Department of Microbiology, Technical University of Denmark, 2800 Lyngby, Denmark1
School of Microbiology and Immunology, University of New South Wales, Sydney, Australia2
Centre for Marine Biofouling and Bio-Innovation, University of New South Wales, Sydney, Australia3
Lehrstuhl fur Mikrobiologie, Technische Universitat Munchen, Freising Munich, D-85350, Germany4
Marine Chemistry Section, H. C. Ørsted Institute, University of Copenhagen, 2100 Copenhagen, Denmark5
Author for correspondence: Michael Givskov. Tel: +45 45252769. Fax: +45 45932809. e-mail: immg{at}pop.dtu.dk
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: swarming, Serratia liquefaciens, signalling, furanones
Abbreviations: AHL, N-acyl-L-homoserine lactone; C4-HSL, N-butanoyl-L-homoserine lactone; C6-HSL, N-hexanoyl-L-homoserine lactone; C8-HSL, octanoyl-L-homoserine lactone; 3-oxo-C6-HSL, N-(3-oxohexanoyl)-L-homoserine lactone; 3-oxo-C12-HSL, N-(3-oxododecanoyl)-L-homoserine lactone
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The formation of a swarm colony requires environmental as well as intercellular signals, of which viscosity, surface contact and the quorum size of the bacterial population are considered the most important (Eberl et al., 1996a , b
). We hypothesize that exposure of cells to surfaces with a certain viscosity is recognized by an unknown sensor and that signal transduction then progresses via the products of the flhDC master operon (Eberl et al., 1996b
; Tolker-Nielsen et al., 2000
). This leads to swarm-cell differentiation, which involves development of characteristic traits such as cell elongation, multinucleation and increased flagellation. Swarm cells never travel alone, and only a dense bacterial population is able to display swarming motility. Sensing of the population density is accomplished by a quorum-sensing system that is constituted by the swrR and swrI genes and the two N-acyl-L-homoserine lactones (AHL) signal molecules N-butanoyl-L-homoserine lactone (C4-HSL) and N-hexanoyl-L-homoserine lactone (C6-HSL) (Eberl et al., 1996a
, 1999
). According to two-dimensional PAGE analysis, at least 28 genes are under the control of this system (Givskov et al., 1998
); one of these target genes, swrA, was identified and found to encode a putative peptide synthase which in turn gives rise to production of an extracellular bio-surfactant serrawettin W2 (Lindum et al., 1998
). The extracellular localization of this surfactant is crucial for expansion of the growing and differentiated cell mass (Lindum et al., 1998
).
Brominated furanones, produced as secondary metabolites by the benthic marine macro-alga Delisea pulchra, inhibit swarming motility of S. liquefaciens (Givskov et al., 1996 ) and Proteus mirabilis (Gram et al., 1996
), and antagonize surface colonization in general (Kjelleberg et al., 1997
). These compounds did not abolish the developmental process leading to differentiated swarm cells nor did they affect growth rate of S. liquefaciens (Givskov et al., 1996
). The inhibitory effect of the furanones on swarming motility was reversible by the addition of increasing concentrations of exogenous C4-HSL (Givskov et al., 1996
). One interpretation of these data are that furanones act by mimicking the native prokaryotic AHL signal, presumably by occupying the binding site on the putative regulatory protein, SwrR (Eberl et al., 1999
). The inhibitory effect of these furanones on bioluminescence in a Vibrio fischeri LuxR-based AHL monitor system supports this and strongly suggests that the inhibitory activity extends to a range of AHL-controlled phenotypes. Using tritium labelled N-(3-oxo)-hexanoyl-L-homoserine lactone (3-oxo-C6-HSL) and different furanones, Manefield et al. (1999)
showed that the furanones are capable of displacing 3-oxo-C6-HSL from an Escherichia coli strain overproducing LuxR, thus providing direct evidence for the specific activity of the furanones. In this study we demonstrate that furanones, in competition with C4-HSL, control transcription of the important quorum-sensing target gene swrA, reduce the amount of serrawettin W2 produced and thereby abolish swarming motility. Furthermore, it is demonstrated that the furanones can interfere with not only intraspecies cellcell communication but also interspecies communication in the process of colonization of bacterial communities.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Drop-collapsing test for surfactant production.
One millilitre of an overnight culture of S. liquefaciens MG1 was washed with fresh medium, resuspended in 1 ml medium and 200 µl was plated out on each of two swarm plates. One of the plates contained 20 µM compound 2, the other had no addition of furanone. The plates were incubated overnight at 30 °C and the cultures were washed off using 3 ml 0·9% NaCl in water. Cells were removed by centrifugation and 5 µl was put on top of a Petri dish. If the droplet contains a surfactant it collapses after a few minutes. Assessment of surface tension was performed as described by Lindum et al. (1998) .
Extraction and identification of signal molecules.
AHL molecules were extracted from two swarm plates, one in which the medium contained 20 µM compound 2 and 3 µg serrawettin W2 ml-1 and one with no addition. Prior to extraction, the swarm plates were inoculated with S. liquefaciens MG1 and incubated for 16 h at 30 °C. After incubation the entire agar slab was transferred to a 250 ml flask containing 25 ml ethyl acetate. The flask was shaken vigorously several times during a period of 20 min. The ethyl acetate was evaporated on a rotary evaporator and the remnant was dissolved in 50 µl ethyl acetate and 20 µl were subjected to TLC analysis as described by Gram et al. (1999) .
Mixed swarm assays.
Overnight cultures of the relevant strains were washed in fresh medium and then mixed in equal ratio. Five microlitres of the mixture was inoculated on swarm plates containing various amounts of furanones and surfactant. After incubation the cultures were visualized using bright field and epifluorescence microscopy as described by Eberl et al. (1999) .
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
One simple way to assess the effect of furanones on serrawettin W2 synthesis was by means of a drop-collapsing test (Lindum et al., 1998 ). Serrawettin W2 was extracted from cultures grown in the presence or absence of 20 µM compound 2. This analysis showed that the culture grown in the absence of compound 2 produced an extracellular molecule that lowered the surface tension of water. In contrast, extracts from the culture that was grown in the presence of compound 2 displayed a much reduced ability to lower the surface tension. Performed this way, a positive drop-collapsing test correlates with the presence of at least 0·8 µg serrawettin W2 ml-1. This is also the minimum requirement for scoring a positive swarm phenotype with S. liquefaciens PL10 (swrI, swrA::luxAB) which carries an inactivated swrA gene (Lindum et al., 1998
). This strongly suggests that exogenous addition of compound 2 leads to a reduction in serrawettin W2 production.
Effect of furanone on swrA transcription
The chromosomally located transcriptional swrA::luxAB fusion was used to demonstrate that transcription of swrA is stimulated by external addition of C4-HSL (Lindum et al., 1998 ). PL10 swarm cells grown in the presence of serrawettin W2 were washed off from the edge of swarming colonies and resuspended in fresh growth medium prior to subsequent measurements of bioluminescence and optical density. Maximum induction of the swrA::luxAB fusion was recorded when the mutant was grown as a swarming colony on plates containing 400 nM C4-HSL (Fig. 3
). In the absence of exogenous C4-HSL, swrA::luxAB expression was 10 relative light units (OD450 unit)-1 (data not shown). Activity was induced upon addition of exogenous C4-HSL, from a ninefold induction in presence of 100 nM C4-HSL up to a 45-fold induction at 400 nM C4-HSL. The presence of 20 µM compound 2 resulted in a significantly lower swrA::luxAB expression. Whether 100, 200 or 400 nM C4-HSL was present, the swrA activity was markedly lowered. The data presented in Fig. 3
indicate that transcription of the swrA gene is inhibited by compound 2. Similar results were obtained with compound 1 (data not shown). Taken together these results suggest that compounds 1 and 2 function as competitive inhibitors to the cognate signal molecule C4-HSL.
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study we have utilized swarming motility as a model system for a quorum-sensing-controlled colonization phenotype and to explore the genetic effects exerted on this system by AHL antagonists. The model system was used previously to demonstrate the existence of natural quorum-sensing antagonists being produced by a eukaryote (Givskov et al., 1996 ). Halogenated furanones produced by Delisea pulchra were found to reduce the motility of the swarm cells by means other than influencing flagellar synthesis, cell differentiation or growth rate (Givskov et al., 1996
). Instead, surfactant deficiency would explain the lack of swarming motility, as the addition of at least 0·8 µg serrawettin W2 ml-1 is needed to achieve a sufficient reduction of surface tension and allow for swarming motility. There are two likely sites of action of the furanone to accomplish the inhibition of surfactant production. Either the furanone inhibits the activity of the serrawettin W2 peptide synthase or it interferes with the putative C4-HSL receptor and thereby displaces the AHL molecule. In this case, however, the first option is unlikely because at the same time, both transcription of the swrA gene and production of serrawettin W2 were severely reduced in the presence of compound 2. Lack of SwrA activity is not due to interference with AHL production, as demonstrated in Fig. 4
. Taken together with the observation that addition of exogenous surfactant can restore swarming motility when compound 2 is present at high concentrations as seen in Figs 2
, 5
, 6 and Table 2
, it strongly suggests that furanones inhibit the communication system. The findings of Manefield et al. (1999)
, indicating displacement of 3-oxo-C6-HSL from a strain overproducing LuxR by furanones, support this hypothesis.
The information available on bacteria expressing quorum-sensing-regulated phenotypic traits for colonization and pathogenesis is growing. It has been proposed that different species, co-existing in nature, are able to co-operate using AHL signal molecules (Hardman et al., 1998 ). One such example is demonstrated by Bassler et al. (1997)
who found that several species, including Vibrio parahaemolyticus and Vibrio cholerae, were able to induce bioluminescence in Vibrio harveyi. Another example is the ability of natural, non-swarming, non-AHL producers (such as E. coli K-12 and the majority of Pseudomonas putida strains) that harbour a swrI+-containing plasmid as well as non-swarming AHL producers to form swarming colonies in conjunction with the S. liquefaciens swrI mutant (Eberl et al., 1999
). In this study, intercellular communication has been visualized by means of the AHL sensor plasmid carried by the S. liquefaciens MG44 mutant. The presence of green fluorescent cells tells us that AHL target gene expression is turned on in the presence of the incoming AHL molecules (J. B. Andersen and others, unpublished). This strongly suggests that AHL signals originating from the AHL producers trigger surfactant synthesis in the population of S. liquefaciens swrI cells. Thus, the organisms interact by means of chemical signals originating from the AHL producers and reception by the other members of the population. The combined action of the different species enables the mixed culture to express the biological phenomenon, in this case swarming motility. The ability of halogenated furanones to interfere with such co-operative behaviour is demonstrated in a similar scenario. The presence of non-fluorescent S. liquefaciens MG44 cells indicates that the algal metabolites shut down the process of intercellular communication. Reception of the signal and thereby activation of target genes is hindered; thus, the biological phenomenon, swarming, is not observed in the presence of furanones.
As shown in Fig. 5 and Table 2
, swarming motility was inhibited by 20 µM compound 2 but the expression of the PluxI::gfp(ASV) reporter was not. These results suggest that higher concentrations of furanones are needed to inhibit the plasmid-borne system. This may be due to the artificially high copy number of the reporter promoter. It is also likely that the two quorum-sensing systems have different sensitivities towards AHLs and furanones. Another possible explanation for this phenomenon is the nature of the AHL molecules being produced. Both P. aeruginosa PAO1 and S. ficaria produce 3-oxo-C6-HSL, which is the native AHL for the lux system on which the sensor is based (J. B. Andersen and others, unpublished). 3-oxo-C6-HSL is much less capable of activating the swr system than the lux system the binding affinity is probably much lower (Eberl et al., 1996a
). Hence, it is possible that compound 2 can displace 3-oxo-C6-HSL from SwrR more easily than LuxR, resulting in different sensitivities of the two systems towards the furanone.
The data presented in Fig. 5 and Table 2
also reveal that the signal molecules enter the cells in the presence of compound 2. This rules out the possibility that the inhibitory effect of furanones on swarming motility is caused by interference with the uptake of AHL molecules. However, in the two cases with 3-oxo-C6-HSL-producing strains and no surfactant added, a dense colony was formed in which it was impossible to shut down the AHL monitor. This can be explained by assuming that the internal concentration of 3-oxo-C6-HSL is very high in the dense colony. By employing a new, quantitative method for AHL analysis, a high concentration of 3-oxo-AHLs were found in biofilms of P. aeruginosa (T. Charlton & S. Kjelleberg, unpublished data). It is likely that the high sensitivity of the LuxR-based AHL sensor and the competitive nature of the inhibition renders the furanone unable to shut down the sensor.
Work in progress in our laboratories suggests that S. liquefaciens employs signals to successfully accomplish three distinct stages of colonization: initial attachment, swarming and biofilm formation (M. Labatte & S. Kjelleberg, unpublished data). This paper has demonstrated that signals and signal antagonists control the second stage in this series of colonization events. Furthermore, the inhibition of intercellular communication and hence swarming is not limited to one species but extends to cover interspecies communication. This may have great impact in natural habitats where different species, which can co-operate using AHL signal molecules, are present in microniches (McKenney et al., 1995 ). Moreover, these data suggest a means by which quorum-sensing-antagonist producing eukaryotes control the development of surface communities (Kjelleberg et al., 1997
). It would appear that swarming on surfaces represent a trait common to many bacteria and hence can be presumed to facilitate the colonization of living organisms (Gram et al., 1996
).
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Allison, C., Jones, P., Coleman, N. & Hughes, C. (1992b). Ability of Proteus mirabilis to invade human urothelial cells is coupled to motility and swarming differentiation. Infect Immun 60, 4740-4746.[Abstract]
Andersen, J. B., Sternberg, C., Poulsen, L. K., Bjorn, 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.
Bassler, B. L., Greenberg, P. & Stevens, A. M. (1997). Cross-species induction of luminescence in the quorum-sensing bacterium Vibrio harveyi. J Bacteriol 179, 4043-4045.[Abstract]
Costerton, J. W., Lewandowski, Z., Caldwell, D. E., Korber, D. R. & Lappin-Scott, H. M. (1995). Microbial biofilms. Annu Rev Microbiol 49, 711-747.[Medline]
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.
Eberl, L., Winson, M. K., Sternberg, C. & 7 other authors (1996a). Involvement of N-acyl-L-homoserine lactone autoinducers in control of multicellular behaviour of Serratia liquefaciens. Mol Microbiol 20, 127136.
Eberl, L., Christiansen, G., Molin, S. & Givskov, M. (1996b). Differentiation of Serratia liquefaciens into swarm cells is controlled by the expression of the flhD master operon. J Bacteriol 178, 554-559.[Abstract]
Eberl, L., Molin, S. & Givskov, M. (1999). Surface motility of Serratia liquefaciens MG1. J Bacteriol 181, 1703-1712.
Geisenberger, O., Givskov, M., Riedel, K., Høiby, N., Tümmler, B. & Eberl, L. (2000). Production of N-acyl-L-homoserine lactones by Pseudomonas aeruginosa isolates from chronic lung infections associated with cystic fibrosis. FEMS Microbiol Lett 184, 273-277.[Medline]
Givskov, M., Olsen, L. & Molin, S. (1988). Cloning and expression in Escherichia coli of the gene for extracellular phospholipase A1 from Serratia liquefaciens. J Bacteriol 170, 5855-5862.[Medline]
Givskov, M., Eberl, L., Christiansen, G., Benedik, M. J. & Molin, S. (1995). Induction of phospholipase- and flagellar synthesis in Serratia liquefaciens is controlled by expression of the flagellar master operon flhD. Mol Microbiol 15, 445-454.[Medline]
Givskov, M., de Nys, R., Manefield, M., Gram, L., Maximilien, R., Eberl, L., Molin, S., Steinberg, P. & Kjelleberg, S. (1996). Eukaryotic interference with homoserine lactone mediated prokaryotic signaling. J Bacteriol 178, 6618-6622.[Abstract]
Givskov, M., Eberl, L. & Molin, S. (1997). Control of exoenzyme production, motility and cell differentiation in Serratia liquefaciens. FEMS Microbiol Lett 148, 115-122.
Givskov, M., Östling, J., Lindum, P. W., Eberl, L., Christiansen, G., Molin, S. & Kjelleberg, S. (1998). The participation of two separate regulatory systems in controlling swarming motility of Serratia liquefaciens. J Bacteriol 180, 742-745.
Gram, L., de Nys, R., Maximilien, R., Givskov, M., Steinberg, P. & Kjelleberg, S. (1996). Inhibitory effects of secondary metabolites from the red alga Delisea pulchra on swarming motility of Proteus mirabilis. Appl Environ Microbiol 62, 4284-4287.[Abstract]
Gram, L., Christensen, A. B., Ravn, L., Molin, S. & Givskov, M. (1999). Production of acetylated homoserine lactones by psychrophilic members of the Enterobacteriaceae isolated from foods. Appl Environ Microbiol 65, 3458-3463.
Hardman, A. M., Stewart, G. S. A. B. & Williams, P. (1998). Quorum sensing and the cell-cell communication dependent regulation of gene expression in pathogenic and non-pathogenic bacteria. Antonie Leeuwenhoek 74, 199-210.[Medline]
Hassett, D. J., Ma, J., Elkins, J. G. & 10 other authors (1999). Quorum sensing in Pseudomonas aeruginosa controls expression of catalase and superoxide dismutase genes and mediates biofilm susceptibility to hydrogen peroxide. Mol Microbiol 34, 10821093.[Medline]
Kjelleberg, S., Steinberg, P. D., Givskov, M., Manefield, M. & de Nys, R. (1997). Do marine products interfere with procaryotic AHL regulatory systems? Aquat Microbiol Ecol 13, 85-93.
Koch, C. & Høiby, N. (1993). Pathogenesis of cystic fibrosis. Lancet 341, 1065-1069.[Medline]
Lindum, P. W., Anthoni, U., Christoffersen, C., Eberl, L., Molin, S. & Givskov, M. (1998). -acyl-L-homoserine lactone autoinducers control production of an extracellular surface active lipopeptide required for swarming motility of Serratia liquefaciens MG1. J Bacteriol 180, 6384-6388.
McKenney, D., Brown, K. & Allison, D. G. (1995). Influence of Pseudomonas aeruginosa exoproducts on virulence factor production in Burkholderia cepacia: evidence of interspecies communication. J Bacteriol 177, 6989-6992.[Abstract]
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, 283-292.[Abstract]
Pearson, J. P., Gray, K. M., Passador, L., Tucker, K. D., Eberhard, A., Iglewski, B. H. & Greenberg, E. P. (1994). Structure of the autoinducer required for the expression of Pseudomonas aeruginosa virulence genes. Proc Natl Acad Sci USA 91, 197-201.[Abstract]
Pearson, J. P., Pesci, E. C. & Iglewski, B. H. (1997). Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in control of elastase and rhamnolipid biosynthesis genes. J Bacteriol 179, 5756-5767.[Abstract]
Shapiro, J. A. (1998). Thinking about bacterial populations as multicellular organisms. Annu Rev Microbiol 52, 81-104.[Medline]
Swift, S., Karlyshev, A., Fish, L., Durant, E., Winson, M., Chhabra, S., Williams, P., Macintyre, S. & Stewart, G. (1997). Quorum sensing in Aeromonas hydrophila and Aeromonas salmonicida: identification of the LuxRI homologs AhyRI and AsaRI and their cognate N-acylhomoserine lactone signal molecules. J Bacteriol 179, 5271-5281.[Abstract]
Swift, S., Williams, P. & Stewart, G. S. A. B. (1999). -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.
Telford, G., Wheeler, D., Williams, P., Tomkins, P. T., Appleby, P., Sewell, H., Stewart, G. S. A. B., Bycroft, B. W. & Pritchard, D. I. (1998). The Pseudomonas aeruginosa quorum-sensing signal molecule N-(3-oxododecanoyl)-L-homoserine lactone has immunomodulatory activity. Infect Immun 66, 36-42.
Tolker-Nielsen, T., Christensen, A. B., Eberl, L., Rasmussen, T. B., Sternberg, C., Molin, S. & Givskov, M. (2000). Assessment of flhDC mRNA levels in individual Serratia liquefaciens swarm cells. J Bacteriol 182, 2680-2686.
Winson, M., Camara, M., Latifi, A. & 10 other authors (1995). Multiple N-acyl-L-homoserine lactone signal molecules regulate production of virulence determinants and secondary metabolites in Pseudomonas aeruginosa. Proc Natl Acad Sci USA 92, 94279431.
Received 12 July 2000;
revised 7 September 2000;
accepted 14 September 2000.