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Applied and Environmental Microbiology, January 2007, p. 535-544, Vol. 73, No. 2
0099-2240/07/$08.00+0     doi:10.1128/AEM.01451-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Synthesis of N-Acyl Homoserine Lactone Analogues Reveals Strong Activators of SdiA, the Salmonella enterica Serovar Typhimurium LuxR Homologue ^,

Joost C. A. Janssens,^1,2 Kristine Metzger,¹ Ruth Daniels,¹ Dave Ptacek,¹ Tine Verhoeven,¹ Lothar W. Habel,² Jos Vanderleyden,¹ Dirk E. De Vos,² and Sigrid C. J. De Keersmaecker^1*

Centre of Microbial and Plant Genetics, Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, B-3001 Leuven, Belgium,¹ Centre for Surface Chemistry and Catalysis, Katholieke Universiteit Leuven, Kasteelpark Arenberg 23, B-3001 Leuven, Belgium²

Received 23 June 2006/ Accepted 27 October 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
N-Acyl homoserine lactones (AHLs) are molecules that are synthesized and detected by many gram-negative bacteria to monitor the population density, a phenomenon known as quorum sensing. Salmonella enterica serovar Typhimurium is an exceptional species since it does not synthesize its own AHLs, while it does encode a LuxR homologue, SdiA, which enables this bacterium to detect AHLs that are produced by other species. To obtain more information about the specificity of the ligand binding by SdiA, we synthesized and screened a limited library of AHL analogues. We identified two classes of analogues that are strong activators of SdiA: the N-(3-oxo-acyl)-homocysteine thiolactones (3O-AHTLs) and the N-(3-oxo-acyl)-trans-2-aminocyclohexanols. To our knowledge, this is the first report of compounds (the 3O-AHTLs) that are able to activate a LuxR homologue at concentrations that are lower than the concentrations of the most active AHLs. SdiA responds with greatest sensitivity to AHTLs that have a keto modification at the third carbon atom and an acyl chain that is seven or eight carbon atoms long. The N-(3-oxo-acyl)-trans-2-aminocyclohexanols were found to be less sensitive to deactivation by lactonase and alkaline pH than the 3O-AHTLs and the AHLs are. We also examined the activity of our library with LuxR of Vibrio fischeri and identified three new inhibitors of LuxR. Finally, we performed preliminary binding experiments which suggested that SdiA binds its activators reversibly. These results increase our understanding of the specificity of the SdiA-ligand interaction, which could have uses in the development of anti-quorum-sensing-based antimicrobials.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacteria are able to communicate with each other by means of different classes of signal molecules. In general, each bacterial cell produces a small amount of one or more signal molecules, which are subsequently released into the environment. When the total amount of the signal molecule(s) increases, the concentration reaches a detection limit, causing activation or repression of certain target genes. This mechanism, which enables the population to respond collectively to a changing environment, is referred to as quorum sensing (15, 41, 43, 46).

In gram-negative bacteria, the classical QS system is the LuxR/LuxI system of Vibrio fischeri, in which LuxI is the signal synthase that synthesizes N-(3-oxo-C₆)-L-homoserine lactone, while LuxR is the signal receptor (13). LuxI homologues in other bacterial species synthesize different N-acyl homoserine lactones, which have various acyl chain lengths (C₄ to C₁₈), degrees of saturation, or modifications at the third carbon of the acyl chain (20, 43). Following the interaction with the AHL signal molecule, the LuxR homologue is activated or inhibited as a transcription factor which modulates expression of the target genes, thereby regulating a number of important biological functions, including bioluminescence, biofilm formation, and virulence (15, 23). Indeed, many pathogens, such as Pseudomonas aeruginosa, Burkholderia cenocepacia, and Erwinia carotovora, regulate the expression of virulence factors required for pathogenicity via AHL-mediated QS systems (35, 36, 44).

Species of the genera Salmonella, Escherichia, and Klebsiella are a unique case, since they encode a LuxR homologue, designated SdiA, while they do not have a LuxI homologue or a member of another family of AHL synthases (1). It has been shown that the pathogen Salmonella enterica serovar Typhimurium does not produce detectable concentrations of AHLs but uses SdiA to detect AHLs produced by other species (22, 32). SdiA subsequently activates two Salmonella-specific loci, srgE (SdiA-regulated gene), which is carried in the chromosome and has an unknown function, and the rck (resistance to complement killing) operon, which is located on the Salmonella virulence plasmid (2, 32). Interestingly, three genes in the rck operon play a role in adhesion to host tissues and resistance to complement killing (1). Until now, these two loci have been the only known targets of SdiA. Michael et al. showed that a plasmid-based Prck-lux fusion (pBA428) could be activated in an sdiA-dependent manner in response to synthetic AHLs and that 3O-C₆-HSL and 3O-C₈-HSL were the strongest activators at concentrations of 1 to 10 nM (22). Consistent with these results, Smith and Ahmer reported that a plasmid-encoded PsrgE-lux fusion (pJNS25) is responsive to C₆-HSL and 3O-C₈-HSL (32). Despite the regulation of genes that presumably play a role in host interactions, the exact biological function of SdiA in Salmonella remains unclear. Some authors have suggested that SdiA may have evolved into a system for detecting acidic environments and responding to iron, although the reasons for this are currently unknown (28, 40).

Until now, there have been no reports concerning the Salmonella SdiA response to molecules other than the unsubstituted AHLs and the 3O-AHLs with even numbers of carbon atoms in their chains that were used by Michael et al. (22). However, there have been several reports concerning the activity of synthetic AHL analogues with LuxR of V. fischeri and the LuxR homologues of Aeromonas hydrophila, Agrobacterium tumefaciens, Chromobacterium violaceum, E. carotovora, and P. aeruginosa (7, 9, 14, 17, 21, 24, 26, 30, 37, 52). These studies generated substantial information about the structure-function relationships of AHL signal molecules and showed that the most potent activators of all of the LuxR homologues studied were the cognate AHLs. Small changes in the acyl chain reduced the activating capacity of the compounds, and most modifications of the homoserine lactone ring totally deactivated the molecules. Interestingly, a change in the HSL structure to a homocysteine thiolactone ring is permitted in some QS systems, while such a change deactivates the molecule in other systems (14, 21, 24). Smith et al. identified some strong activators of the QS systems of P. aeruginosa by modification of the HSL ring structure of 3O-C₁₂-HSL (33, 34). However, until now, there have been no reports concerning a really strong agonist, a molecule which is detected by a LuxR-type receptor at concentrations lower than the concentration of the native AHL signal.

Since timing is an important issue in pathogen infection, molecules that activate the expression of virulence factors too early might be able to attenuate the virulence of the pathogen. In addition to inhibitors, strong activators of QS might be interesting tools for interfering with the QS systems of pathogens. Since the exact relationship between SdiA and virulence in Salmonella is still unclear, we are interested in both activators and inhibitors of SdiA.

To obtain more information about the structure-activity relationship of AHL analogues, we synthesized and screened a limited library of AHLs and AHL analogues to determine their activities with LuxR of V. fischeri and SdiA of S. enterica serovar Typhimurium. We were able to identify two types of AHL analogues that are strong activators of SdiA, as well as three new inhibitors of LuxR. We show here that SdiA indeed has exceptional properties since it is more sensitive to N-(3-oxo-acyl)-homocysteine thiolactones than to AHLs, while 3O-C₆-HTL showed little activity with LuxR. The second class of SdiA activators, the N-(3-oxo-acyl)-trans-2-aminocyclohexanols, was found to be more stable than the lactones. We also describe preliminary results indicating that SdiA binds its activators reversibly.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abbreviations.
QS, quorum sensing; LB, Luria-Bertani; EC₅₀, concentration required for half-maximal induction of the reporter strain; X-Gal, 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside; TLC, thin-layer chromatography; AHL, N-acyl homoserine lactone; HSL, homoserine lactone; HTL, homocysteine thiolactone; ACH, trans-2-aminocyclohexanol; AHTL, N-acyl homocysteine thiolactone; ANT, anthranilamide; 3O-AHL, N-(3-oxo-acyl)-homoserine lactone; 3O-AHTL, N-(3-oxo-acyl)-homocysteine thiolactone. The length of the acyl chain is indicated as follows: C_n, where n is the number of carbon atoms in the chain [e.g., 3O-C₁₀-HSL is N-(3-oxo-decanoyl)-homoserine lactone].

Bacterial strains, plasmids, and media.
The bacterial strains used in this study were A. tumefaciens NT1 (42), Escherichia coli JM109 (48), E. coli DH5, S. enterica serovar Typhimurium wild-type strain 14028 (American Type Culture Collection), and the isogenic sdiA mutant S. enterica serovar Typhimurium BA612 (2). The plasmids used were pBA428 (Prck-luxCDABE) (22), pJM749 (tra-lacZ749) (25), pJNS25 (PsrgE-luxCDABE) (32), pMIR101 (aiiA_soil) (6), pSB401 (luxR⁺ PluxI-luxCDABE) (45), and pSVB33 (traR) (25). S. enterica serovar Typhimurium and E. coli were grown with aeration at 37°C in Luria-Bertani medium (29) or on LB medium plates containing 1.5% agar (Invitrogen) unless indicated otherwise. A. tumefaciens was grown with aeration at 30°C in TY medium (3) or on TY plates containing 1.5% agar. Carbenicillin, kanamycin, and tetracycline were used at concentrations of 100, 50, and 10 µg/ml, respectively, when appropriate.

Synthesis of AHLs and analogues.
The unsubstituted AHLs compounds 10 to 16 (Table 1) were purchased from Sigma-Aldrich, DL-homocysteine thiolactone hydrochloride (compound 27) (Table 2) was purchased from Acros Organics, and the fatty acids (compounds 22 and 23) were purchased from Janssen Chimica. Compounds 1 to 7, 17 to 21, 24 to 26, 28, and 29 were synthesized as previously described (8), and the overall yields were 5 to 70%. The 3-hydroxy derivatives compounds 8 and 9 were synthesized by reduction of the 3-oxo derivatives compounds 5 and 6 using the procedure of Chhabra et al. (9) except for the final extraction step, in which dichloromethane/water (1:1, vol/vol) was used instead of ethyl acetate. All compounds were purified by preparative chromatography on a silica gel column using ethyl acetate or dichloromethane/ethanol (95:5, vol/vol) as the eluent. Nuclear magnetic resonance spectra were recorded with a Bruker AMX 300 at 300 MHz (¹H) and 75.5 MHz (¹³C). Analytical details for the new molecules compounds 19 to 21 can be found in the supplemental material. All synthesized compounds were stored as dry powder at –20°C, remained stable for at least 2 years, and were added to the media as dilutions from 10 mM stock solutions in acetonitrile. Using breathable sealing membranes (BREATHSEAL; Greiner Bio-One N.V.) during incubation, the final concentration of acetonitrile could be as high as 9% (vol/vol) without influencing the growth or the AHL response of the reporters.

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TABLE 1. Chemical structures, abbreviations, and EC50 values of compounds in the reporter strains S. enterica serovar Typhimurium 14028/pJNS25, S. enterica serovar Typhimurium 14028/pBA428, and E. coli JM109/pSB401

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TABLE 2. Chemical structures, abbreviations, and EC50 values of compounds in the reporter strains S. enterica serovar Typhimurium 14028/pJNS25 and S. enterica serovar Typhimurium 14028/pBA428

Dose-response experiments.
The inducing activities of the compounds were monitored using the E. coli JM109/pSB401, S. enterica serovar Typhimurium 14028/pBA428, and S. enterica serovar Typhimurium 14028/pJNS25 biosensor strains. Starting with a 10 mM stock solution of each compound in acetonitrile, threefold serial dilutions in 100 µl LB broth per well were prepared in triplicate in sterile white microplates (Cliniplate; Thermo Life Sciences). Subsequently, ca. 10⁹ bacteria from an overnight culture of the biosensor strain were inoculated into 10 ml (for E. coli) or 100 ml (for S. enterica serovar Typhimurium) of LB broth, and 100 µl was added to each well of the microplates, resulting in a total of 200 µl medium per well. The microplates were covered with Breathable Sealing Membranes (Greiner Bio-One N.V.) and incubated with aeration for 6 h at 37°C (for S. enterica serovar Typhimurium) or for 4 h at 30°C (for E. coli). Following this incubation, the luminescence (in counts/second of light) was determined using a charge-coupled device camera (Berthold Night Owl; PerkinElmer Life Science), and the optical density at 620 nm was determined with a microtiter plate reader (VERSAmax; Molecular Devices). Gene expression was normalized by dividing the luminescence value by the A₆₂₀ value of each sample.

Some preliminary experiments with strain 14028/pJNS25 were performed with LB semisolid agar (0.3% agar) as previously described (22). Liquid LB medium was used in all further experiments, because although the maximal response in agar was greater, the liquid medium was more effective because it was easier to handle and it did not change the structure-activity relationship (32).

It should be noted that vigorous shaking for 2 h was required to dissolve C₁₂-HSL (compound 15) and C₁₄-HSL (compound 16) in acetonitrile. Although no precipitation of the compounds was observed upon dilution of these stock solutions into LB broth, the lower activities of these compounds might have been due to their lower solubilities in the aqueous medium.

Competition assays.
The abilities of the compounds to inhibit the induction of bioluminescence by 3O-C₆-DL-HSL (compound 3) or 3O-C₈-DL-HSL (compound 5) were determined using a protocol similar to the protocol used in the dose-response experiments described above, with following modification. Together with the compound tested, compound 3 or 5 was added to each well of the microplate at the concentration required for half-maximal induction of the reporter strain (Table 1).

TLC.
Thin-layer chromatography (TLC) analyses were performed as previously described (31). Briefly, 1-µl samples were separated by TLC on analytical C₁₈ reversed-phase TLC plates. The plates were developed with methanol-water (60:40, vol/vol), and the active compound was detected by using an overlay of soft agar with 20 µg/ml X-Gal and the A. tumefaciens NT1/pJM749,pSVB33 biosensor, which was grown for 3 days at 30°C (25). After detection, the plates were dried, and the images were archived with a digital camera.

Lactonase experiments.
Lactonase sensitivity tests were performed as previously described (6). Briefly, ca. 10⁶ E. coli DH5/pMIR101 cells from an overnight culture, which produced the lactonase AiiA_soil, were inoculated into 1 ml of fresh medium containing the compound tested at a concentration of 25 µM. To prevent ring opening under alkaline conditions (5, 50), the medium was buffered to pH 6.5 with 15 mM KH₂PO₄/K₂HPO₄. After incubation at 25°C for 2 h, 6 h, and 24 h, 10-µl portions of the culture medium were spotted onto TLC plates for detection of activity as described above. In addition, the culture medium was centrifuged for 5 min at 3,000 x g, the cell-free supernatant was diluted 1/5 in LB medium, and the ability of the supernatant to activate the bioreporter S. enterica serovar Typhimurium 14028/pJNS25 was assayed. As positive controls, samples not inoculated with lactonase-expressing E. coli were used in all assays.

pH stability experiments.
Buffered aqueous solutions with a range of pH values (pH 6 to 12) were prepared as previously described (27). The compounds tested were dissolved in these solutions at a final concentration of 10 mM and incubated at 37°C for 1 h. Portions (2 µl) of the solutions were spotted on a TLC plate (0.2 mm of silica gel on aluminum; Fluka) and developed with 100% ethyl acetate. TLC spots were visualized as described in the supplemental material. Since the differences between the migration distances of the open and closed forms of the AHLs and the AHTLs on the TLC plate were large (more than 0.3), this was a fast and convenient method for determination of the pH sensitivity of lactones. In addition, the solutions were spotted on a C₁₈ reversed-phase TLC plate and assayed with the A. tumefaciens NT1/pJM749,pSVB33 biosensor strain as described above.

Preparation of cell lysates and extraction of intracellular compounds.
In general, preparation of cell lysates and extraction of intracellular compounds were performed as described by Luo et al. (18). Briefly, ca. 10⁸ cells from overnight cultures of S. enterica serovar Typhimurium strains 14028/pJNS25 and BA612/pJNS25 were inoculated into 10 ml LB medium containing the activating compound at a final concentration of 100 µM. After incubation for 6 h at 37°C, cells were harvested by centrifugation at 2,000 x g for 5 min and extensively washed five times with 10 ml fresh LB medium. The supernatant from each washing step was separated on a TLC plate. After the final wash, portions of the cultures were diluted into fresh LB medium and incubated for 13 h. At 0, 4, and 13 h after the final wash, the cells were collected by centrifugation, and each pellet was resuspended in 200 µl lysis buffer (18). Cells were disrupted by sonication, and cell debris was removed by centrifugation at 50,000 x g for 15 min at 4°C. Lysates of unwashed 14028/pJNS25 cells were used as positive controls in all experiments. The cell lysates were extracted twice with 200 µl ethyl acetate (acidified with acetic acid), and the organic phases were dried via evaporation. The residues were dissolved in 25 µl ethyl acetate and diluted 1/1,000 in LB medium containing the bioreporter strain S. enterica serovar Typhimurium 14028/pJNS25. Bioluminescence was measured after 6 h of incubation at 37°C.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Synthesis of AHLs and analogues.
To study the relationship between the structure of AHL analogues and their activities with the Salmonella LuxR homologue, SdiA, a limited library of 23 synthetic AHLs and AHL analogues (compounds 1 to 23) was created (Table 1). AHLs and 3O-AHLs with even numbers of carbon atoms in their side chains were previously screened to determine their inducing activities with the S. enterica serovar Typhimurium reporter 14028/pBA428 by Michael et al. (22). Besides these compounds, three AHLs with odd numbers of carbon atoms in their side chains (compounds 2, 4, and 12) and two 3-hydroxy-AHLs (compounds 8 and 9) were included. The synthesis of the AHL analogues compounds 17 to 21 was based on previous reports. Compound 17 has been reported to be a strong inhibitor of the 3O-C₆-HSL (compound 3) activity with LuxR of V. fischeri (7, 26). Since 3O-C₆-HSL (compound 3) is also a strong activator of the 14028/pBA428 reporter (22), compound 17 was included in our tests. In the AHL analogues compounds 18 to 21, the HSL ring structure of 3O-C₆-HSL (compound 3) was replaced by other structures based on the report of Smith et al. (34). Of special interest is the replacement of HSL by homocysteine thiolactone, since 3O-C₆-HTL (compound 18) has antagonizing effects on the LuxRI system of V. fischeri (14), while 3O-C₁₂-HTL (compound 26) is an activator of the LasRI system of P. aeruginosa (24, 34). One might therefore hypothesize that 3O-C₆-HTL (compound 18) could be an activator or an inhibitor of SdiA. Finally, hexanoic acid (compound 22) and octanoic acid (compound 23) were included to investigate the possible effects of fatty acids on SdiA.

Inducing activity with LuxR and SdiA.
Compounds 1 to 23 were first screened in dose-response experiments to determine their abilities to activate the known AHL reporter system of E. coli JM109/pSB401, which contains LuxR of V. fischeri (26, 45). Subsequently, the molecules were screened to determine their activities with SdiA using the S. enterica serovar Typhimurium reporter strains 14028/pJNS25 (containing a PsgrE-lux fusion) and 14028/pBA428 (containing a Prck-lux fusion).

Table 1 shows the EC₅₀ of each molecule with the three reporter strains. The 3O-AHLs with chains between six and eight carbon atoms long (compounds 3 to 5) were the strongest activators of E. coli JM109/pSB401. All other modifications, including replacement of the HSL ring with an HTL ring structure, resulted in a strong decrease in activity with V. fischeri LuxR.

The observed activities (Table 1) with the S. enterica serovar Typhimurium reporters was SdiA dependent since none of the molecules was able to activate the isogenic sdiA mutant strains BA612/pJNS25 and BA612/pBA428 (data not shown). Although the reporter 14028/pJNS25 is more sensitive than 14028/pBA428, the relative activities of the molecules were similar with these two reporter strains. Table 1 shows that 3O-AHLs with chains between six and eight carbon atoms long (compounds 3 to 5) were the most active AHLs, confirming the results of Michael et al. (22). SdiA was also strongly activated by unsubstituted AHLs with chains between six and eight carbon atoms long (compounds 11 to 13), while the presence of a 3-hydroxyl substituent (compounds 8 and 9) instead of a 3-oxo group (compounds 5 and 6) resulted in a distinct decrease in activity. The AHL analogues compounds 17 and 19 exhibited good activities, while high concentrations (µM range) of compounds 20 and 21 were required to activate the reporters. The fatty acids (compounds 22 and 23) did not show activity at the highest concentration tested (300 µM). However, an interesting result was obtained with 3O-C₆-HTL (compound 18), which activated SdiA at a 2.5-fold-lower concentration than its HSL counterpart, 3O-C₆-HSL (compound 3), activated SdiA.

N-(3-Oxo-acyl)-homocysteine thiolactones are stronger activators of SdiA than N-(3-oxo-acyl)-homoserine lactones.
In the first series of experiments we identified 3O-C₆-HTL (compound 18) and 3O-C₆-ACH (compound 19) as good activators of SdiA. Since neither of these compounds contained an HSL ring structure, their activities were remarkable, and we decided to proceed by studying the activities of these modifications with SdiA. Therefore, some additional N-(3-oxo-acyl)-homocysteine thiolactones (compounds 24 to 26) and N-(3-oxo-acyl)-2-aminocyclohexanols (compounds 28 and 29) were synthesized. Table 2 and Fig. 1A show that 3O-C₇-ACH (compound 28) and 3O-C₈-ACH (compound 29) were both stronger activators of the reporters 14028/pJNS25 and 14028/pBA428 than 3O-C₆-ACH (compound 19), but they exhibited less activity than their HSL counterparts (compounds 4 and 5). Figure 1A shows that the activities of all synthesized N-(3-oxo-acyl)-2-aminocyclohexanols (compounds 19, 28, and 29) at a concentration of 1 µM were comparable to the activity of 1 µM 3O-C₆-HSL (compound 3).

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FIG. 1. Dose-response curves showing the activities with the reporter strain S. enterica serovar Typhimurium 14028/pJNS25. (A) x, acetonitrile; , 3O-C6-HSL (compound 3); , , and , chemically synthesized N-(3-oxo-acyl)-trans-2-aminocyclohexanols with chains that are six carbon atoms long (compound 19), seven carbon atoms long (compound 28), and eight carbon atoms long (compound 29), respectively. (B) , , and +, chemically synthesized 3O-AHLs (dotted lines) and 3O-AHTLs (solid lines) with chains that are 6 carbon atoms long (compounds 3 and 18), 8 carbon atoms long (compounds 5 and 25), and 12 carbon atoms long (compounds 7 and 26), respectively. The standard deviations are not shown for clarity but did not exceed 11% of the means. The data are the results of one experiment that was representative of five independent replicates. The gene expression was normalized by dividing the luminescence value by the A620 of each sample. RLU, relative light units.

Interestingly, the 3O-AHTLs compounds 24 to 26 were all 2.5-fold-stronger activators of SdiA than their HSL counterparts, compounds 4, 5, and 7, respectively (Table 2). The dose-response curves for three 3O-AHTLs with chains 6, 8, and 12 carbon atoms long (compounds 18, 25, and 26) and the corresponding 3O-AHLs (compounds 3, 5, and 7) with the 14028/pJNS25 reporter are shown in Fig. 1B. The dose-response curves for 3O-C₇-HSL (compound 4) and 3O-C₇-HTL (compound 24) are comparable to those for the 3O-C₈ derivatives compounds 5 and 25 (data not shown). Since DL-homocysteine thiolactone (compound 27) itself did not exhibit activity at the highest concentration tested (300 µM), we concluded that the acyl chain is crucial for the activity of the AHTLs. Of all molecules that have been tested, 3O-C₇-HTL (compound 24) and 3O-C₈-HTL (compound 25) are the strongest activators of SdiA.

N-(3-Oxo-acyl)-2-aminocyclohexanols are less sensitive to lactonase and alkaline pH conditions than 3O-AHLs and 3O-AHTLs.
It has been shown previously that AHL molecules undergo lactonolysis under alkaline conditions (5, 50) and are degraded by lactonases, such as AiiA_204B1 from Bacillus sp. strain 204B1 (11, 12). Therefore, the 3O-AHTLs (compounds 18, 24, and 25) and the ACH derivatives (compounds 19, 28, and 29) could be particularly interesting as SdiA activators if they prove to be less sensitive to alkaline conditions and lactonases.

As described in Materials and Methods, a series of experiments were performed to determine the sensitivities of 3O-C₇-HSL (compound 4), 3O-C₇-HTL (compound 24), and 3O-C₇-ACH (compound 28) to the lactonase AiiA_soil, a Bacillus sp. AiiA_204B1 homologue (6). Figure 2 shows that the activities of 3O-C₇-HSL (compound 4) and 3O-C₇-HTL (compound 24) decreased 80% and 55%, respectively, after 24 h of incubation in the presence of lactonase. As expected, since it does not contain a lactone moiety, 3O-C₇-ACH (compound 28) remained as active as a control without lactonase. Similar results were obtained for molecules having other chain lengths (data not shown). From these experiments, we concluded that N-(3-oxo-acyl)-2-aminocyclohexanols are not degraded by AiiA_soil, while the 3O-AHTLs are sensitive to lactonase degradation. The inactivation of 3O-AHTLs by a Bacillus sp. lactonase has not been described previously.

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FIG. 2. Degradation of lactone and thiolactone structures in the presence of the lactonase AiiAsoil. 3O-C7-HSL (compound 4), 3O-C7-HTL (compound 24), and 3O-C7-ACH (compound 28) at a concentration of 25 µM were incubated in the absence (100% activity) (open bars) or presence (shaded bars) of AiiAsoil-producing E. coli DH5/pMIR101. After 24 h, the cultures were centrifuged, and each cell-free supernatant was assayed to determine its ability to activate the bioreporter S. enterica serovar Typhimurium 14028/pJNS25. The data are the results of one experiment that was representative of two independent repeats, and the error bars indicate standard deviations for nine measurements.

We subsequently conducted a set of experiments in which the pH sensitivities of 3O-C₇-HSL (compound 4), 3O-C₇-HTL (compound 24), and 3O-C₇-ACH (compound 28) were compared. For this, pure compounds were dissolved in buffered aqueous solutions with different pH values and analyzed using a hydrophilic TLC to determine the intactness of the structure. Figure 3 shows that 3O-C₇-HSL (compound 4) and 3O-C₇-HTL (compound 24) were both completely degraded after 1 h of incubation at 37°C in solutions with pH values greater than 8. This result is in accordance with previously reported data for 3O-C₆-HSL, which becomes unstable at pH values between 7 and 8 (5, 50). As expected from its structure, 3O-C₇-ACH (compound 28) remained intact at high pH values (Fig. 3). The activity of all molecules with closed ring structures and the inactivity of the opened structures of the lactones were confirmed on a C₁₈ reversed-phase TLC plate using the biosensor strain A. tumefaciens NT1/pJM749/pSVB33 (data not shown).

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FIG. 3. Degradation of lactone and thiolactone structures at alkaline pH values. Aqueous solutions (10 mM) of pure 3O-C7-HSL (compound 4), 3O-C7-HTL (compound 24), and 3O-C7-ACH (compound 28) at different pH values were incubated for 1 h at 37°C, spotted on a hydrophilic TLC plate, and developed with 100% ethyl acetate. The spots were visualized as described in the supplemental material. The results are the results for 3O-C7-HSL (compound 4) (lanes 2 and 3) and 3O-C7-HTL (compound 24) (lanes 5 and 6) after incubation at pH 8 and 9, respectively, as well as the results for 3O-C7-ACH (compound 28) after incubation at pH 8, 9, and 12 (lanes 8, 9, and 10). As positive controls, 10 mM stock solutions of the molecules in acetonitrile were run in parallel (lanes 1, 4, and 7). Since the opened lactone and thiolactone structures had higher polarity, they migrated more slowly than the intact compounds and remained at the bottom of the plate. The results show that 3O-C7-HSL (compound 4) and 3O-C7-HTL (compound 24) were both completely degraded at pH 9, while 3O-C7-ACH (compound 28) remained active at pH 12. Similar results were obtained after 24 h of incubation. The results are representative of three independent repeats.

These results indicate that changing the HSL ring structure of an AHL into an ACH structure results in a strong SdiA activator which is less sensitive to lactonase and alkaline conditions.

Inhibitor activity with LuxR and SdiA.
Some of the compounds that showed little or no activity during the induction experiments with LuxR and SdiA were screened to determine their activities as inhibitors. In the first set of experiments, these molecules (compounds 1, 8, 14 to 17, and 20 to 23) were tested in competition assays with E. coli JM109/pSB401 to determine their abilities to inhibit the activity of 40 nM 3O-C6-HSL (compound 3). As reported by Reverchon et al. (26), compound 17 is a strong inhibitor of LuxR, since 10 µM compound 17 decreases the activity of 40 nM 3O-C₆-HSL by 75% (Fig. 4). The inhibiting activities of 3O-C₄-HSL (compound 1) and C₁₀-HSL (compound 14) with LuxR were reported previously (14, 30), whereas 3OH-C₈-HSL (compound 8), C₁₂-HSL (compound 15), and 3O-C₆-ANT (compound 20) were identified as new inhibitors of LuxR (Fig. 4). 3OH-C₈-HSL (compound 8) was a strong inhibitor, since 10 µM 3OH-C₈-HSL decreased the activity of 40 nM 3O-C₆-HSL by 70%. 3O-C₆-ANT (compound 20) was the weakest inhibitor, but its activity is interesting since 3O-C₁₂-ANT has been reported to be an inhibitor of LasR (34). C₁₄-HSL (compound 16) showed little activity, with 40 µM C₁₄-HSL inhibiting the activity of 40 nM 3O-C₆-HSL by 20%, while compounds 21 to 23 at a concentration of 40 µM had no activity under the conditions used (data not shown).

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FIG. 4. Inhibiting effects of several compounds on E. coli JM109/pSB401, activated by 40 nM 3O-C6-HSL (100%). The concentrations required for 50% inhibition are indicated in parentheses. The experiments were done in triplicate, and the standard deviations (not shown) did not exceed 9% of the means.

Exploratory inhibition experiments with the reporter S. enterica serovar Typhimurium 14028/pJNS25 using the molecules that were weak activators of SdiA, however, did not reveal an SdiA inhibitor (data not shown).

SdiA does not retain detectable amounts of the activating compounds after extensive washing.
It has been reported that TraR, the LuxR analogue of A. tumefaciens, binds its native signal molecule in an irreversible way, while LuxR binds 3O-C₆-HSL reversibly (18, 38, 53). Since none of the compounds tested showed any inhibiting effect on S. enterica serovar Typhimurium 14028/pJNS25, we speculated that SdiA might bind in an irreversible manner with the activating molecule. To test this hypothesis, an assay was performed to investigate the ability of SdiA to retain 3O-C₆-HSL (compound 3) after removal of this molecule from the environment, as described in Materials and Methods. During washing of the cells, the amount of extracellular 3O-C₆-HSL (compound 3) decreased, and the compound could not be detected after the fifth washing step (Fig. 5A). To assay the remaining intracellular levels of the compounds, the cell pellets obtained after the final washing step were lysed and extracted with ethyl acetate. Portions of the cells that were washed five times were reincubated and lysed after 4 and 13 h. Figure 5B shows that none of the extracts was able to activate the S. enterica serovar Typhimurium 14028/pJNS25 reporter more strongly than the negative control, indicating that SdiA is unable to retain detectable amounts of 3O-C₆-HSL (compound 3) after extensive washing and extraction of possible remaining molecules. Similar results were obtained for C₆-HSL (compound 11) and 3O-C₆-HTL (compound 18) (data not shown).

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FIG. 5. Analysis of the intracellular AHL remaining after extensive washing. S. enterica serovar Typhimurium strains 14028/pJNS25 and BA612/pJNS25 were incubated for 6 h in the presence of 100 µM 3O-C6-HSL (compound 3). The cells were harvested and extensively washed five times with fresh LB medium. (A) Samples of the cell-free supernatant of 14028/pJNS25 obtained after each washing step were separated on a TLC plate and subsequently analyzed using an A. tumefaciens NT1/pJM749/pSVB33 AHL biosensor overlay. Lanes 1 to 5 show that the amount of signal decreased with extensive washing and almost disappeared after the fifth washing step. (B) After the final wash, the cells were lysed, and extracts of the lysates were assayed to determine the AHL activities using the bioreporter S. enterica serovar Typhimurium 14028/pJNS25 (0 h). A portion of the cells that were washed five times was resuspended in fresh LB medium and incubated for an additional 13 h. Samples were taken after 4 h and 13 h and lysed, and extracts were analyzed to determined their AHL activities. Lysates of unwashed 14028/pJNS25 cells were used as positive controls in all experiments. The data show the AHL activities of lysates of the sdiA mutant BA612/pJNS25 grown in the presence of 3O-C6-HSL (open bars) and of wild-type strain 14028/pJNS25 grown in the presence (cross-hatched bars) or absence (negative control) (solid bars) of 3O-C6-HSL. The activity is expressed as a percentage of the activity of the positive control (100%). We found that none of the extracts activated the reporter more strongly than the negative controls, indicating that SdiA is not able to retain detectable amounts of 3O,C6-HSL after extensive washing. The data are the results of one experiment that was representative of three independent repeats, and the error bars indicate standard deviations for four measurements.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Recent reports have shown that interference with the AHL-mediated cell-to-cell communication systems of certain bacterial pathogens can attenuate the virulence of the pathogens in vivo (4, 11, 16, 19, 47). Therefore, the presence of a LuxR homologue, SdiA, in the gastrointestinal pathogen S. enterica serovar Typhimurium is of interest (2). However, since S. enterica serovar Typhimurium does not produce a native AHL, it is unlikely that Salmonella uses SdiA for detection of its own population density. As it has been shown that SdiA can be used for the detection of AHLs that are produced by other bacteria, it has been hypothesized that Salmonella might use this kind of "eavesdropping" to sense that it has entered the gastrointestinal tract (32). Since SdiA regulates the activity of the rck operon, which is involved in adhesion to extracellular matrix and resistance to complement killing, there is a possible link between SdiA and virulence. This link stimulated us to investigate the possibility of interfering with Salmonella SdiA. We therefore constructed a limited library consisting of 23 molecules, which were also used to investigate some differences between V. fischeri LuxR and Salmonella SdiA. The results for the activities of compounds 1 to 17 with LuxR were generally consistent with the results described in previous publications (14, 26, 30, 45). However, in our experiments 3O-C₇-HSL (compound 4) was a stronger activator of LuxR than the native signal 3O-C₆-HSL. Additionally, we concluded that SdiA is more sensitive than LuxR, since the reporters 14028/pJNS25 and 14028/pBA428 were both activated by concentrations of compounds 1 to 21 that were lower than the concentrations that activated E. coli JM109/pSB401. SdiA also detects a broader range of AHLs and is more sensitive to the unsubstituted AHLs compounds 11 to 13 than LuxR. These differences in the AHL spectra are not surprising, since Salmonella SdiA is only 26% identical and 47% similar to LuxR (2).

Interestingly, we identified 3O-C₆-HTL (compound 18) as a stronger activator of SdiA than its AHL counterpart, 3O-C₆-HSL (compound 3), while 3O-C₆-HTL is known to be an inhibitor of LuxR (14). Passador et al. showed that replacement of the HSL ring structure by an HTL ring in 3O-C₁₂-HSL, the native ligand of P. aeruginosas LasR, does not change the activity of the molecule with LasR (24). However, we demonstrated here that replacement of the HSL moiety by a thiolactone clearly increased the activities of the compounds with Salmonella SdiA (Fig. 1B). To our knowledge, this is the first report about a structure that activates a LuxR homologue at lower concentrations than the most active AHLs do. Smith et al. reported that N-(3-oxo-C₁₂)-2-aminocyclohexanol is a good activator of P. aeruginosa LasR, since the inducing activity of 100 µM 3O-C₁₂-ACH was comparable to the activity of 100 µM 3O-C₁₂-HSL (33). However, this compound is not a stronger activator of LasR than 3O-C₁₂-HSL, because 1 µM 3O-C₁₂-ACH showed only 50% of the activity exhibited by the native signal at a concentration of 1 µM (33). In contrast, we demonstrated here that all N-(3-oxo-acyl)-homocysteine thiolactones tested were stronger activators of SdiA than their AHL counterparts, even at concentrations lower than 1 nM (Fig. 1B). Of all the molecules that have been tested, 3O-C₇-HTL (compound 24) and 3O-C₈-HTL (compound 25) are the strongest activators of SdiA.

Since Salmonella does not produce a native AHL, there has been no selection pressure on the AHL receptor during evolution, which might explain why SdiA can be activated as strongly by a compound that is not a genuine AHL. Collins et al. recently reported that AHL-dependent QS systems can evolve rapidly to respond to a broad range of AHL molecules by only a few point mutations (10). One could speculate that AHTLs are the natural ligands of SdiA. However, so far there have been no reports concerning the occurrence of AHTLs in nature.

As noted above, 3O-C₁₂-ACH is a good activator of P. aeruginosa LasR. P. aeruginosa also encodes a second LuxR homologue, RhlR, which detects the native ligand C₄-HSL (33). Since C₄-ACH had very little inducing activity with RhlR, the researchers concluded that LuxR homologues, such as LasR and RhlR, do not necessarily recognize the ring moiety of their ligands in the same manner (33). We demonstrated here that modification of the HSL ring of compounds 3, 4, and 5 into an ACH structure resulted in the SdiA activators compounds 19, 28, and 29 (Fig. 1A). These compounds at a concentration of 300 nM exhibited the same activity as 300 nM 3O-C₆-HSL (compound 3), while they were less active at lower concentrations. It is remarkable that both modifications (HTL and ACH) of the HSL moiety that have shown strong inducing activity with LasR are also strong activators of SdiA, while this activity was not observed with LuxR and RhlR. These results suggest that there is a strong resemblance between the HSL-recognizing parts of LasR and SdiA. Therefore, we examined the effect of 7-(3-oxo-hexanoyl)-anthranilamide (compound 20) on SdiA in a competition experiment, since 3O-C₁₂-ANT is an inhibitor of LasR (34). We were surprised to observe that even a 10,000-fold excess of 3O-C₆-ANT over 3O-C₆-HSL (compound 3) had no effect on the activity of SdiA (data not shown), while 40 µM 3O-C₆-ANT inhibited the activity of 40 nM 3O-C₆-HSL (compound 3) with LuxR by 70% (Fig. 4).

Since information about the affinity of a receptor for its ligands is important for determining the possibility of identifying a competitive inhibitor of the receptor, we were stimulated by these unexpected results to investigate the affinity of SdiA for the activators 3O-C₆-HSL (compound 3), 3O-C₆-HTL (compound 18), and C₆-HSL (compound 11). Yao et al. showed in a recent report on the crystal structure of SdiA of E. coli, which is 69% identical at the amino acid level to its Salmonella homologue (2), that C₈-HSL bound to this SdiA is sequestered in a deep pocket in the hydrophobic core of the protein (49). Since this kind of binding core is similar to that of A. tumefaciens TraR (39, 49, 51), we hypothesized that SdiA might bind its activators as strongly as TraR binds 3O-C₈-HSL (18). To study the reversibility of the AHL binding by SdiA, we adapted one of the experiments that Luo et al. used to demonstrate that TraR binds 3O-C₈-HSL irreversibly (18). However, based on our experiments we concluded that SdiA does not retain detectable amounts of AHLs after extensive washing, suggesting that SdiA might bind its ligands reversibly. However, more experiments are needed before further conclusions can be drawn about the binding properties of SdiA and to explain why none of the compounds tested acts as an inhibitor of SdiA activity.

Our findings concerning the activities of AHL analogues with SdiA demonstrate that it is difficult and perhaps even impossible to make predictions about the activity of a certain compound with an AHL receptor based on its activity with other LuxR homologues. It is tempting to conclude that every AHL receptor is unique, since this means that it might be possible to identify compounds that selectively activate or inhibit a certain LuxR homologue without interfering with the QS systems of other species. Identification of this kind of selective compound might be interesting because of possible applications for control of microbial activity via interference with the quorum-sensing systems of bacteria. In conclusion, we identified two types of AHL derivatives that are strong activators of Salmonella SdiA. Both types have interesting properties, since the 3O-AHTLs are stronger SdiA activators than the 3O-AHLs, while the ACH derivatives are much more stable than the lactones. Since the exact biological function of SdiA in Salmonella is still unclear, we are currently using these selectively activating compounds to study their effects not only on S. enterica serovar Typhimurium planktonic cells but also on S. enterica serovar Typhimurium biofilm structures.

 
This work was supported by the Industrial Research Fund of K.U. Leuven (KP/06/014) and by the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen) through scholarship SB/41310 to J.J. and projects GBOU-SQUAD-20160 and BLO-20428 (Ministry of the Flemish Community). S.D.K. is a Research Associate of the Belgian Fund for Scientific Research—Flanders (FWO-Vlaanderen).

We thank A. Eberhard (Cornell University, Ithaca, NY) and H. Suga (University of Tokyo, Tokyo, Japan) for interesting suggestions during the initial stage of this work. For kindly providing the S. enterica serovar Typhimurium strains and E. coli DH5/pMIR101, we thank B. Ahmer (Ohio State University, Columbus, OH) and D. Faure (Centre National de la Recherche Scientifique, Gif-sur-Yvette, France), respectively.

 
* Corresponding author. Mailing address: Centre of Microbial and Plant Genetics, K. U. Leuven, Kasteelpark Arenberg 20, B-3001 Leuven, Belgium. Phone: 32-16321631. Fax: 32-16321966. E-mail: Sigrid.DeKeersmaecker{at}biw.kuleuven.be.

Published ahead of print on 3 November 2006.

Supplemental material for this article may be found at http://aem.asm.org/.

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Ahmer, B. M. 2004. Cell-to-cell signalling in Escherichia coli and Salmonella enterica. Mol. Microbiol. 52:933-945.[CrossRef][Medline]
2
2. Ahmer, B. M., J. van Reeuwijk, C. D. Timmers, P. J. Valentine, and F. Heffron. 1998. Salmonella typhimurium encodes an SdiA homolog, a putative quorum sensor of the LuxR family, that regulates genes on the virulence plasmid. J. Bacteriol. 180:1185-1193.[Abstract/Free Full Text]
3
3. Beringer, J. E. 1974. R factor transfer in Rhizobium leguminosarum. J. Gen. Microbiol. 84:188-198.[Abstract/Free Full Text]
4
4. Bjarnsholt, T., P. O. Jensen, T. B. Rasmussen, L. Christophersen, H. Calum, M. Hentzer, H. P. Hougen, J. Rygaard, C. Moser, L. Eberl, N. Hoiby, and M. Givskov. 2005. Garlic blocks quorum sensing and promotes rapid clearing of pulmonary Pseudomonas aeruginosa infections. Microbiology 151:3873-3880.[Abstract/Free Full Text]
5
5. Byers, J. T., C. Lucas, G. P. Salmond, and M. Welch. 2002. Nonenzymatic turnover of an Erwinia carotovora quorum-sensing signaling molecule. J. Bacteriol. 184:1163-1171.[Abstract/Free Full Text]
6
6. Carlier, A., S. Uroz, B. Smadja, R. Fray, X. Latour, Y. Dessaux, and D. Faure. 2003. The Ti plasmid of Agrobacterium tumefaciens harbors an attM-paralogous gene, aiiB, also encoding N-acyl homoserine lactonase activity. Appl. Environ. Microbiol. 69:4989-4993.[Abstract/Free Full Text]
7
7. Castang, S., B. Chantegrel, C. Deshayes, R. Dolmazon, P. Gouet, R. Haser, S. Reverchon, W. Nasser, N. Hugouvieux-Cotte-Pattat, and A. Doutheau. 2004. N-Sulfonyl homoserine lactones as antagonists of bacterial quorum sensing. Bioorg. Med. Chem. Lett. 14:5145-5149.[CrossRef][Medline]
8
8. Chhabra, S. R., C. Harty, D. S. Hooi, M. Daykin, P. Williams, G. Telford, D. I. Pritchard, and B. W. Bycroft. 2003. Synthetic analogues of the bacterial signal (quorum sensing) molecule N-(3-oxododecanoyl)-L-homoserine lactone as immune modulators. J. Med. Chem. 46:97-104.[CrossRef][Medline]
9
9. Chhabra, S. R., P. Stead, N. J. Bainton, G. P. Salmond, G. S. Stewart, P. Williams, and B. W. Bycroft. 1993. Autoregulation of carbapenem biosynthesis in Erwinia carotovora by analogues of N-(3-oxohexanoyl)-L-homoserine lactone. J. Antibiot. 46:441-454.[Medline]
10
10. Collins, C. H., F. H. Arnold, and J. R. Leadbetter. 2005. Directed evolution of Vibrio fischeri LuxR for increased sensitivity to a broad spectrum of acyl-homoserine lactones. Mol. Microbiol. 55:712-723.[CrossRef][Medline]
11
11. Dong, Y. H., L. H. Wang, J. L. Xu, H. B. Zhang, X. F. Zhang, and L. H. Zhang. 2001. Quenching quorum-sensing-dependent bacterial infection by an N-acyl homoserine lactonase. Nature 411:813-817.[CrossRef][Medline]
12
12. Dong, Y. H., and L. H. Zhang. 2005. Quorum sensing and quorum-quenching enzymes. J. Microbiol. 43:101-109.
13
13. Eberhard, A., A. L. Burlingame, C. Eberhard, G. L. Kenyon, K. H. Nealson, and N. J. Oppenheimer. 1981. Structural identification of autoinducer of Photobacterium fischeri luciferase. Biochemistry 20:2444-2449.[CrossRef][Medline]
14
14. Eberhard, A., C. A. Widrig, P. McBath, and J. B. Schineller. 1986. Analogs of the autoinducer of bioluminescence in Vibrio fischeri. Arch. Microbiol. 146:35-40.[CrossRef][Medline]
15
15. Fuqua, C., M. R. Parsek, and E. P. Greenberg. 2001. Regulation of gene expression by cell-to-cell communication: acyl-homoserine lactone quorum sensing. Annu. Rev. Genet. 35:439-468.[CrossRef][Medline]
16
16. Hentzer, M., H. Wu, J. B. Andersen, K. Riedel, T. B. Rasmussen, N. Bagge, N. Kumar, M. A. Schembri, Z. Song, P. Kristoffersen, M. Manefield, J. W. Costerton, S. Molin, L. Eberl, P. Steinberg, S. Kjelleberg, N. Hoiby, and M. Givskov. 2003. Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors. EMBO J. 22:3803-3815.[CrossRef][Medline]
17
17. Kline, T., J. Bowman, B. H. Iglewski, T. de Kievit, Y. Kakai, and L. Passador. 1999. Novel synthetic analogs of the Pseudomonas autoinducer. Bioorg. Med. Chem. Lett. 9:3447-3452.[CrossRef][Medline]
18
18. Luo, Z. Q., S. Su, and S. K. Farrand. 2003. In situ activation of the quorum-sensing transcription factor TraR by cognate and noncognate acyl-homoserine lactone ligands: kinetics and consequences. J. Bacteriol. 185:5665-5672.[Abstract/Free Full Text]
19
19. Mae, A., M. Montesano, V. Koiv, and E. T. Palva. 2001. Transgenic plants producing the bacterial pheromone N-acyl-homoserine lactone exhibit enhanced resistance to the bacterial phytopathogen Erwinia carotovora. Mol. Plant-Microbe Interact 14:1035-1042.[Medline]
20
20. Marketon, M. M., M. R. Gronquist, A. Eberhard, and J. E. Gonzalez. 2002. Characterization of the Sinorhizobium meliloti sinR/sinI locus and the production of novel N-acyl homoserine lactones. J. Bacteriol. 184:5686-5695.[Abstract/Free Full Text]
21
21. McClean, K. H., M. K. Winson, L. Fish, A. Taylor, S. R. Chhabra, M. Camara, M. Daykin, J. H. Lamb, S. Swift, B. W. Bycroft, G. S. Stewart, and P. Williams. 1997. Quorum sensing and Chromobacterium violaceum: exploitation of violacein production and inhibition for the detection of N-acylhomoserine lactones. Microbiology 143:3703-3711.[Abstract/Free Full Text]
22
22. Michael, B., J. N. Smith, S. Swift, F. Heffron, and B. M. Ahmer. 2001. SdiA of Salmonella enterica is a LuxR homolog that detects mixed microbial communities. J. Bacteriol. 183:5733-5742.[Abstract/Free Full Text]
23
23. Parsek, M. R., and E. P. Greenberg. 2005. Sociomicrobiology: the connections between quorum sensing and biofilms. Trends Microbiol. 13:27-33.[CrossRef][Medline]
24
24. Passador, L., K. D. Tucker, K. R. Guertin, M. P. Journet, A. S. Kende, and B. H. Iglewski. 1996. Functional analysis of the Pseudomonas aeruginosa autoinducer PAI. J. Bacteriol. 178:5995-6000.[Abstract/Free Full Text]
25
25. Piper, K. R., V. B. Beck, and S. K. Farrand. 1993. Conjugation factor of Agrobacterium tumefaciens regulates Ti plasmid transfer by autoinduction. Nature 362:448-450.[CrossRef][Medline]
26
26. Reverchon, S., B. Chantegrel, C. Deshayes, A. Doutheau, and N. Cotte-Pattat. 2002. New synthetic analogues of N-acyl homoserine lactones as agonists or antagonists of transcriptional regulators involved in bacterial quorum sensing. Bioorg. Med. Chem. Lett. 12:1153-1157.[CrossRef][Medline]
27
27. Robinson, R. A. 1989. Buffer solutions, p. D144-D148. In R. C. Weast, D. R. Lide, M. J. Astle, and W. H. Beyer (ed.), CRC handbook of chemistry and physics, 70th ed. CRC Press Inc., Boca Raton, FL.
28
28. Rychlik, I., and P. A. Barrow. 2005. Salmonella stress management and its relevance to behaviour during intestinal colonisation and infection. FEMS Microbiol. Rev. 29:1021-1040.[CrossRef][Medline]
29
29. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
30
30. Schaefer, A. L., B. L. Hanzelka, A. Eberhard, and E. P. Greenberg. 1996. Quorum sensing in Vibrio fischeri: probing autoinducer-LuxR interactions with autoinducer analogs. J. Bacteriol. 178:2897-2901.[Abstract/Free Full Text]
31
31. Shaw, P. D., G. Ping, S. L. Daly, C. Cha, J. E. Cronan, Jr., K. L. Rinehart, and S. K. Farrand. 1997. Detecting and characterizing N-acyl-homoserine lactone signal molecules by thin-layer chromatography. Proc. Natl. Acad. Sci. USA 94:6036-6041.[Abstract/Free Full Text]
32
32. Smith, J. N., and B. M. Ahmer. 2003. Detection of other microbial species by Salmonella: expression of the SdiA regulon. J. Bacteriol. 185:1357-1366.[Abstract/Free Full Text]
33
33. Smith, K. M., Y. Bu, and H. Suga. 2003. Induction and inhibition of Pseudomonas aeruginosa quorum sensing by synthetic autoinducer analogs. Chem. Biol. 10:81-89.[CrossRef][Medline]
34
34. Smith, K. M., Y. Bu, and H. Suga. 2003. Library screening for synthetic agonists and antagonists of a Pseudomonas aeruginosa autoinducer. Chem. Biol. 10:563-571.[CrossRef][Medline]
35
35. Smith, R. S., and B. H. Iglewski. 2003. P. aeruginosa quorum-sensing systems and virulence. Curr. Opin. Microbiol. 6:56-60.[CrossRef][Medline]
36
36. Sokol, P. A., U. Sajjan, M. B. Visser, S. Gingues, J. Forstner, and C. Kooi. 2003. The CepIR quorum-sensing system contributes to the virulence of Burkholderia cenocepacia respiratory infections. Microbiology 149:3649-3658.[Abstract/Free Full Text]
37
37. Swift, S., M. J. Lynch, L. Fish, D. F. Kirke, J. M. Tomas, G. S. Stewart, and P. Williams. 1999. Quorum sensing-dependent regulation and blockade of exoprotease production in Aeromonas hydrophila. Infect. Immun. 67:5192-5199.[Abstract/Free Full Text]
38
38. Urbanowski, M. L., C. P. Lostroh, and E. P. Greenberg. 2004. Reversible acyl-homoserine lactone binding to purified Vibrio fischeri LuxR protein. J. Bacteriol. 186:631-637.[Abstract/Free Full Text]
39
39. Vannini, A., C. Volpari, C. Gargioli, E. Muraglia, R. Cortese, R. De Francesco, P. Neddermann, and S. D. Marco. 2002. The crystal structure of the quorum sensing protein TraR bound to its autoinducer and target DNA. EMBO J. 21:4393-4401.[CrossRef][Medline]
40
40. Volf, J., M. Sevcik, H. Havlickova, F. Sisak, J. Damborsky, and I. Rychlik. 2002. Role of SdiA in Salmonella enterica serovar Typhimurium physiology and virulence. Arch. Microbiol. 178:94-101.[CrossRef][Medline]
41
41. Waters, C. M., and B. L. Bassler. 2005. Quorum sensing: cell-to-cell communication in bacteria. Annu. Rev. Cell Dev. Biol. 21:319-346.[CrossRef][Medline]
42
42. Watson, B., T. C. Currier, M. P. Gordon, M. D. Chilton, and E. W. Nester. 1975. Plasmid required for virulence of Agrobacterium tumefaciens. J. Bacteriol. 123:255-264.[Abstract/Free Full Text]
43
43. Whitehead, N. A., A. M. Barnard, H. Slater, N. J. Simpson, and G. P. Salmond. 2001. Quorum-sensing in Gram-negative bacteria. FEMS Microbiol. Rev. 25:365-404.[CrossRef][Medline]
44
44. Whitehead, N. A., J. T. Byers, P. Commander, M. J. Corbett, S. J. Coulthurst, L. Everson, A. K. Harris, C. L. Pemberton, N. J. Simpson, H. Slater, D. S. Smith, M. Welch, N. Williamson, and G. P. Salmond. 2002. The regulation of virulence in phytopathogenic Erwinia species: quorum sensing, antibiotics and ecological considerations. Antonie Leeuwenhoek 81:223-231.[CrossRef][Medline]
45
45. Winson, M. K., S. Swift, L. Fish, J. P. Throup, F. Jorgensen, S. R. Chhabra, B. W. Bycroft, P. Williams, and G. S. Stewart. 1998. Construction and analysis of luxCDABE-based plasmid sensors for investigating N-acyl homoserine lactone-mediated quorum sensing. FEMS Microbiol. Lett. 163:185-192.[CrossRef][Medline]
46
46. Withers, H., S. Swift, and P. Williams. 2001. Quorum sensing as an integral component of gene regulatory networks in Gram-negative bacteria. Curr. Opin. Microbiol. 4:186-193.[CrossRef][Medline]
47
47. Wu, H., Z. Song, M. Hentzer, J. B. Andersen, S. Molin, M. Givskov, and N. Hoiby. 2004. Synthetic furanones inhibit quorum-sensing and enhance bacterial clearance in Pseudomonas aeruginosa lung infection in mice. J. Antimicrob. Chemother. 53:1054-1061.[Abstract/Free Full Text]
48
48. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103-119.[CrossRef][Medline]
49
49. Yao, Y., M. A. Martinez-Yamout, T. J. Dickerson, A. P. Brogan, P. E. Wright, and H. J. Dyson. 2006. Structure of the Escherichia coli quorum sensing protein SdiA: activation of the folding switch by acyl homoserine lactones. J. Mol. Biol. 355:262-273.[CrossRef][Medline]
50
50. Yates, E. A., B. Philipp, C. Buckley, S. Atkinson, S. R. Chhabra, R. E. Sockett, M. Goldner, Y. Dessaux, M. Camara, H. Smith, and P. Williams. 2002. N-acylhomoserine lactones undergo lactonolysis in a pH-, temperature-, and acyl chain length-dependent manner during growth of Yersinia pseudotuberculosis and Pseudomonas aeruginosa. Infect. Immun. 70:5635-5646.[Abstract/Free Full Text]
51
51. Zhang, R. G., T. Pappas, J. L. Brace, P. C. Miller, T. Oulmassov, J. M. Molyneaux, J. C. Anderson, J. K. Bashkin, S. C. Winans, and A. Joachimiak. 2002. Structure of a bacterial quorum-sensing transcription factor complexed with pheromone and DNA. Nature 417:971-974.[CrossRef][Medline]
52
52. Zhu, J., J. W. Beaber, M. I. More, C. Fuqua, A. Eberhard, and S. C. Winans. 1998. Analogs of the autoinducer 3-oxooctanoyl-homoserine lactone strongly inhibit activity of the TraR protein of Agrobacterium tumefaciens. J. Bacteriol. 180:5398-5405.[Abstract/Free Full Text]
53
53. Zhu, J., and S. C. Winans. 2001. The quorum-sensing transcriptional regulator TraR requires its cognate signaling ligand for protein folding, protease resistance, and dimerization. Proc. Natl. Acad. Sci. USA 98:1507-1512.[Abstract/Free Full Text]

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Applied and Environmental Microbiology, January 2007, p. 535-544, Vol. 73, No. 2
0099-2240/07/$08.00+0     doi:10.1128/AEM.01451-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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