC cultures. Interestingly, the reporter strain Rm41 sinI::lacZ (with or without the pJNSinR plasmid) is activated in the presence of C12-HL and C14-HL when grown in minimal medium, irrespective of the carbon source used, providing a broader detection for long-chain AHLs. The specificity for the detection of long-chain AHLs shown by the indicator strain remained unchanged irrespective of the medium (minimal or complete) used in the assay. Rm41 sinI AHL crude extracts, which contain only short-chain AHLs, did not induce any activity.



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  		FIG. 7. ß-Galactosidase activity of Rm41 sinI::lacZ(pJNSinR) in different media containing 1% L-arabinose (LB/MC and MM Gly) measured after addition of different long-chain AHLs. Rm41 and Rm41 sinI crude extracts (10 µl) and 5 nmol of the different long-chain AHLs (50 and 250 nmol for C18-HL) were added to 2-ml cultures.

Recognition of long-chain AHLs produced by unrelated bacteria.
Once we analyzed the AHL induction profiles of Rm41 sinI::lacZ and Rm41 sinI::lacZ(pJNSinR), we tested whether these indicator strains can be activated by the presence of long-chain AHL produced by unrelated bacteria. Since the detection of the long-chain AHL C16-HL produced by R. capsulatus and P. denitrificans was achieved by the use of a radioactive incorporation assay (31), we cross-streaked both putative indicator strains, Rm41 sinI::lacZ and Rm41 sinI::lacZ(pJNSinR), against these two microorganisms on LB/MC plates containing X-Gal. We found that only the indicator strain Rm41 sinI::lacZ(pJNSinR) developed a blue color in response to the AHLs made by both bacteria (Fig. 8). We also confirmed this result by measuring the ß-galactosidase activity. Ten-microliter portions of both R. capsulatus and P. denitrificans AHL crude extracts were individually added to Rm41 sinI::lacZ and Rm41 sinI::lacZ(pJNSinR) cultures. Both indicator strains were activated in the presence of these signal molecules, but they showed different levels of ß-galactosidase expression (Fig. 6). Strain Rm41 sinI::lacZ(pJNSinR) expressed around twofold-more (R. capsulatus AHLs) and threefold-more (P. denitrificans AHLs) ß-galactosidase activity than the strain that expresses wild-type levels of SinR. The ease and specific detection of the long-chain AHLs provide evidence of the potential use of Rm41 sinI::lacZ(pJNSinR) as a long-chain AHL indicator strain.



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  		FIG. 8. Detection of long-chain AHLs produced by different bacteria using Rm41 sinI::lacZ(pJNSinR) as an indicator strain. Rm41 sinI::lacZ(pJNSinR) was streaked across P. denitrificans (A), Rm1021 (B), and R. capsulatus (C) onto LB/MC plates containing X-Gal.


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DISCUSSION
 
The studies performed in our laboratory on the symbiotic relationship between the soil bacterium S. meliloti and the leguminous alfalfa plant have revealed the presence of two and three different quorum-sensing systems in the two common wild-type S. meliloti strains Rm1021 and Rm41, respectively. One of them, the Sin system, located on the chromosome of both S. meliloti strains, is responsible for the exclusive production of long-chain AHLs, including the longest acyl chain reported to date (C18-HL). A few microorganisms have been reported as long-chain AHL producers, including P. denitrificans (C16-HL), R. capsulatus (C14-HL and C16-HL) (31), Rhodobacter sphaeroides (C14:1-HL) (31), and Rhizobium leguminosarum (3-OH-C14:1) (9). The detection of the autoinducer molecules with 14 carbons in these organisms has been achieved using a P. aeruginosa strain that contains a lacZ reporter fusion (26). However, the long-chain C16-HL was hardly detected by any of the biosensors described to date, and the use of a radioactive incorporation assay was necessary for their detection (31). The difficulties in the detection of long-chain AHLs can be attributed to the fact that there are no available specific indicator organisms for them and can also be related to their low permeability through the cell membrane. It has been reported that long-chain AHLs accumulate in the cells and may need to be actively transported across the cell membrane by efflux pumps such as MexAB-OprD (27, 31). Consequently, AHL extraction in S. meliloti has always been performed using the whole culture (cells and supernatant) (17) instead of spent supernatants (2). We have identified the production of several sinI-encoded long-chain AHLs such as C12-HL, C14-HL, oxo-C14-HL, C16-HL, C16:1-HL, oxo-C16:1-HL, and C18-HL in S. meliloti strains Rm41 and Rm1021 (18; Marketon et al., submitted). The ability of S. meliloti to synthesize a broad range of long-chain AHLs makes this organism a logical candidate for the development of a new indicator strain.

In an attempt to develop a new tool for the specific detection of long-chain AHLs, we disrupted the Sin autoinducer synthase gene (sinI) by constructing a lacZ transcriptional fusion in Rm41 and Rm1021, using a suicide plasmid. The analysis of AHL extracts from these S. meliloti sinI::lacZ (Rm1021 and Rm41) derivatives by TLC confirmed that none of the sinI-encoded long-chain AHLs was made (Fig. 2, lanes 2 and 4). We selected the Rm41 sinI::lacZ derivative for further study based on the 10-fold-higher AHL production in comparison with the Rm1021 derivatives (17). The analysis of AHLs that can activate the Rm41 sinI::lacZ transcriptional fusion showed that this strain recognizes only long-chain AHLs (Fig. 4). This feature makes the strain a very specific indicator for the detection of long-chain AHLs, in contrast to the classical indicator strain NTL4(pZLR4), which can be induced by the presence of medium- and long-chain AHLs (Fig. 4) (20). In addition, we also analyzed the particular AHLs detected by the Rm41 sinI::lacZ reporter strain. The indicator strain was used as an overlay to detect different long-chain AHLs previously spotted onto a TLC plate (Fig. 5), and we also measured the ß-galactosidase activity expressed after addition of each AHL individually to Rm41 sinI::lacZ cultures (Fig. 6). In both cases, we found that higher levels of expression were achieved in the presence of 3-oxo-C16:1-HL, 3-oxo-C14-HL, C16:1-HL, and C16-HL. C12-HL and C14-HL can be detected in ß-galactosidase assays when the cultures are grown in minimal medium instead of LB/MC broth. C18-HL was not detected by the sinI::lacZ indicator strain under any of the conditions tested (Fig. 7).

In order to improve the response of the putative indicator Rm41 sinI::lacZ in the detection of long-chain AHLs, we constructed a strain that overexpresses the SinR regulator from an L-arabinose-inducible PBAD promoter (13). The expression of the PBAD promoter in A. tumefaciens A136 has been shown to be induced in the presence of 0.5% (wt/vol) arabinose and affected by other nutritional supplements present in the medium (23). We found that in S. meliloti, maximal pJNSinR expression was achieved in the presence of 1% (wt/vol) L-arabinose (data not shown). The overexpression of SinR provided an increase in the response of the indicator strain Rm41 sinI::lacZ to long-chain AHLs. The ß-galactosidase activity in LB/MC cultures increased about twofold more in comparison with the values shown by the Rm41 sinI::lacZ strain, which contains wild-type levels of SinR (Fig. 6). We also analyzed the effects of different nutritional supplements on sinI::lacZ expression in the Rm41 sinI::lacZ (pJNSinR) indicator and found that the highest ß-galactosidase activity was in minimal medium supplemented with 1% (wt/vol) arabinose (Fig. 7). Interestingly, the long-chain AHL induction profile shown with the Rm41 sinI::lacZ indicator strain was very similar to the profile shown by the Rm41 sinI::lacZ(pJNSinR) derivative, which overexpresses the SinR regulator in LB/MC cultures (Fig. 6). The specificity for the detection of long-chain AHLs was also demonstrated in cultures grown in minimal medium. The short-chain AHLs present in the Rm41 sinI AHL crude extract did not induce any activity in either the Rm41 sinI::lacZ or Rm41 sinI::lacZ(pJNSinR) strain. In contrast, it has been shown that the overexpression of a LuxR-type regulator can affect the specificity for the detection of AHLs in organisms such as E. coli (25) and A. tumefaciens (39). Recently, Zhu et al. (38) developed an ultrasensitive indicator strain of A. tumefaciens (WCF47) that overproduces the TraR regulator protein by using the T7 expression system. This biosensor strain has the ability to detect with extreme sensitivity (picomolar concentrations) a broad range of autoinducers ranging in sizes from C4-HL to C18-HL.

In this report, we also show evidence that the putative indicator strain Rm41 sinI::lacZ(pJNSinR) is able to detect long-chain AHLs produced by unrelated bacteria (Fig. 6). For instance, the autoinducer compounds made by P. denitrificans (C16-HL) and R. capsulatus (C14-HL and C16-HL) have been previously detected only by radioactive incorporation techniques (31). This indicator strain, in spite of the light blue color background, can detect long-chain AHL producers by simple cross-streaking, resulting in the development of a dark blue color (Fig. 8). Furthermore, the induction of the indicator Rm41 sinI::lacZ(pJNSinR) by the presence of AHL crude extracts from long-chain AHL producers could easily be measured by ß-galactosidase assays (Fig. 6).

In conclusion, we propose that strain Rm41 sinI::lacZ(pJNSinR) can be used as an indicator for the exclusive detection of long-chain AHLs ranging in size from C12-HL to C16-HL. This indicator can be induced by a broad range of long-chain autoinducer molecules, including those molecules lacking substituent groups or substituted in the third carbon with an oxo group (oxo-C14) or with double bonds (C16:1 and oxo-C16:1). These findings suggest that S. meliloti will serve as a tool for future detection of long-chain-AHL-producing microorganisms.


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ACKNOWLEDGMENTS
 
We thank the members of our laboratory for their helpful discussions and critical reading of the manuscript. We also thank Peter Greenberg, Clay Fuqua, and Stephen Winans for providing strains and Anatol Eberhard for providing the synthetic AHLs used in this study.

This work was supported by National Science Foundation grant MCB-9733532 to J.E.G. and the Texas Advanced Research Program under grant 009741-0022-2001 to J.E.G. I.L. was supported by a postdoctoral grant from the Scientific Foundation Ramón Areces.


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FOOTNOTES
 
* Corresponding author. Mailing address: FO 3.1, Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, TX 75083-0688. Phone: (972) 883-2526. Fax: (630) 604-3093. E-mail: jgonzal{at}utdallas.edu. Back


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Applied and Environmental Microbiology, June 2004, p. 3715-3723, Vol. 70, No. 6
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.6.3715-3723.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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