Lehrstuhl für Mikrobiologie, Technische Universität München, Am Hochanger 4, 85350 Freising, Germany1
Department of Microbiology, DTU, Building 301, 2800 Lyngby, Denmark2
Author for correspondence: Leo Eberl. Tel: +49 8161 715446. Fax: +49 8161 715475. e-mail: EBERL{at}mikro.biologie.tu-muenchen.de
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ABSTRACT |
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Keywords: cystic fibrosis, N-acylhomoserine lactone, biosurfactant
Abbreviations: AHL, N-acylhomoserine lactone; CF, cystic fibrosis; CLSM, confocal laser scanning microscopy; GFP, green fluorescent protein; C8-HSL, N-octanoylhomoserine lactone; C6-HSL, N-hexanoylhomoserine lactone; 3-oxo-C6-HSL, N-(3-oxohexanoyl)homoserine lactone
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INTRODUCTION |
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The opportunistic pathogenic bacterium Pseudomonas aeruginosa is capable of chronically colonizing the lungs of patients suffering from cystic fibrosis (CF), the most common lethal inherited disease among the Caucasian population (Koch & Høiby, 1993 ; Govan & Deretic, 1996
; Tümmler & Kiewitz, 1999
). During chronic infection, P. aeruginosa produces copious amounts of alginate, which forms a matrix completely embedding the cells, and becomes highly resistant to antibiotic treatment. These observations led to the suggestion that P. aeruginosa may exist as a biofilm in the CF lung (Lam et al., 1980
; Costerton et al., 1999
). This hypothesis was recently corroborated through profiling of N-acylhomoserine lactone (AHL) signal molecules (Singh et al., 2000
). Burkholderia cepacia has been recognized as another important pathogen in patients with CF. Infection with B. cepacia often occurs in patients who are already colonized with P. aeruginosa. In fact, it has been suggested that P. aeruginosa exoproducts may modify the epithelial cell surface of the lung such that attachment of B. cepacia is facilitated (Saiman et al., 1990
). Co-colonization can result in three clinical outcomes: asymptomatic carriage, slow and continuous decline in lung function, or, for approximately 20% of the patients, fulminant and fatal pneumonia, the so-called cepacia syndrome (Isles et al., 1984
).
In both P. aeruginosa (for reviews see de Kievit & Iglewski, 2000 ; Parsek & Greenberg, 2000
; Williams et al., 2000
) and B. cepacia (Lewenza et al., 1999
), expression of various virulence factors is controlled by AHL-dependent quorum-sensing systems. These regulatory systems ensure that pathogenic traits are only expressed when the bacterial population density is high enough to overwhelm the host before it is able to mount an efficient response. Interestingly, for P. aeruginosa it has been demonstrated that the architecture of biofilms formed on an abiotic surface is also quorum-sensing-controlled (Davies et al., 1998
). These results argue in favour of functional overlaps between factors necessary for biofilm formation and pathogenicity. The quorum-sensing system of B. cepacia K56-2 (genomovar III) has been recently identified (Lewenza et al., 1999
). This density-dependent regulatory system relies on two proteins: the AHL synthase CepI, which directs the synthesis of N-octanoylhomoserine lactone (C8-HSL) and, as a minor product, N-hexanoylhomoserine lactone (C6-HSL) (Gotschlich et al., 2001
), and CepR, which after binding of C8-HSL is thought to activate or repress transcription of target genes. The cep system was demonstrated to positively regulate protease production and to repress synthesis of the siderophore ornibactin (Lewenza et al., 1999
). Since the two bacteria not only form mixed biofilms in CF lungs but also utilize the same chemical language, it appears likely that the two species synergistically enhance the others virulence (McKenney et al., 1995
).
The recent development of a simple biofilm assay has greatly facilitated the analysis of the genetic mechanisms underlying biofilm formation. In this assay, bacteria are grown in the wells of microtitre dishes in which the cells attach to the abiotic surface. Following removal of planktonic cells, the established biofilm is quantified after staining with crystal violet. Over the past few years, this assay has been extensively used to identify genes involved in biofilm formation in a number of bacteria, including Escherichia coli (Pratt & Kolter, 1998 ), Pseudomonas fluorescens (OToole & Kolter, 1998a
), P. aeruginosa (OToole & Kolter, 1998b
), Vibrio cholerae (Watnick & Kolter, 1999
) and Streptococcus gordonii (Loo et al., 2000
). In the present study we have employed this assay to isolate random transposon insertion mutants in B. cepacia H111 that are defective in biofilm formation on a polystyrene surface. One of these mutants is demonstrated to bear the transposon within the cepR gene. This finding prompted us to investigate the role of the cep quorum-sensing system in the strains ability to form biofilms. It is shown that both biofilm formation and swarming motility are cep-regulated phenotypes.
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METHODS |
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DNA manipulations and nucleotide sequencing.
Cloning, restriction enzyme analysis and transformation of E. coli were performed essentially as described by Sambrook et al. (1989) . PCR was performed using the TaKaRa rTaq DNA polymerase (TaKaRa Shuzo). Plasmid DNA was isolated with the QIAprep Spin Miniprep kit and chromosomal DNA from B. cepacia was purified with the DNeasy Tissue kit. DNA fragments were purified from agarose gels using the QIAquick Gel Extraction kit (all kits were from Qiagen).
For complementation of B. cepacia H111-R, we constructed plasmid pBAH27 (cepR+) as follows. The cepR gene was PCR-amplified using primers cepR-R (5'-GGGGTACCAACCTGACAAGTATGACAGCG-3') and cepR-OV (5'-GGGGTACCGGATGAGCATGGAGAAAAGC-3') (KpnI restriction sites are underlined). Following digestion with KpnI, the PCR fragments were inserted into the broad-host-range vector pBBR1MCS-5 cut with the same enzyme. The plasmid containing the insert in the orientation placing the cepR gene downstream of the Plac promoter of the cloning vector was chosen and this construct was designated pBAH27. For flow-chamber experiments, the strains were tagged with green fluorescent protein (GFP). This was accomplished by the insertion of a PA1/04/03-gfp-T0-T1 transposon cassette (Andersen et al., 1998 ) into the chromosomes of target strains using the suicide construct pMH94 (M. Hentzer & M. R. Parsek, unpublished results). Plasmid pMH94 was delivered to target strains by conjugative transfer and integrants were selected on PIA medium (Becton Dickinson Biosciences) containing tellurite.
Sequencing was performed by the dideoxynucleotide chain-termination method (Sanger et al., 1977 ) in a LI-COR 4200 DNA sequencer. The primer 5'-CAGATCTGATCAAGAGACAG-3', which binds to the I-end of the Tn5 transposon, was used for determination of the transposon insertion point in B. cepacia m64. DNA sequences were compared to other sequences in GenBank using the on-line BLAST search engine at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
Construction of a B. cepacia H111 mutant bank.
The hybrid transposon mini-Tn5Km2-luxCDABE was randomly inserted into the chromosome of B. cepacia H111 by the triparental mating procedure described above. Transconjugants were selected on LB medium containing kanamycin and tetracycline. These random insertion mutants were picked and grown in 150 µl LB medium in the wells of polypropylene MicroWell dishes (Nunc). For storage, 75 µl 50% (v/v) glycerol was added and the dishes were frozen at -80 °C.
Screen for mutants defective in biofilm formation.
Biofilm formation in polystyrene microtitre dishes was assayed essentially as described by OToole & Kolter (1998a ) and Pratt & Kolter (1998
) with a few modifications. Cells were grown in the wells of the microtitre dishes in 100 µl AB medium supplemented with 10 mM citrate for 48 h at 30 °C. The medium was then removed and 100 µl of a 1% (w/v) aqueous solution of crystal violet was added. Following staining at room temperature for 20 min, the dye was removed and the wells were washed thoroughly. For quantification of attached cells, the crystal violet was solubilized in a 80:20 (v/v) mixture of ethanol and acetone and the absorbance was determined at 570 nm.
Detection and characterization of AHLs.
Production of AHLs was investigated with the aid of the bioluminescent plasmid sensor pSB403 (Winson et al., 1998b ). This sensor plasmid contains the Photobacterium fischeri luxR gene together with the luxI promoter region as a transcriptional fusion to the bioluminescence genes luxCDABE of Photorhabdus luminescens. The quorum-sensing system of Photobacterium fischeri relies on N-(3-oxohexanoyl)homoserine lactone (3-oxo-C6-HSL) and the sensor plasmid consequently exhibits the highest sensitivity for this AHL molecule. However, several other AHL molecules are detected by the sensor, albeit with somewhat reduced sensitivity (Winson et al., 1998b
; Geisenberger et al., 2000
). Bioluminescence was detected either with the highly sensitive photon-counting camera C2400-40 (Hamamatsu Photonics) or by exposure to an X-ray film. For more detailed analysis, the AHL molecules were extracted from spent culture supernatants of the strains, separated by TLC and AHL spots were visualized by overlaying the TLC plates with soft agar seeded with the sensor strain E. coli MT102(pSB403) as described previously (Shaw et al., 1997
; Geisenberger et al., 2000
). Routinely, AHLs were extracted twice with dichloromethane (250:100 supernatant/dichloromethane) from 250 ml sterile-filtered supernatants of B. cepacia cultures grown in AB minimal medium containing 10 mM citrate at 30 °C to an OD600 of 1·0. The combined extracts were dried over anhydrous magnesium sulfate, filtered and evaporated to dryness. Residues were dissolved in 250 µl ethyl acetate. Samples (10 µl) were then applied to C18 reversed-phase TLC plates (Merck no. 1.15389) and dried with a stream of cold air. Samples were separated by using methanol in water (60%, v/v) as the mobile phase. For detection of AHLs, the TLC plate was overlaid with a thin film of LB agar (143 ml) seeded with 7 ml of an exponentially grown AHL biosensor and was then incubated at 30 °C for 24 h. The tentative identification of AHLs present in spent culture supernatant extracts was achieved by comparison of mobilities (RF values) relative to those for the synthetic AHL standards.
For quantification of AHL signal molecules, 100 µl of filter-sterilized supernatants of cultures grown in LB medium to an OD600 of 3·0 was added to 100 µl of an exponential culture of E. coli MT102(pSB403) in the wells of a FluoroNunc Polysorp microtitre dish. Following incubation at 30 °C for 6 h, bioluminescence was measured with a Lamda Fluoro 320 Plus reader (Bio-Tek Instruments).
Exoenzyme and siderophore production.
Exoenzyme and siderophore production was tested by streaking strains on appropriate indicator plates. Proteolytic activity was determined on LB medium supplemented with 2% skim milk, chitinolytic activity on ethylene glycol chitin agar (Connell et al., 1998 ) and lipolytic activity on tributyrin agar base containing 1% glycerol tributyrate (both Merck). Clear haloes around the colonies after incubation at 37 °C overnight indicated exoenzyme activity. Siderophore production was tested by growing strains on CAS agar (Schwyn & Neilands, 1987
) for 24 h. Siderophores remove the iron from the CAS dye complex, resulting in a colour change around the colonies from blue to orange.
Construction of B. cepacia H111 cepI and cepR mutant strains.
Defined cep mutants (Fig. 1) were constructed by the gene replacement method described by Hoang et al. (1998)
. For the construction of a cepR mutant, two DNA fragments were PCR-amplified: an 850 bp EcoRISacI fragment spanning the intergeneric region plus the first 75 bp of cepR using the primer pair intercep-f-Eco (5'-GGAATTCGAGATCCGCCGCGAGTTCG-3') and intercep-r-Sac (5'-GATCCGCTGGAAGAGCTCC-3'), and a 760 bp SphIHindIII fragment containing the 3' region of the cepR gene using the primer pair cepR-f-Sph (5'-ACATGCATGCGCTCGGATTCGAATACTGC-3') and cepR-r-Hind (5'-CCCAAGCTTAGAAGCTCGAGCAGATCGC-3'). Using the restriction sites introduced by the PCR primers (respective sites are underlined), these two DNA fragments were successively inserted into the compatible sites of the gene replacement vector pEX19Gm (Hoang et al., 1998
). Next, the npt gene from transposon Tn903 (Oka et al., 1981
), which confers resistance to kanamycin, was cloned as a 1·7 kb BamHI fragment into the vector cut with the same enzyme. The final construct, which was designated pBAH33, was transferred to B. cepacia H111 and integrants were selected on LB medium containing kanamycin and tetracycline. To screen for gene replacement mutants, Kanr clones were tested for gentamicin sensitivity as the gentamicin resistance gene is lost in the case of a double crossover event. One mutant, which was designated B. cepacia H111-R, was chosen and the correct genetic structure of the strain was confirmed by Southern Blot analysis. Construction of a defined cepI mutant was performed as described for the cepR mutant, except that the gene replacement vector pEX18Gm and two different PCR fragments were used. An 800 bp EcoRISacI fragment spanning the intergeneric region plus 100 bp of cepR was amplified with the primer pair igR-f-Eco (5'-GGAATTCCCAGTATTCGAATCCGAGCCGC-3') and igR-r-Sac (5'-CGAGCTCGGGATGTCCTCGGATCTGTGC3'), and a 650 bp BamHIHindIII fragment containing the 5' region of cepI was amplified using the primer pair cepI-f-Bam (5'-CGGGATCCCGCCTTCGTTCACGAGGAAGGG-3') and cepI-r-Hind (5'-CCCAAGCTTGGGCGCGCGTTCCGGCTCAGG-3'). The final gene replacement construct was designated pAG and the respective B. cepacia H111 cepI mutant was named H111-I.
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Microscopic inspection and image acquisition were performed on a confocal laser scanning microscope (TCS4D; Leica Lasertechnik) equipped with a 63x/1.32-0.6 oil objective. For statistical evaluation of biofilm structures, a 40x/0·75 air objective was used. Image scanning was carried out with the 488 nm laser line of an Ar/Kr laser. Captured images were visualized using the IMARIS software package (Bitplane) running on a Silicon Graphics Indigo 2 workstation.
For statistical evaluation of biofilm structures, three independent rounds of biofilm experiments were performed, and in each round, each strain was grown in two separate channels. Seven image stacks were taken of each channel every 24 h for 7 d after inoculation. These images were analysed by the computer program COMSTAT, which comprises various features for quantifying three-dimensional biofilm image stacks (Heydorn et al., 2000 ). The parameters used for characterization of biofilm architecture included biomass, substratum coverage, mean thickness and roughness coefficient.
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RESULTS |
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The suicide vector pUT (de Lorenzo & Timmis, 1994 ) was used to deliver the hybrid transposon mini-Tn5Km2-luxCDABE (Winson et al., 1998a
) into the B. cepacia H111 chromosome. A collection of 5000 random insertion mutants was screened for ability to form biofilms. A total of eighteen mutants which were to different degrees defective in biofilm formation was obtained (data not shown). During the course of a detailed phenotypical characterization of these mutants we noticed that one, m64, was deficient in the production of AHL signal molecules. As shown in Fig. 2
, the wild-type strain H111 strongly activated the bioluminescent AHL sensor plasmid pSB403 while no activation was observed with m64. This result is reminiscent of the situation found with P. aeruginosa, for which it has been shown that development of a mature biofilm is quorum-sensing-regulated (Davies et al., 1998
). We therefore focused our further investigations on the analysis of this mutant.
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Construction and characterization of defined B. cepacia H111 cep mutants
To investigate the role of the cepIR genes in biofilm formation in greater detail, we constructed site-directed insertion mutations in the two genes by using a gene replacement method (see Methods for details and Fig. 1). The genetic structure of the two mutants, which were designated H111-I and H111-R, respectively, was confirmed by Southern blot analysis (data not shown).
As expected, neither the cepI mutant H111-I nor the cepR mutant H111-R produced detectable amounts of AHLs (Fig. 2). However, production of AHLs was restored to wild-type levels when H111-R was complemented with plasmid pBAH27, which contains the cepR gene inserted into the broad-host-range vector pBBR1MCS-5.
B. cepacia produces different siderophores and a number of exoenzymes that are thought to be pathogenesis factors in humans as well as in plants (Lonon et al., 1988 ; McKevitt et al., 1989
; Gessner & Mortensen, 1990
; Yohalem & Lorbeer, 1994
; Darling et al., 1998
). In a recent study it was shown that the cep system of B. cepacia K56-2 is involved in the regulation of the synthesis of extracellular enzymes and siderophores (Lewenza et al., 1999
). We therefore tested the B. cepacia H111 wild-type and the two mutants H111-I and H111-R for the production of extracellular protease, lipase, chitinase and siderophores on appropriate indicator plates. The results of these investigations are summarized in Table 2
. Consistent with the results reported by Lewenza et al. (1999)
, both mutants showed a clear reduction in protease activity. Furthermore, proteolytic activities of the mutants were completely restored when mutant H111-I was grown in the presence of 200 nM C8-HSL or when plasmid pBAH27 (cepR+) was transferred to mutant H111-R. Both mutants were found to produce significantly lowered amounts of siderophores as assessed on CAS indicator plates. As for proteolytic activity, these defects were restored to wild-type levels by the external addition of 200 nM C8-HSL to H111-I or by complementation of H111-R with plasmid pBAH27 (cepR+). These data are in contrast to the results of the above-mentioned study, which showed that inactivation of either cepI or cepR results in an up-regulation of siderophore production in B. cepacia K56-2. Most likely, this apparent discrepancy can be attributed to the different strains used in the studies. Likewise, while Lewenza et al. (1999)
observed reduced lipase activity with the cepR but not with the cepI mutant of K56-2, we were unable to detect any difference in the lipase activities of H111, H111-I and H111-R. Chitinase activity was slightly reduced in the two mutants when compared with the wild-type, and since complementation (as described above) restored the defects we suggest that chitinase production in B. cepacia H111 is, at least in part, regulated by quorum sensing.
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Parallel flow chambers were inoculated with each of the three GFP-tagged strains and biofilm development was monitored on a daily basis for 7 d. Visual inspection of CLSM images revealed that the biofilms formed by the two mutants not only differed in their substratum coverage and thickness, as had been anticipated from the microtitre plate assays, but also exhibited strikingly different structures (Fig. 4 and data not shown). Both wild-type and mutant strains formed characteristic microcolonies after initial surface attachment. However, while wild-type biofilms rapidly matured and covered most of the available surface space within 24 h, mutant biofilms were arrested in the microcolony stage and never colonized the entire surface during the course of the experiment.
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Swarming motility of B. cepacia H111 is regulated by the cep system
Genetic studies have shown that the formation of a mature biofilm proceeds through an ordered series of steps (for recent reviews see Pratt & Kolter, 1999 ; Watnick & Kolter, 2000
; OToole et al., 2000
). In this model, motility plays a major role in biofilm formation. Flagella-mediated motility is believed to be required to overcome repulsive forces at the surface of the substratum. Furthermore, once the initial contact to the surface is established, cells are thought to move on top of the substratum to form microcolonies. Finally, these microcolonies undergo a differentiation process which leads to the development of a typical three-dimensional biofilm architecture.
For P. aeruginosa, it has been shown that aggregation of the cells to microcolonies is dependent on twitching motility, a special form of surface translocation that depends on type IV pili (OToole & Kolter, 1998b ). For V. cholerae El Tor and E. coli, it has been suggested that flagella-driven motility is not only important for initial attachment of cells to the substratum but also for translocation along the surface in a process that leads to the formation of microcolonies (Pratt & Kolter, 1998
; Watnick & Kolter, 1999
). The importance of motility for biofilm formation, together with the fact that different forms of bacterial motility, including swimming of Yersinia pseudotuberculosis (Atkinson et al., 1999
), twitching of P. aeruginosa (Glessner et al., 1999
) and swarming of Serratia liquefaciens (Eberl et al., 1996
) and P. aeruginosa (Köhler et al., 2000
) are quorum-sensing-regulated, prompted us to investigate whether the cep system of B. cepacia H111 is involved in the control of motility.
When cells of B. cepacia H111 are point-inoculated into AB minimal medium which is supplemented with 10 mM citrate and solidified with 0·3% agar, they swim through the water channels in the agar giving rise to typical chemotactic rings. Swimming behaviour of the cep mutants and the wild-type was completely indistinguishable, indicating that swimming motility is not quorum-sensing-regulated (data not shown). We also tested the strain for twitching motility under various conditions, but were unable to demonstrate this form of motility for strain H111. However, during the course of these experiments we observed that, when medium containing 0·4% agar was supplemented with 0·1% Casamino acids, cells also spread as a thin layer on the top of the agar surface. Microscopic inspection revealed that the cells migrate in a co-ordinated fashion that is characteristic of swarming motility (for reviews see Allison & Hughes, 1991 ; Harshey, 1994
; Eberl et al., 1999
). The migration front of the expanding colony is preceded by a visible layer of slime-like material giving the colony a glistening appearance, a phenomenon that is typical for this form of motility. After incubation for 36 h, B. cepacia H111 colonized the entire surface of the agar plate (Fig. 6
). By contrast, the two cep mutants were unable to swarm. Moreover, the mutants were also deficient in the production of extracellular slime. Addition of 200 nM C8-HSL to the medium restored both swarming motility and slime production of mutant H111-I (data not shown). Both phenotypes were also restored when mutant H111-R was complemented with plasmid pBAH27 (cepR+) (Fig. 6
). These results show that swarming motility of B. cepacia H111 is under control of the cep quorum-sensing system. Previously, it has been shown that AHL-mediated cellcell communication is also required for swarming motility of S. liquefaciens MG1 (Eberl et al., 1996
) and P. aeruginosa (Köhler et al., 2000
). In both bacteria the quorum-sensing systems control the production of biosurfactants, namely rhamnolipids in the case of P. aeruginosa and serrawettin W2 in the case of S. liquefaciens, which are essential for swarming motility (Ochsner & Reiser, 1995
; Lindum et al., 1998
; Köhler et al., 2000
). A S. liquefaciens mutant defective in the synthesis of AHL molecules is unable to swarm unless the medium is supplemented with either AHLs or a compound capable of lowering the surface tension of the medium such as serrawettin W2, surfactin or trace amounts of SDS (Lindum et al., 1998
; Eberl et al., 1999
). We therefore tested the two cep mutants of B. cepacia for their ability to swarm on low-agar plates supplemented with either surfactin or serrawettin W2. The two cep mutants swarmed on this medium (Fig. 6
and data not shown), suggesting that production of a biosurfactant in B. cepacia H111 is controlled by the cep quorum-sensing system, which in turn is required for swarming motility of this bacterium.
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DISCUSSION |
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During an attempt to identify cep-regulated factors required for biofilm formation by B. cepacia, we noticed that the strains ability to swarm on suitable surfaces is quorum-sensing-regulated. Swarming motility is an intrinsically surface-dependent mode of translocation, which, to the best of our knowledge, has not been reported earlier for members of the genus Burkholderia but has been described for many other bacteria (Allison & Hughes, 1991 ; Harshey, 1994
). Swarming motility of quorum-sensing-defective mutants of B. cepacia H111 could be fully restored by supplementing the media with different surfactants. We therefore propose that the cep system of B. cepacia controls the production of a biosurfactant, which is required for swarming motility of the strain. Noteworthy in this context is the recent finding that another genomovar III B. cepacia strain (J2315) produces a lipopeptide of unknown structure which exhibits strong surface-active properties (Hutchison et al., 1998
). Our results are reminiscent of the situation found with S. liquefaciens and P. aeruginosa. These two bacteria have been demonstrated to employ quorum-sensing systems to control the synthesis of the surface-active compounds serrawettin W2 and rhamnolipids, respectively (Ochsner & Reiser, 1995
; Lindum et al., 1998
). Since the ability to swarm is strictly dependent on the production of biosurfactants, swarming motility is a quorum-sensing-regulated phenomenon in both bacteria.
The addition of surfactants to the medium, at concentrations sufficiently high to restore swarming motility of the cep mutants to the level of the wild-type, only weakly, if at all, increased biofilm formation by the B. cepacia H111 cep mutants. This suggests that locomotion via swarming motility is not required for biofilm formation. On the other hand, the cep-regulated production of the biosurfactant itself may affect biofilm formation as previous results have demonstrated that various surface-active compounds have the capability of regulating the attachment and detachment of bacteria to and from surfaces (Rosenberg & Ron, 1999 ; Ahimou et al., 2000
). There are several possible explanations as to why our attempts to substitute the missing biosurfactant with surfactin, serrawettin W2 or SDS failed to restore biofilm formation by the cep mutants: (i) the physical properties of the surfactants used and the one produced by the strain are substantially different, (ii) production of the surfactant has to follow a specific temporal and/or spatial expression pattern within the biofilm, or (iii) other cep-regulated, as yet unidentified, factors may be required for biofilm formation. Work currently under way aims to test these possibilities.
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ACKNOWLEDGEMENTS |
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This work was supported by grants from the BMBF and the DFG (EB 2051/1-2) to L.E. and the Danish Medical Research Council to M.G.
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Received 3 March 2001;
revised 2 May 2001;
accepted 8 May 2001.