School of Microbiology and Immunology, University of New South Wales, Sydney, Australia1
Department of Microbiology, Technical University of Denmark, 2800 Lyngby, Denmark2
Centre for Marine Biofouling and Bio-Innovation, University of New South Wales, Sydney, Australia3
Author for correspondence: Michael Givskov. Tel: +45 45252769. Fax: +45 45932809. e-mail: immg{at}pop.dtu.dk
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: Delisea pulchra, homoserine lactone, intercellular signal
Abbreviations: AHL, N-acyl-L-homoserine lactone; 3-oxo-C6-HSL, N-3-oxo-hexanoyl-homoserine lactone; RFU, relative fluorescence unit
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The diverse range of AHL regulated phenotypes includes the production of degradative extracellular enzymes by Pseudomonas aeruginosa and Erwinia carotovora (Jones et al., 1993 ), bioluminescence in Vibrio fischeri (Sitnikov et al., 1995
) and Vibrio harveyi (Bassler et al., 1993
), plasmid transfer in Agrobacterium tumefaciens (Piper et al., 1993
), antibiotic production in E. carotovora (Bainton et al., 1992
), and more complex phenotypes such as surface motility in Serratia liquefaciens (Eberl et al., 1999
) and development of biofilm architecture in P. aeruginosa (Davies et al., 1998
)
AHLs are synthesized by homologues from either the AinS or LuxI family of AHL synthases and mediate transcription of various target genes through an interaction with, in most cases, a homologue of the LuxR protein of V. fischeri (reviewed by Fuqua et al., 1996 ). AHLs show variation in the length, degree of saturation and adjoining substitutions of the acyl chain (reviewed by Fuqua & Eberhard, 1999
). These structural variations account for the different responses elicited by different AHLs in quorum sensing assays (McClean et al., 1997
; Zhu et al., 1998
). The molecular mechanism by which AHLs trigger the transcriptional activation of target promoters via an interaction with LuxR homologues remains to be fully elucidated (see Discussion), but appears to involve AHL binding to and induction of conformational changes in the regulatory protein which lead to multimerization and DNA binding (Choi & Greenberg, 1992
; Qin et al., 2000
; Welch et al., 2000
). Other cellular components are involved in the expression of AHL-regulated genes including the cAMP receptor protein (Nealson et al., 1972
), the H-NS protein (Ulitzur et al., 1997
), and the molecular chaperones GroES and GroEL (Adar et al., 1992
).
The discovery of quorum sensing has afforded a novel opportunity to control unwanted microbial activity without the use of growth inhibitory agents such as antibiotics, preservatives and disinfectants that select for resistant organisms. A means of interfering with AHL-mediated gene expression not only has potential in a number of applied contexts, including the treatment of lung infections in cystic fibrosis patients, but would also constitute an evolutionary advantage for plant and animal species under selective pressure from quorum sensing pathogens.
Gram-negative bacteria engage in AHL-dependent phytopathogenic (Barras et al., 1994 ; Zhang et al., 1993
) and phytosymbiotic (Rodelas et al., 1999
) relationships with terrestrial plants. Whilst there are no known examples of such relationships in the marine environment, marine plants are at once rich in secondary metabolite chemistry and, in the absence of more advanced immune systems, prone to disease (Correa, 1996
; Fenical, 1997
). For these reasons marine plants are likely candidates for the evolution of AHL antagonist activity (Kjelleberg & Steinberg, 2001
). The marine macroalga Delisea pulchra produces a range of lactones, known more specifically as halogenated furanones, which inhibit quorum sensing (Givskov et al., 1996
; Manefield et al., 1999
; Rasmussen et al., 2000
). It has previously been proposed that the production of the halogenated furanones in specialized cells, which migrate to the surface of the alga to release the compounds (Dworjanyn et al., 1999
), is likely to have evolved in response to the negative impacts of AHL-dependent colonization of its surfaces by marine bacterial species (Givskov et al., 1996
; Kjelleberg et al., 1997
; Kjelleberg & Steinberg, 2001
).
It has been demonstrated that halogenated furanones have inhibitory effects in a variety of biological assays designed to measure AHL-mediated gene expression (Givskov et al., 1996 ). Such inhibition was found to be partially relieved by increasing AHL concentrations in the bioasssays, indicative of competition for a regulatory function (Manefield et al., 1999
). Furthermore, halogenated furanones were found to have activity in an in vivo ligand-binding assay employed to monitor the displacement of AHLs from the LuxR protein (Manefield et al., 1999
). These results have been in accordance with a model in which halogenated furanones compete with AHLs for a common binding site on LuxR and LuxR homologues. In this study we tested directly for an interaction between a halogenated furanone and the LuxR protein, and discovered that the furanones inhibit AHL-mediated gene expression through accelerated degradation of the transcriptional activator.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
AHL and furanone preparation.
Synthetic N-oxohexanoyl-L-homoserine lactone was prepared according to the method of Eberhard et al. (1981) and stored in ethyl acetate at 20 mg ml-1 (approx. 100 µM, depending on the compound) at -20 °C in a non-frost free freezer. The halogenated furanones compound 2, compound 8 and compound 56 were synthesized as previously described by Manny et al. (1997)
. Compound 4 was extracted from D. pulchra and purified by HPLC according to protocols established by de Nys et al. (1993)
. Compound 30 was synthesized according to protocols reported by Wells (1963)
. Tritiated compound 4 was prepared by reducing a ketone group at the C(1') position on the alkyl chain of a compound 4 precursor with tritiated borane producing an enantiomeric mixture of the S and R forms of compound 4 with a tritium atom replacing the hydrogen atom at the C(1') position. The furanones were stored in ethanol at 20 mg ml-1 at -20 °C in a non-frost free freezer.
Binding analysis.
A variation on the method of Hanzelka & Greenberg (1995) was employed to assay for an in vivo affinity between the LuxR protein and 3H-labelled compound 4. LuxR, GroES and GroEL overproduction was induced in exponential phase E. coli XL-1 Blue(pHK724)(pGroESL) and E. coli XL-1 Blue(pGroESL) cultures (OD600 0·3) by incubation with 100 µM IPTG for 2 h. Various concentrations of the labelled halogenated furanone were then added to 1 ml aliquots of the induced cultures. After 10 min incubation, the cells were washed, resuspended in scintillation fluid and monitored for tritium as previously described for 3H-3-oxo-C6-HSL (Manefield et al., 1999
; Hanzelka & Greenberg, 1995
). A standard curve describing the relationship between the quantity of the labelled furanone and its relative activity in c.p.m., obtained by diluting known amounts of 3H-labelled compound 4 in 100 µl scintillation fluid, was used to convert the raw data obtained from c.p.m. to nmol.
Determination of GFP expression.
The effect of halogenated furanones on expression of a 3-oxo-C6-HSL stimulated, LuxR controlled PluxIgfp(ASV) fusion [encoding an unstable Gfp protein with half life ranging from 40 min to 110 min in exponential and stationary phase cells, respectively (Andersen et al., 1998 , 2001
)] was determined as follows. Aliquots (200 µl) of exponential phase E. coli MT102(pJBA89) cultures were distributed to the wells of microtitre dishes in which 3-oxo-C6-HSL and furanone compounds in the required concentrations were already present. After 2 h incubation at 37 °C the relative fluorescence units (RFU) of each sample were captured with a Wallac Victor2, I420 Multilabel Counter using a 485 nm excitation filter and a 535 nm emission filter. The OD450 was also determined for each sample to monitor growth of the bacterial strain.
LuxR Western analysis.
E. coli XL-1 Blue(pHK724)(pGroESL) was grown in the presence of 100 µM IPTG to OD600 0·3. Cells were harvested by centrifugation at 10000 g, 4 °C for 5 min and resuspended in an equal volume of fresh media. The washed culture was split into 5 ml aliquots and treated with various concentrations of halogenated furanones and/or 3-oxo-C6-HSL (N-3-oxo-hexanoyl-homoserine lactone). Samples were taken over time for determination of optical density and for Western analysis. Samples taken for Western analysis were frozen immediately and kept at -20 °C. Prior to SDS-gel electrophoresis, the samples were adjusted to the same absorbance. Western analysis of the separated proteins was performed using an anti-LuxR antibody from Quorum Sciences as the primary antibody and an anti-rabbit horseradish peroxidase conjugated antibody from Amersham Pharmacia Biotech as the secondary antibody, according to the manufacturers recommendations. Localization of the secondary antibody was visualized using chemiluminescent detection reagents from Amersham Pharmacia Biotech and a Hamamatsu C2400-47 double intensified CCD camera (Hamamatsu). Colour images were saved in 16-bit format (the scale has a resolution of 16 colours) using ARGUS-50 software (Hamamatsu). The relative amounts of LuxR protein in each sample were estimated from a standard curve relating colour to relative LuxR content constructed from a two-fold dilution series of fully IPTG induced cells. Prior to SDS-gel electrophoresis, the samples were adjusted to the same absorbance.
To assess whether the translation of persistent luxR mRNA during the above described treatment contributed to the cellular pool of LuxR measured in the Western procedure, 100 µg chloramphenicol ml-1 was administered after the removal of IPTG, just prior to furanone treatment, to disrupt the ribosomal activity required for LuxR production. Cells treated thus were assessed by Western blotting as above.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Fig. 1 describes the dose response relationships obtained for the two strains. The E. coli culture overproducing the LuxR protein retained between 0·4% and 1·5% of the tritiated furanone added, depending on the concentration added. By comparison, the control culture overproducing the chaperone proteins alone retained from 0·4% to 1·1% of the labelled compound added. Whilst this small difference may represent an association between the algal metabolite and the LuxR protein, these results suggest that any LuxRcompound 4 complex formed is unstable in nature.
|
To address this hypothesis we examined the amounts of LuxR protein in cells treated with furanones. To assess the in vivo stability of LuxR, XL-1 Blue(pHK724)(pGroESL) cells were induced with 100 µM IPTG, washed free of the inducer and sampled at different time points after the wash. The amounts of LuxR protein in the different samples were then visualized by an SDS-PAGE-Western blot procedure using LuxR antibody and a chemiluminescence-based detection system. A CCD camera reported chemiluminescence as colour digital images with increasingly lighter colours relating to increases in band intensity (Fig. 2a).
|
A correlation between furanone activity, cellular LuxR concentration and expression of the luxI promoter
To determine whether furanone-dependent decay of the LuxR protein was the mechanism by which halogenated furanones inhibit expression from the promoter of the luxI gene (PluxI) we assessed the relationship between furanone activity, cellular LuxR concentration and PluxI expression. We tested a series of compounds (Table 1) for their ability to inhibit 3-oxo-C6-HSL-induced LuxR-dependent expression of fluorescence from a PluxIgfp(ASV) fusion in the AHL monitor strain E. coli MT102(pJBA89). The effect of two natural and three synthetic halogenated furanones (Table 1
) on 3-oxo-C6-HSL stimulated LuxR PluxIgfp(ASV) expression was determined at eight different furanone concentrations (ranging from 0 to 40 µM) and three different 3-oxo-C6-HSL concentrations (ranging from 25 to 100 nM). Incubation with 100 nM 3-oxo-C6-HSL and no furanone addition was defined as having 100% RFU. A total of 24 RFU values obtained for each furanone compound were used to calculate an inhibition index expressing the quantity of furanone (µmol) per quantity of 3-oxo-C6-HSL (nmol) required to inhibit PluxIgfp(ASV) expression to an arbitrary level (40%). This is termed the ID40 value. The three values obtained, one for each 3-oxo-C6-HSL concentration, were plotted as a function of 3-oxo-C6-HSL concentration (illustrated for compound 30 in Fig. 3
) and the gradient of the best straight line passing through the origin was taken as the inhibition index (IIX40). The IIX40 expresses the number of moles furanone per mmole 3-oxo-C6-HSL required to inhibit expression of fluorescence to 40% of the untreated sample. A low IIX40 value therefore indicates that a compound is an efficient quorum-sensing inhibitor. Using this method, it was found that the most active compounds tested are compounds 30 (synthetic) and 4 (natural). Compounds 2 (natural) and 56 (synthetic) have medium activity whereas compound 8 (synthetic) has poor activity (Table 1
).
|
|
|
|
The presence of 3-oxo-C6-HSL protects the LuxR protein against furanone promoted degradation
Whilst analysing the expression of the LuxR controlled PluxIgfp(ASV) fusion we realized that increasing 3-oxo-C6-HSL concentrations reduced the inhibitory activity of the furanones on the fluorescence phenotype (data not shown). To assess whether this was a manifestation of the protection of LuxR by 3-oxo-C6-HSL we tested the effect of the AHL on compound 30 induced LuxR decay. Fig. 6 (c) illustrates that the addition of 2 µM 3-oxo-C6-HSL approximately 60 s after the addition of 10 µM compound 30 did not afford LuxR any protection from furanone-induced degradation. Fig. 6
(d) however, illustrates that if 2 µM 3-oxo-C6-HSL was added 60 s prior to the addition of 10 µM compound 30, the furanone-induced degradation was retarded by at least 10 min. Note that in the fluorescence assay employed to calculate the inhibition index for each compound, halogenated furanones and 3-oxo-C6-HSL were added simultaneously.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Qin et al. (2000) have proposed a model in which the TraR protein is monomeric and membrane associated in the absence of 3-oxo-C8-HSL but dimeric, cytoplasmic and capable of transcriptional activation in its presence. Zhu & Winans (2001)
have recently presented another model in which the TraR protein is monomeric and vulnerable to proteolysis in the absence of 3-oxo-C6-HSL but dimeric, resistant to proteolysis and capable of transcriptional activation in its presence. These models both draw attention to the relevance of the cytoplasmic concentration of TraR in the activation of target promoters.
In the present communication we have sought to further define the molecular mechanism by which halogenated furanones inhibit the AHL-mediated transcriptional activation of target genes. We have been unable to detect the formation of a stable complex between a tritiated halogenated furanone and the LuxR protein overproduced in E. coli. We have however demonstrated that the cytoplasmic concentration of the LuxR protein is decreased in the presence of halogenated furanones. In the light of both these results and the demonstration that 3-oxo-C8-HSL protects the TraR protein in E. coli from proteolytic digestion (Zhu & Winans, 2001 ) it is suggested that halogenated furanones interact with the LuxR protein but that this interaction causes conformational changes that enlist the furanoneLuxR complex into rapid proteolytic degradation. This model is consistent both with the observed effects of furanones on the formation of the AHLLuxR complex (Manefield et al., 1999
) and with the inability to detect a long-lived furanoneLuxR complex.
Is the loss of LuxR the result of proteolytic degradation? The effect of furanones on the LuxR concentration was comparable in a wild-type E. coli strain and a clpP and a lon E. coli strain. Zhu & Winans (2001) found that a substantial change in the rate of TraR degradation in E. coli required the simultaneous crippling of both the clp and lon proteases. We found no significant change in the inhibition index for compound 30 in the different strains. This indicated that not even the Clp and Lon proteases in concert could severely affect the LuxR stability. This however, does not rule out the possibility that other proteases could be involved in the proteolytic turnover of LuxR. The possibility that the LuxR protein becomes compartmentalized in a manner analogous to the proposal of Qin et al. (2000)
in the presence of the furanones is unlikely given that whole cells (i.e. including membranes) were used in the Western procedure employed here.
We did not reproducibly detect an increase in LuxR stability in the presence of 3-oxo-C6-HSL as was demonstrated by Zhu & Winans (2001) for the TraR protein in the presence of 3-oxo-C8-HSL. In our experiments however 3-oxo-C6-HSL was always added after the cessation of stimulation of the luxR promoter (i.e. after removal of IPTG). Zhu & Winans (2001)
found that the AHL-induced protection of TraR was dependent on the presence of 3-oxo-C8-HSL during synthesis of the protein and that addition of 3-oxo-C8-HSL to E. coli cells already harbouring the TraR monomer did not afford the protein any protection against proteolysis. Our observations with LuxR are therefore not inconsistent with those of Zhu & Winans (2001)
.
The ability of 3-oxo-C6-HSL to protect the LuxR protein from furanone-induced degradation was dependent on the addition of the AHL before the furanone. This result suggests that, unlike 3-oxo-C8-HSL and TraR, 3-oxo-C6-HSL binds mature LuxR and that in this state the halogenated furanones are less able to compete for the AHL binding site. It is possible that 3-oxo-C6-HSL is binding and protecting low levels of freshly translated LuxR from persistent luxR mRNA transcript. However, the addition of chloramphenicol to block translation after cessation of transcription did not affect LuxR levels, indicating that residual translation was not occurring. Either way it is clear that 3-oxo-C6-HSL can protect the LuxR protein from furanone-induced degradation when present before the furanone, but is unable to rescue the protein if the furanone is present first and has already initiated degradation.
The superior activity of compounds lacking a carbon chain extending from the furan ring structure in the control of both PluxIgfp(ASV) expression and LuxR concentration was somewhat unexpected because the homoserine lactone ring without the acyl chain has been shown not to interact with the LuxR homologue CarR (Welch et al., 2000 ). The significance of this will remain unresolved until more structural information regarding LuxR homologues and their AHL binding sites is available.
AHLs are required for the expression of Gram-negative bacterial phenotypes involved in many cases in an interaction with a higher organism. Amongst some of the best studied examples, including elastase production in P. aeruginosa, pectate lyase production in E. carotovora and conjugation of the plant-tumour-inducing Ti plasmid of A. tumefaciens, are behaviours with central roles in the success of bacterial infections of medical, agricultural and therefore economic significance. This investigation has demonstrated that halogenated furanones produced naturally by the marine alga D. pulchra can modulate the cellular concentration of the LuxR protein responsible for the reception of, and response to, AHLs. While this study is directed at the effects of furanones on the LuxR protein of V. fischeri, the findings highlight the potential for the use of halogenated furanones in the control of unwanted bacterial activity. Additionally these results lend support to a model of AHL function in which the metabolite regulates the steady state concentration of LuxR homologues by shielding the regulator from proteolytic degradation.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Albus, A. M., Pesci, E. C., Runyen-Janecky, L. J., West, S. E. & Iglewski, B. H. (1997). Vfr controls quorum sensing in Pseudomonas aeruginosa. J Bacteriol 179, 3928-3935.[Abstract]
Andersen, J. B., Sternberg, C., Poulsen, L. K., Bjørn, S. P., Givskov, M. & Molin, S. (1998). New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl Environ Microbiol 64, 2240-2246.
Andersen, J. B., Heydorn, A., Hentzer, M., Eberl, L., Geisenberger, O., Molin, S. & Givskov, M. (2001). gfp based N-acyl-homoserine-lactone monitors for detection of bacterial communication. Appl Environ Microbiol 67, 575-585.
Bainton, N. J., Bycroft, B. W., Chhabra, S. R. & 8 other authors (1992). A general role for the lux autoinducer in bacterial cell signalling: control of antibiotic biosynthesis in Erwinia. Gene 116, 8791.[Medline]
Barras, F., van Gijsegem, F. & Chatterjee, A. K. (1994). Extracellular enzymes and pathogenesis of soft rot Erwinia. Annu Rev Phytopathol 32, 201-234.
Bassler, B. L., Wright, M., Showalter, R. E. & Silverman, M. R. (1993). Intercellular signaling in Vibrio harveyi: sequence and function of genes regulating expression of luminescence. Mol Microbiol 9, 773-786.[Medline]
Choi, S. H. & Greenberg, E. P. (1992). Genetic evidence for the multimerization of LuxR, the transcriptional activator of Vibrio fischeri luminescence. Mol Mar Biol Biotechnol 1, 408-413.
Clark, D. J. & Maaløe, O. (1967). DNA replication and division cycle in Escherichia coli. J Mol Biol 23, 99-112.
Correa, J. A. (1996). Diseases in seaweeds: an introduction. Hydrobiologia 326, 87-88.
Davies, D. G., Parsek, M. R., Pearson, J. P., Iglewski, B. H., Costerton, J. W. & Greenberg, E. P. (1998). The involvement of cell-to-cell signals in the development of a bacterial biofilm. Science 280, 295-298.
de Nys, R., Wright, A. D., Konig, G. M. & Sticher, O. (1993). New halogenated furanones from the marine alga Delisea pulchra (cf. Fimbriata). Tetrahedron 49, 11213-11220.
Dworjanyn, S. A., de Nys, R. & Steinberg, P. D. (1999). Localisation and surface quantification of secondary metabolites in the red alga Delisea pulchra. Mar Biol 133, 727-736.
Eberhard, A., Burlingame, A. L., Eberhard, C., Kenyon, G. L., Nealson, K. H. & Oppenheimer, N. J. (1981). Structural identification of autoinducer of Photobacterium fischeri luciferase. Biochemistry 20, 2444-2449.[Medline]
Eberl, L. (1999). N-Acyl homoserine lactone-mediated gene regulation in Gram-negative bacteria. Syst Appl Microbiol 22, 493-506.[Medline]
Eberl, L., Molin, S. & Givskov, M. (1999). Surface motility in Serratia liquefaciens. J Bacteriol 181, 1703-1712.
Fenical, W. (1997). New pharmaceuticals from marine organisms. Trends Biotechnol 15, 339-341.[Medline]
Fuqua, C. & Eberhard, A. (1999). Signal generation in autoinduction systems: synthesis of acylated homoserine lactones by LuxI-type proteins. In CellCell Signaling in Bacteria , pp. 211-230. Edited by G. M. Dunny & S. C. Winans. Washington, DC:American Society for Microbiology.
Fuqua, C., Winans, S. C. & Greenberg, E. P. (1996). Census and consensus in bacterial ecosystems: the LuxRLuxI family of quorum-sensing transcriptional regulators. Annu Rev Microbiol 50, 727-751.[Medline]
Givskov, M., de Nys, R., Manefield, M., Gram, L., Maximilien, R., Eberl, L., Molin, S., Steinberg, P. D. & Kjelleberg, S. (1996). Eukaryotic interference with homoserine lactone mediated prokaryotic signaling. J Bacteriol 178, 6618-6622.[Abstract]
Goloubinoff, P., Gatenby, A. A. & Lorimer, G. H. (1989). GroE heat-shock proteins promote the assembly of foreign prokaryotic ribulose biphosphate carboxylase oligomers in Escherichia coli. Nature 337, 44-47.[Medline]
Guyer, M. S., Reed, R. E., Steitz, T. & Low, K. B. (1981). Identification of a sex-factor-affinity site in E. coli as gamma delta. Cold Spring Harbor Symp Quant Biol 45, 135-140.[Medline]
Hanzelka, B. L. & Greenberg, E. P. (1995). Evidence that the N-terminal region of the Vibrio fischeri LuxR protein constitutes an autoinducer-binding domain. J Bacteriol 177, 815-817.[Abstract]
Jones, S., Yu, B., Bainton, N. J. & 11 other authors (1993). The lux autoinducer regulates the production of exoenzyme virulence determinants in Erwinia carotovora and Pseudomonas aeruginosa. EMBO J 12, 24772482.[Abstract]
Kievit, T. R. & Iglewski, B. (2000). Bacterial quorum sensing in pathogenic relationships. Infect Immun 68, 4839-4849.
Kjelleberg, S. & Steinberg, P. (2001). Defences against bacterial colonisation of marine plants. In Phyllosphere Microbiology , pp. 157-172. Edited by S. E. Lindow, E. Hecht-Poinar & V. Elliot. St Paul, MN:American Phytopathological Society.
Kjelleberg, S., Steinberg, P., Givskov, M., Gram, L., Manefield, M. & de Nys, R. (1997). Do marine natural products interfere with prokaryotic AHL regulatory systems? Aquat Microbial Ecol 13, 85-93.
Manefield, M., de Nys, R., Kumar, N., Read, R., Givskov, M., Steinberg, P. & Kjelleberg, S. (1999). Evidence that halogenated furanones from Delisea pulchra inhibit acylated homoserine lactone (AHL)-mediated gene expression by displacing the AHL signal from its receptor protein. Microbiology 145, 283-291.[Abstract]
Manny, A. J., Kjelleberg, S., Kumar, N., de Nys, R., Read, R. W. & Steinberg, P. (1997). Reinvestigation of the sulfuric acid-catalysed cyclisation of brominated 2-alkyllevulinic acids to 3-alkyl-5-methylene-2(5H)-furanones. Tetrahedron 53, 15813-15826.
McClean, K. H., Winson, M. K., Fish, L. & 9 other authors (1997). Quorum sensing and Chromobacterium violaceum: exploitation of violacein production and inhibition for the detection of N-acylhomoserine lactones. Microbiology 143, 37033711.[Abstract]
Nealson, K. H., Eberhard, A. & Hastings, J. W. (1972). Catabolite repression of bacterial bioluminescence: functional implications. Proc Natl Acad Sci USA 69, 1073-1076.[Abstract]
Piper, K., Beck von Bodman, S. & Farrand, S. K. (1993). Conjugation factor of Agrobacterium tumefaciens regulates Ti plasmid transfer by autoinduction. Nature 362, 448-450.[Medline]
Qin, Y., Luo, Z., Smyth, A. J., Gao, P., Beck von Bodman, S. & Farrand, S. K. (2000). Quorum-sensing signal binding results in dimerization of TraR and its release from membranes into the cytoplasm. EMBO J 19, 5212-5221.
Rasmussen, T. B., Manefield, M., Andersen, J. B., Eberl, L., Anthoni, U., Christophersen, C., Steinberg, P., Kjelleberg, S. & Givskov, M. (2000). How Delisea pulchra furanones affect quorum sensing and swarming motility in Serratia liquefaciens MG1. Microbiology 146, 3237-3244.
Rodelas, B., Lithgow, J. K., Wisniewski-Dye, F., Hardman, A., Wilkinson, A., Economou, A., Williams, P. & Downie, J. A. (1999). Analysis of quorum-sensing-dependent control of rhizosphere-expressed (rhi) genes in Rhizobium leguminosarum bv. viciae. J Bacteriol 181, 3816-3823.
Sitnikov, D. M., Schineller, J. B. & Baldwin, T. O. (1995). Transcriptional regulation of bioluminesence genes from Vibrio fischeri. Mol Microbiol 17, 801-812.[Medline]
Swift, S., Williams, P. & Stewart, G. S. A. B. (1999). N-Acylhomoserine lactones and quorum sensing in proteobacteria. In CellCell Signaling in Bacteria , pp. 291-313. Edited by G. M. Dunny & S. C. Winans. Washington DC:American Society for Microbiology.
Ulitzur, S., Matin, A., Fraley, C. & Meighen, E. (1997). H-NS protein represses transcription of the lux systems of Vibrio fischeri and other luminous bacteria cloned into Escherichia coli. Curr Microbiol 35, 336-342.[Medline]
Welch, M., Todd, D. E., Whitehead, N. A., McGowan, S. J., Bycroft, B. A. & Salmond, G. P. C. (2000). N-acyl homoserine lactone binding to the CarR receptor determines quorum-sensing specificity in Erwinia. EMBO J 19, 631-641.
Wells, P. R. (1963). Enol lactones of dibromoacetylacrylic acid. Aust J Chem 16, 165-169.
Zhang, L., Murphy, P. J., Kerr, A. & Tate, M. E. (1993). Agrobacterium conjugation and gene regulation by N-acyl-L-homoserine lactones. Nature 362, 446-448.[Medline]
Zhu, J. & Winans, S. C. (1999). Autoinducer binding by the quorum-sensing regulator TraR increases affinity for target promoters in vitro and decreases TraR turnover rates in whole cells. Proc Natl Acad Sci USA 96, 4832-4837.
Zhu, J. & Winans, S. C. (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.
Zhu, J., Beaber, J. W., More, M. I., Fuqua, C., Eberhard, A. & Winans, S. C. (1998). Analogs of the autoinducer 3-oxooctanoyl-homoserine lactone strongly inhibit activity of the TraR protein of Agrobacterium tumefaciens. J Bacteriol 180, 5398-5405.
Received 2 July 2001;
revised 7 December 2001;
accepted 19 December 2001.