Autoinducer 2 Controls Biofilm Formation in Escher
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细菌学杂志 2006年第1期
Departments of Chemical Engineering and Molecular & Cell Biology, University of Connecticut, 191 Auditorium Rd., Storrs, Connecticut 06269-3222,Department of Chemical and Biomolecular Engineering, University of Maryland, College Park, Center for Biosystems Research, UMBI, College Park, Maryland 20742
ABSTRACT
The cross-species bacterial communication signal autoinducer 2 (AI-2), produced by the purified enzymes Pfs (nucleosidase) and LuxS (terminal synthase) from S-adenosylhomocysteine, directly increased Escherichia coli biofilm mass 30-fold. Continuous-flow cells coupled with confocal microscopy corroborated these results by showing the addition of AI-2 significantly increased both biofilm mass and thickness and reduced the interstitial space between microcolonies. As expected, the addition of AI-2 to cells which lack the ability to transport AI-2 (lsr null mutant) failed to stimulate biofilm formation. Since the addition of AI-2 increased cell motility through enhanced transcription of five motility genes, we propose that AI-2 stimulates biofilm formation and alters its architecture by stimulating flagellar motion and motility. It was also found that the uncharacterized protein B3022 regulates this AI-2-mediated motility and biofilm phenotype through the two-component motility regulatory system QseBC. Deletion of b3022 abolished motility, which was restored by expressing b3022 in trans. Deletion of b3022 also decreased biofilm formation significantly, relative to the wild-type strain in three media (46 to 74%) in 96-well plates, as well as decreased biomass (8-fold) and substratum coverage (19-fold) in continuous-flow cells with minimal medium (growth rate not altered and biofilm restored by expressing b3022 in trans). Deleting b3022 changed the wild-type biofilm architecture from a thick (54-μm) complex structure to one that contained only a few microcolonies. B3022 positively regulates expression of qseBC, flhD, fliA, and motA, since deleting b3022 decreased their transcription by 61-, 25-, 2.4-, and 18-fold, respectively. Transcriptome analysis also revealed that B3022 induces crl (26-fold) and flhCD (8- to 27-fold). Adding AI-2 (6.4 μM) increased biofilm formation of wild-type K-12 MG1655 but not that of the isogenic b3022, qseBC, fliA, and motA mutants. Adding AI-2 also increased motA transcription for the wild-type strain but did not stimulate motA transcription for the b3022 and qseB mutants. Together, these results indicate AI-2 induces biofilm formation in E. coli through B3022, which then regulates QseBC and motility; hence, b3022 has been renamed the motility quorum-sensing regulator gene (the mqsR gene).
INTRODUCTION
There is an explosive amount of research on biofilms with the ultimate aim of their control (24); however, little is known about the regulation of this complex process of cell attachment leading to exquisite architecture (11). Since 65% of human bacterial infections involve biofilms (31), understanding the genetic basis of biofilm formation to find effective ways to prevent biofilms is important for combating disease and for engineering applications. To this end, we have studied the whole bacterial genome with DNA microarrays by two complementary approaches: studying biofilm gene expression relative to planktonic cells (34, 35) and studying plant-derived biofilm inhibitors that do not alter the bacterial growth rate, such as ursolic acid (38) and (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone (furanone) of the alga Delisea pulchra (36, 37). We found that furanone inhibits Escherichia coli biofilm formation and that 80% of the genes that were repressed by furanone were induced by cross-species quorum-sensing signal autoinducer 2 (AI-2) (36); hence, AI-2 should stimulate biofilm formation.
Bacteria use quorum sensing to regulate some forms of gene expression by sensing their population density via small signaling compounds that are secreted into the environment (3). AI-2 is produced by LuxS, is a species-nonspecific signal used by both gram-negative and gram-positive bacteria (47), and has been found in at least 55 strains (4). Three groups have used DNA microarrays to show AI-2 controls 166 to 404 genes, including those for chemotaxis, flagellar synthesis, motility, and virulence factors in E. coli (15, 36, 44). However, the species-nonspecific signal AI-2 has not been shown directly to control biofilms.
Quorum sensing has been linked to biofilms previously, since a species-specific signal, N-(3-oxododecanoyl)-L-homoserine lactone, has been shown to influence biofilm formation in Pseudomonas aeruginosa (14). In addition, quorum sensing controls biofilm formation by controlling exopolysaccharide synthesis in Vibrio cholerae (which has homoserine lactone and AI-2 signals) (19), by controlling cell aggregation in Serratia liquefaciens (which has a homoserine lactone signal) (24), and by controlling genetic competence in Streptococcus mutans (which has a peptide signal) (25). AI-2 has been found to influence biofilm formation in a mixed-species biofilm between Streptococcus gordonii and Porphyromonas gingivalis (26) and has been shown to impact slightly the architecture of Klebsiella pneumoniae (although no effect of AI-2 on biofilm formation was found using a luxS mutant for intestinal colonization and colonization on polystyrene) (2) and to affect attachment in Helicobacter pylori (a luxS homolog has been found that negatively regulates biofilm formation) (10). For these few AI-2 results with biofilms, mutants or conditioned media were used rather than the signal itself and the role of AI-2 was not clear; indeed, a recent report indicates that LuxS has no effect on biofilm formation of Haemophilus influenzae (12). Here, we show conclusively that synthesized AI-2 directly stimulates biofilm formation in E. coli, that it controls biofilm architecture, that it controls this phenotype by stimulating bacterial motility, and that it does this through the uncharacterized protein MqsR (B3022).
Although the E. coli locus mqsR (b3022) was found to be induced eightfold in biofilms (35), there is little information about the function of MqsR. MqsR appears to be a conserved regulator protein (98 amino acids), since it has >50% homology with hypothetical proteins from Yersinia pseudotuberculosis, Yersinia pestis, Cupriavidus oxalaticus, Bordetella bronchiseptica, Pseudomonas fluorescens, and Bordetella pertussis (8, 27, 29, 43, 48). As part of the 300-gene, quorum-sensing regulon in E. coli (15, 36, 44), qseBC (b3025 and b3026) are organized in an operon in the E. coli chromosome with QseB playing a role as a response regulator and QseC playing a role as the sensor kinase (45). Flagellum expression is temporally regulated, and the operons are divided into early, middle, and late genes. QseBC regulates transcription of the master regulon flhDC and therefore expression of the middle operon (e.g., fliA encoding sigma factor 28) and late operon (e.g., fliC encoding flagellin and motA encoding the proton exchange conductor for flagellum movement) (9). Here, we determined that MqsR controls biofilm formation in E. coli by positively regulating qseBC; hence, MqsR is the mediator between AI-2 and motility.
MATERIALS AND METHODS
Bacterial strains, growth media, and toxicity testing. The strains and plasmids of this study are listed in Table 1. Luria-Bertani medium (LB) (41) was used to preculture all the E. coli cells. LB, minimal M9 medium with 0.4% (wt/vol) Casamino Acids and 0.8-g/liter sodium citrate (M9C citrate) (40), LB supplemented with 0.2% (wt/vol) glucose (LB glu), and M9 supplemented with 0.4% (wt/vol) glucose and 0.4% (wt/vol) Casamino Acids (M9C glu) were used for the 96-well biofilm experiments. For the flow cell experiments, M9C glu medium was used for MG1655 and MG1655 mqsR, and LB was used for ATCC 25404. For purification of Pfs and LuxS, E. coli DH5 carrying pTrcHis-pfs or pTrcHis-luxS from E. coli W3110 (21) was cultured in SOB medium (20 g of tryptone/liter, 5 g of yeast extract/liter, 0.5 g of sodium chloride/liter, 2.5 mM potassium chloride, and 10 mM magnesium chloride) (41).
Construction of the complementation plasmid pVLT31 mqsR+. To show that the mqsR gene is responsible for the altered motility phenotype, a complementation plasmid was constructed using the low-copy-number plasmid pVLT31 (16). The whole coding sequence of mqsR was amplified using the primer pair pA (front primer, 5'-GGTTATAACTGAATTCACAGGGAGGCGGGG-3') and pB (rear primer, 5'-GCCAGAAACCATTTCTAGATGGTGGCAAACCGG-3'). The PCR product was digested with EcoR I and XbaI and cloned into the multiple cloning site of pVLT31 that was digested with the same two enzymes to create the complementation plasmid pVLT31 mqsR+. This recombinant plasmid was confirmed through DNA sequencing upstream of the ptac promoter using primer pC (5'-GAGCGGATAACAATTTCACACAGG-3'). Since pVLT31 carries lacIq, expression of the mqsR+ gene requires isopropyl- D-thiogalactopyranoside (IPTG; Sigma, St. Louis, Mo.), which was added at 0.4 mM for complementing motility and at 0.2 mM for complementing biofilm formation of the mqsR mutant.
Synthesis of AI-2. At an optical density at 600 nm (OD600) of 0.4 to 0.6, 1 mM IPTG was added to induce expression of His-Pfs or His-LuxS. After 4 h of induction, cells were collected by centrifugation at 14,000 x g for 20 min at 4°C. The cells were stored at –80°C and lysed using BugBuster solution (Novagen) (300 μl and 2.5 ml culture pellets for His-Pfs or 1.5 ml and 50 ml culture pellets for His-LuxS) for 20 min at room temperature. Soluble cell extracts were collected by centrifugation at 14,000 x g for 20 min at 4°C, mixed with Co2+ affinity resin (BD TALON; BD Biosciences), and washed with equilibration-wash buffer (50 mM sodium phosphate, 0.3 M sodium chloride [pH 7.0]). Twenty microliters and 600 μl of Co2+ resin suspension were mixed with the soluble cell extracts from the 2.5-ml and 50-ml cultures. His-Pfs or His-LuxS was bound to the Co2+ resin, and the Co2+ resin was washed to remove nonspecifically bound proteins as described in the manufacturer's protocol. The bound proteins were eluted with 300 μl of elution buffer (125 mM imidazole in equilibration-wash buffer) containing 100 μM zinc chloride, 10 mM mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride. His-tagged protein purification was performed at 0°C, Co2+ resin was removed by centrifugation, and the supernatant was extracted twice with chloroform. Image analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels indicated Pfs (28,899 Da) and LuxS (23,962 Da) were highly purified (>99%; data not shown), and no other bands were seen. In addition, no other immunoreactive bands were detected by using anti-His immunoblots.
The purified Pfs and LuxS enzymes were used to synthesize AI-2 from 1 mM S-adenosylhomocysteine in 50 mM Tris-HCl, pH 7.8 (41), containing 100 μM zinc chloride and 1 mM phenylmethylsulfonyl fluoride. In vitro AI-2 synthesis reactions were carried out at 37°C overnight. Concentrations of His-Pfs and His-LuxS in the reaction mixtures were 8 μM and 69 μM, respectively. High-performance liquid chromatography showed complete conversion of SAH by Pfs, as well as the complete conversion of S-ribosylhomocysteine by LuxS in the AI-2 samples.
Autoinducer activity assay. Activity of the synthesized AI-2 was assayed as described previously (46). Briefly, the reporter strain Vibrio harveyi BB170 was grown in autoinducer bioassay medium (0.3 M NaCl, 0.05 M MgSO4, 0.2% Casamino Acids, 10 μM KH2PO4, 1 μM L-arginine, 20% glycerol, 0.01 μg/ml riboflavin, and 1 μg/ml thiamine) overnight and diluted 1:5,000 into the fresh AB medium, and then AI-2 was added at 0.2, 0.4, 0.8, or 1.6 μM. The time course of bioluminescence was measured with a 20/20 luminometer (Turner Design, Sunnyvale, CA) and reported as relative light units. The cell density of the V. harveyi reporter strain was measured by spreading the cells on Luria marine medium (20 g/liter NaCl, 10 g/liter Bacto tryptone, and 5 g/liter yeast extract) plates and counting the CFU after 24 h. Each experiment was conducted in duplicate. The optimum concentration of AI-2 for bioluminescence (0.8 μM) was used as the basis for evaluating the effect of AI-2 on E. coli biofilm formation (0.8, 1.6, 3.2, and 6.4 μM).
Crystal violet biofilm assay. This assay was adapted from those reported previously (32). E. coli was grown in polystyrene 96-well plates at 37°C for 2 days without shaking in LB medium, M9C glu, LB glu medium, or M9C citrate supplemented with AI-2. Each data point was averaged from four replicate wells, and the standard deviations were calculated. Plates were processed after 24 h. The experiments were conducted twice using two independent cultures with each culture evaluated in four wells (total of eight wells). Negative controls were wells containing 11 μM (each) adenine and homocysteine.
Flow cell biofilm experiments and image analysis. LB medium was supplemented with 200-μg/ml erythromycin to maintain pCM18 (20) (Table 1), which contains the constitutive green fluorescent protein (GFP) vector and which allows visualization of the biofilm. The biofilm was formed at 37°C in a continuous flow cell that consists of a standard glass microscope slide on one side and a plastic coverslip on the other side with dimensions of 47.5 mm by 12.7 mm with a 1.6-mm gap between the surfaces (BST model FC81; Biosurface Technologies Corp., Bozeman, MT). Overnight cultures in LB medium supplemented with 120- to 200-μg/ml erythromycin (to retain the GFP plasmid pCM18) were centrifuged and resuspended in LB medium with erythromycin. This diluted culture (OD600, 0.05) was used to inoculate the flow cell for 2 h at a flow rate of 10 ml/h before fresh LB or M9C glu medium flow with erythromycin was started at the same flow rate; the initial inoculum was 1.5 x 108 cells/ml. To determine the impact of AI-2, 6.4 μM was added upon inoculation and in the continuous feed or homocysteine and adenine were added (each, 6.4 μM) as the negative control. Biofilm development in the flow cell was monitored with a TCS SP2 scanning confocal laser microscope (Leica Microsystems, Heidelberg, Germany) with a 40x objective at 16 and 24 h.
Color confocal flow cell images were converted to grayscale using Image Converter (Neomesh Microsystems, Wainuiomata, Wellington, New Zealand). Biomass, substratum coverage, surface roughness, and mean thickness were determined with COMSTAT image-processing software (22) written as a script in Matlab 5.1 (The MathWorks) and equipped with the Image Processing Toolbox. Thresholding was fixed for all image stacks. At each time point, nine different positions were chosen for microscope analysis, and 225 images were processed for each time point. Values are means of data from the different positions at the same time point, and standard deviations were calculated based on these mean values for each position. Simulated three-dimensional images were obtained by using IMARIS (BITplane, Zurich, Switzerland). Twenty-five pictures were processed for each three-dimensional image.
Motility assay. LB overnight cultures were used to assay motility in plates containing 1% tryptone, 0.25% NaCl, and 0.3% agar (45). The motility halos were measured at 8 h for ATCC 25404, MG1655, and MG1655 mqsR/pVLT31 mqsR+ and at 16 h for DH5, JM109, and BW25113. Between 3 and 25 plates were used to evaluate motility in each strain. Motility agar plates containing AI-2 (0.8 or 3.2 μM) were used to test the impact of AI-2 on motility, and homocysteine and adenine (each, 0.8 or 3.2 μM) were added to the agar as a negative control. Each experiment was performed in duplicate.
Transcription reporter assays. To determine the effect of AI-2 on the expression of the motility genes, ATCC 25404 cultures with the lacZ fusion transcriptional reporters qseB::lacZ, flhD::lacZ, fliA::lacZ, fliC::lacZ, and motA::lacZ (45) were cultured overnight in LB ampicillin (100 μg/ml), diluted 1:100 in LB medium, and then grown to stationary phase to an OD600 of 3, since internalization of AI-2 takes place primarily in stationary phase (53). Once cells reached stationary phase, AI-2 was added at 6.4 μM for 2 h (adenine and homocysteine were added, each at a concentration of 6.4 μM, as a negative control). Galactosidase activity was evaluated as described previously (51). All activities were calculated based on a protein concentration of 0.24 mg of protein/ml/OD600 unit (17).
To determine the effect of mqsR and qseB on the expression of the motility genes, MG1655 and MG1655 mqsR were cultured overnight in LB ampicillin (100 μg/ml), diluted 1:100 in LB medium, and grown to exponential phase to an OD600 of 1. When the effect of AI-2 on qseB expression in MG1655 and MG1655 mqsR was tested, cells were cultured overnight in LB ampicillin (100 μg/ml), diluted 1:100 in M9C glu medium, different concentrations of AI-2 (0, 3.2, and 6.4 μM) were added, and the cells were grown to exponential phase (OD600 of 1). Homocysteine and adenine (each, 6.4 μM) were added to cultures as a negative control. The same procedure was followed for testing qseB expression in MG1655 mqsR complemented with pVLT31 mqsR+, but instead of AI-2, IPTG was added at different concentrations (0, 0.1, 0.2, 0.4, and 1 mM).
Microarray analysis. The strains were cultured in LB medium overnight (with kanamycin added to MG1655 mqsR), diluted 1:100 in LB medium, and grown to exponential phase (OD600, 1); total RNA was isolated as described previously (35). The Affymetrix E. coli GeneChip antisense genome array (catalogue no. 900381), which contains probe sets for all 4,290 open reading frames, rRNA, tRNA, and 1,350 intergenic regions, was used to study the effect of the mqsR deletion on the gene expression profile of E. coli. Briefly, the total RNA samples were first converted into cDNA through a reverse transcription reaction with poly(A) RNA controls spiked into the same reaction mixture to monitor the entire target labeling process. The cDNA was then digested with DNase I (Amersham Biosciences) to produce 50- to 200-bp fragments, which were checked by running the fragmented cDNA on a 2% agarose gel. The fragmented cDNA was labeled at the 3' termini by the Enzo BioArray Terminal Labeling kit with Biotin-ddUTP (catalogue no. 900181; Affymetrix). The biotin-labeled target was hybridized to the Affymetrix GeneChip E.coli antisense array at 45°C for 16 h at 60 rpm using the Hybridization Oven 640 (Affymetrix), and then a three-step fluorescent staining was conducted using the Fluidics Station 450 (Affymetrix) during the washing and staining procedure. This includes binding of streptavidin to the biotin-labeled cDNA in the first staining solution, binding of biotin-conjugated streptavidin antibody to the streptavidin in the second staining solution, and binding of phycoerythrin-conjugated streptavidin to the biotin-labeled antibody in the third staining solution. The microarray was scanned at 570 nm to get an image file with the GeneChip Scanner 3000 (Affymetrix). Using GeneChip Operating Software, individual strain reports for both the wild-type strain and mutant cDNA samples were obtained, as well as reports comparing the mqsR mutant to wild-type E. coli. Total cell intensity was scaled automatically in the software to an average value of 500. Since the standard deviation for the expression ratio for all the genes was 2.7, genes with a 4-fold change in intensity between the two chips and a P value of <0.05 were considered differentially expressed.
RESULTS
AI-2 stimulates E. coli biofilm formation in 96-well plates. We suspected AI-2 was involved directly in biofilm formation, since our microarray data indicated that AI-2 controls motility-related genes (e.g., cheABRWYZ, flgABCDEFGHIJKLMN, fliACDFHKLMNOPQ, and motAB) (36) and since the plant-derived furanone inhibits biofilms and represses the same AI-2-controlled genes (36). To investigate this hypothesis, we synthesized AI-2 (there is no commercial source) using two E. coli enzymes and found it was active via a 2,400-fold increase in bioluminescence in the V. harveyi BB170 reporter (0.8 μM AI-2). Then, this active AI-2 (0.2 to 11 μM) was evaluated for its effect on the biofilm formation of three E. coli wild-type strains (ATCC 25404, MG1655, and BW25113), the LuxS-deficient strains E. coli BW25113 luxS and E. coli DH5, and the well-known laboratory strain E. coli JM109. Biofilm formation was stimulated significantly by 11 μM AI-2 in LB medium at 24 h for ATCC 25404 (26 ± 2 fold), DH5 (29 ± 0.3 fold), and MG1655 (4 ± 2 fold) (Table 2). Biofilm formation was also stimulated in JM109 and BW25113 (2 ± 1 fold at 3.2 μM) and in the luxS mutant of BW25113 (1.7 ± 0.3 fold at 1.25 μM). These results with rich media were corroborated with M9C citrate, where 3.2 μM AI-2 stimulated biofilm formation after 24 h for DH5 (2.2 ± 1 fold) and JM109 (1.7 ± 0.4 fold).
Note that in the absence of AI-2, BW25113 luxS made 50% less biofilm than the isogenic wild-type strain, which indicates again that AI-2 stimulates biofilm formation in E. coli, since LuxS forms AI-2. Biofilm could be restored by adding complementing luxS in trans using plasmid pCA24N luxS+ (46% of the wild-type biofilm was formed at 0 mM IPTG and 110% of the wild-type biofilm was formed at 0.25 mM IPTG in LB medium).
To confirm that AI-2 was the cause of the increase in biofilm formation, we measured biofilm stimulation with the isogenic MG1655 lsrK mutant because this mutation dramatically impairs the AI-2 uptake compared with other mutations in the lsr system (49, 52). As expected, AI-2 was not able to induce biofilm formation of the lsrK mutant at 6.4 μM (Fig. 1A); hence, AI-2 induces biofilm formation through the LsrK transport pathway (49).
E. coli DH5, which is deficient in AI-2 synthesis (36) and which was found here to be completely nonmotile, was also studied using the continuous flow cell to see the effect of the luxS mutation on biofilm formation (Table 3) and architecture (image not shown). Although we recognize that this strain is not isogenic with ATCC 25404, we thought it might be informative to see if it responded to AI-2 and to look at its architecture. Compared with ATCC 25404 without AI-2, DH5 displayed less biomass (15 ± 4 μm3/μm2 versus 8 ± 3 μm3/μm2), less substratum coverage (57% ± 7% versus 41% ± 11%), and less thickness (25 ± 7 μm versus 10 ± 4 μm).
We also tested the effect of AI-2 (6.4 μM) on biofilm formation when strains harbored the derepressed conjugation plasmid R1drd19, which enhances biofilm formation (Fig. 1A) (18). Biofilm formation was not significantly induced with either MG1655 or BW25113 when R1drd19 was present.
AI-2 promotes E. coli biofilm formation in a continuous flow cell. To further investigate the effect of AI-2 on biofilm architecture, as well as to corroborate the 96-well plate crystal violet results, a continuous flow cell with LB medium was used to study the biofilm of ATCC 25404 (harboring the GFP plasmid pCM18). In the absence of AI-2, ATCC 25404 developed regular microcolonies covering 41 and 57% of the surface at 16 and 24 h, respectively (Table 3; Fig. 2A); previously similar structures were seen for E. coli SAR18 and MG1655 (33). The addition of 6.4 μM AI-2 also led to the formation of typical microcolonies (Fig. 2B), but the amount of attached cells was greater, since the biomass and thickness increased 5-fold ± 3-fold and 7-fold ± 2-fold at 16 h and 5-fold ± 1-fold and 4-fold ± 1-fold at 24 h, respectively. The roughness coefficient (3-fold ± 1-fold decrease) (Table 3 and Fig. 2) indicated that there was less heterogeneity when AI-2 is added, since the biofilm had fewer interstitial spaces at both times analyzed.
AI-2 increases motility in E. coli through QseB. To determine how AI-2 stimulates biofilm formation in E. coli, we investigated whether AI-2 addition affected the motility of five strains, since our microarrays (36) indicated that these genes were induced by AI-2 (determined by using a luxS mutant). The motility of both ATCC 25404 and MG1655 increased about 30% upon the addition of 0.8 μM AI-2. It was necessary to increase the AI-2 dose to 3.2 μM to see an effect with DH5 and BW25113 (80% and 43% increases in motility, respectively). JM109 did not respond significantly to AI-2 addition at 3.2 μM.
To discern the genetic basis of this increase in motility upon AI-2 addition, we probed the ability of AI-2 to induce the promoters of motility genes qseB, flhD, fliA, fliC, and motA. Upon addition of 6.4 μM AI-2, the quorum-sensing flagellum regulon qseB (45) was induced 8-fold ± 3-fold. These results corroborated the ones reported by Sperandio et al. (45), who previously found that qseB was induced 17-fold compared with the luxS mutant through DNA microarray studies with E. coli O157:H7 and its isogenic luxS mutant. The induction of qseB here led to a 4.0-fold ± 0.1-fold increase in the transcription of flhD (master controller of the flagellum regulon), a 2.6-fold ± 0.3-fold increase in fliA (sigma factor 28), a 3.6-fold ± 0.8-fold increase in fliC (flagellin), and a 6-fold ± 0.3-fold increase in motA (proton conductor for flagellum movement).
Based on this increase in motility through QseB upon AI-2 addition, we hypothesized that AI-2 induces biofilm formation by inducing motility and that this increase in motility leads to increased attachment. To test our hypothesis, we measured biofilm formation upon the addition of AI-2 to two motility-deficient strains. We added AI-2 to the paralyzed isogenic MG1655 motA mutant (9) (we confirmed that this strain is nonmotile), which has reduced biofilm formation (32), and to the isogenic MG1655 qseB mutant, which we found to have impaired motility as previously reported (45). As expected, biofilm formation (Fig. 1A) was not altered when AI-2 was added to both motility mutants, nor did it affect MG1655 fliA (reduction of motility was corroborated for this strain, too).
Deletion of mqsR decreases biofilm formation in 96-well plates and continuous flow system. Since mqsR is induced eightfold in biofilms (35) and is near qseBC (Fig. 3), we investigated its role in AI-2-controlled biofilm formation. Initially, we confirmed the impact of the mqsR deletion on biofilm formation of MG1655 by using 96-well plates after 24 h. Deletion of mqsR decreased biofilm formation in LB (74%), M9C glu (46%), and LB glu (78%) (Fig. 1B). Biofilm formation in flow cells corroborated these results (Table 3; Fig. 2C and D), since deleting mqsR at 48 h led to an 8-fold ± 14-fold reduction in biomass, a 19-fold ± 12-fold reduction in substratum coverage, and a 4-fold ± 3-fold change in thickness. Deleting mqsR changed the biofilm architecture significantly from a 54-μm-thick film with microcolonies to one with nearly no biomass (few colonies remaining). The 7.5-fold ± 4.6-fold increase in the roughness coefficient (24 h) also indicated that there were few colonies formed after deletion of mqsR. Growth was not altered after mqsR was deleted, so the changes in the biofilm were not a result of growth rate differences; the specific growth rates in LB were 1.64 ± 0.02/h versus 1.720 ± 0.008/h for MG1655 and MG1655 mqsR, respectively, while in M9C glu the specific growth rates were 0.990 ± 0.004/h versus 0.93 ± 0.03/h, respectively.
To corroborate that mqsR actually controls biofilm formation, we complemented mqsR in trans by constructing a low-copy-number plasmid (pVLT31) which expresses mqsR+ upon IPTG addition. By the addition of IPTG to MG1655mqsR/pVLT31 mqsR+ in LB medium, biofilm formation was restored from 30% of MG1655/pVLT31 at 0 mM IPTG to 84% at 0.2 mM IPTG; hence, MqsR regulates biofilm formation.
Note that the deletion of fliA, qseB, and motA also inhibited biofilm formation substantially (Fig. 1A and B). This appears to be the first report about the regulation of biofilm by QseB.
MqsR controls motility by regulating QseBC, FliA, and MotA. QseB and QseC are a two-component, quorum-sensing controlled regulator system for motility (45). We hypothesized that mqsR induces biofilm formation by regulating the two-component regulatory system qseBC, which then regulates the motility master regulon flhD (9). In agreement with this hypothesis, we found that when mqsR was deleted, motility was abolished (Fig. 4). Furthermore, this lack of motility due to the deletion of mqsR was caused by a reduction in transcription of qseB (61-fold ± 27-fold), fliA (2.4-fold ± 2-fold), and motA (18-fold ± 10- fold) (Fig. 5A) in M9C glu. Similar results were found with LB, as qseB, fliA, and motA decreased 2.3-fold ± 1.4-fold, 5-fold ± 1-fold, and 11-fold ± 11-fold, respectively (results not shown). Note that although flhD transcription was not altered substantially in these experiments, its expression was altered greatly in the DNA microarrays (Table 4). To corroborate that mqsR abolishes motility, we complemented mqsR in trans with the low-copy-number plasmid pVLT31, which carries mqsR+ (Fig. 4). Increasing the expression of mqsR by adding IPTG reestablished cell motility in a dose-dependent manner until it reached 50% of wild-type motility at 0.4 mM IPTG. Hence, MqsR regulates biofilm formation by inducing motility through QseBC.
MqsR is a global regulator in E. coli MG1655. Considering the size of MqsR (98 aa), we hypothesized it could be a global regulator in E. coli. To investigate this, differential gene expression was determined for MG1655 and MG1655 mqsR in LB liquid culture. By deleting mqsR, 41 genes were down-regulated >4-fold, while 33 genes were up-regulated >4-fold (Tables 4 and 5). Of the 246 genes that were down-regulated two- to ninefold, 14% (34 genes) were reported to be AI-2 controlled (15, 36, 44), which supplies additional evidence that MqsR is a global mediator between AI-2 and E. coli. Note that the genes that encode the master flagellum regulons flhD and flhC were down-regulated 24- and 7.5-fold, respectively, which also corroborates that MqsR regulates motility by controlling the master flagellum regulon flhDC. It was also found MqsR induced curli expression, based on its 26-fold differential expression of crl (Table 4), a transcriptional regulator of the cryptic csgAB locus for curli surface fibers (6), which has been reported to play a role in E. coli biofilm formation (7). The array results also showed that MqsR regulates motility by controlling not only QseB (Fig. 5A) but also csrA, which is down-regulated in the mqsR mutant (twofold) and which regulates motility master regulon expression in E. coli (50).
MqsR links AI-2 quorum-sensing signal and biofilm formation. We tested the effect of the mqsR deletion on the ability of AI-2 to induce biofilm formation in LB by using microtiter plates. The addition of 6.4 μM AI-2 increased MG1655 biofilm mass by 2.8-fold ± 0.5-fold, while it had no effect when mqsR was deleted (Fig. 1A). Therefore, induction of biofilm formation by increasing motility is mediated by MqsR.
Induction of motility with AI-2 is mediated by MqsR and then QseBC. To corroborate the results obtained by Sperandio et al. (45), we measured the expression of qseB in MG1655 upon the addition of our synthesized AI-2 (0.8 μM). As expected, qseB transcription increased eightfold upon the addition of AI-2 (results not shown), while Sperandio et al. found sixfold induction by using preconditioned Dulbecco's modified Eagle medium. To find if the induction of motility with AI-2 was mediated by both MqsR and QseBC, we then measured motA expression in the qseB and mqsR mutants upon the addition of AI-2 (0.8 μM) and compared it to that of wild-type MG1655 (Fig. 5B). The addition of AI-2 induced motA activity for the wild-type strain but did not induce motA in the mqsR and qseB mutants; therefore, the induction of motility was mediated by both MqsR and QseBC (Fig. 3).
Further evidence that mqsR is first in the cascade was provided by measuring the transcription of qseB with the wild-type strain and the mqsR mutant upon the addition of AI-2. If MqsR is first in the cascade and necessary for the transduction of the AI-2 signal, then the addition of AI-2 should only increase qseB transcription when mqsR is not deleted. We found that adding AI-2 at 6.4 μM induced the expression of qseB 3.2-fold in the wild-type strain in M9C glu but did not induce qseB in the mqsR mutant (Fig. 6A). As expected, the wild-type strain responded to AI-2 addition in a dose-dependent manner. To show further that MqsR is first in the cascade, the expression of qseB from pVS159 was also measured while inducing MqsR expression in trans in the mqsR mutant by adding IPTG to strains harboring pVLT31 mqsR+. As expected if MqsR was required for signal transduction to QseB, expression of qseB was induced fourfold in a dose-dependent manner in M9C glu (Fig. 6B). Hence, MqsR is first in the cascade (Fig. 3).
DISCUSSION
We focused on E. coli biofilms, since this strain is the most thoroughly studied bacterium (5), its genome is sequenced (5), microarrays are available (35, 42), the functions of many of its proteins have been elucidated (39), and many isogenic mutants are available (23). Also, our group has experience in determining the genetic basis of E. coli biofilm formation and its inhibition with natural, plant-derived antagonists (37, 38). Although E. coli is well studied, its biofilm has not received the same scrutiny, since many (but not all) K-12 strains make a poor biofilm if they lack a conjugative plasmid (18, 33).
In this study, we show that cross-species quorum-sensing signal AI-2 stimulates biofilm formation in five different E. coli hosts (ATCC 25404, MG1655, BW25113, DH5, and JM109), in two different media (M9 citrate and LB), and in both batch and continuous flow systems (which enables the biofilm to be studied under two different hydrodynamic conditions, corroborates the results, and allows the biofilm architecture to be examined); hence, stimulation of biofilm formation by AI-2 is a general phenomenon. Our results here serve to explain two results that have been found previously: that AI-2 controls chemotaxis, flagellar synthesis, and motility in E. coli (36, 44) and that the quorum-sensing antagonist furanone was effective in preventing the biofilms of E. coli by repressing these same chemotaxis, flagellar synthesis, and motility genes (36). Therefore, AI-2 stimulates biofilm formation directly, flagellar synthesis and motility are clearly involved in biofilm formation, and furanone inhibits biofilm formation by masking AI-2.
We have also shown here that AI-2 stimulates biofilm formation by increasing motility, since addition of AI-2 stimulated the motility of two strains (ATCC 25404 and MG1655), since AI-2 addition had no effect on the biofilm formation of the motility-impaired mutants motA and qseB (Fig. 1A), since the transcription of flagellar genes is induced by AI-2, and since the biomass, substratum coverage, and biofilm thickness of the luxS mutant E. coli DH5, which has abolished motility, are less than those of E. coli ATCC 25404 with AI-2 (Table 3). Furthermore, by stimulating motility, the addition of AI-2 changes the architecture of the ATCC 25404 biofilm to a flatter phenotype in flow cells (Fig. 2). Previous reports have indicated that motility plays an important role in the attachment of cells to the surface (32), but here we show that motility (stimulated by AI-2) affects the architecture, too.
Previous reports have indicated AI-2 is not necessary for mature biofilm formation when a conjugation plasmid such as R1drd19 is present in minimal AB medium with glucose (33). Here, along with one of the first applications of synthesized AI-2, we used wild-type strains that lack a conjugation plasmid and were cultured in rich medium and found AI-2 plays a surprisingly large role in biofilm formation (25-fold). The difference in results was most likely due to the lack of the conjugation plasmid, as we showed here (Fig. 1A), as well as to the difference in hosts used (that is one reason we verified our results with five familiar strains). Contrary to previous reports (18), the wild-type strain (MG1655) harboring the conjugative plasmid forms less biofilm in the presence of AI-2 than the non-plasmid-carrying strain (Fig. 1A). One explanation may be that we found that the addition of conjugation plasmids induces biofilm formation by inducing cell aggregation, not by changing motility (18a). We believe that cells harboring R1drd19 in the presence of AI-2 have induced motility, which may impede cell aggregation and thereby decrease biofilm formation. The fact that we saw the smallest stimulation of both biofilm formation and motility with AI-2 for JM109 corroborates this, since JM109 contains the F' conjugation plasmid.
We also found that AI-2 stimulates biofilm formation via the uncharacterized protein MqsR by showing that MqsR induces motility (Fig. 4) and biofilm formation in both batch and continuous systems (Fig. 1B and 2), that AI-2 stimulates motility through MotA (Fig. 5B) and biofilm formation through MqsR (Fig. 1A), and that MqsR stimulates QseB (Fig. 5A), which controls motility in E. coli. Previous reports have found relationships between quorum sensing and biofilms (28), but these reports have not found the genetic underpinnings behind the biofilm phenotype. Based on the discovery in the present work that AI-2 stimulates biofilms directly, we propose a genetic model (Fig. 3) for how AI-2 controls biofilm formation in E. coli. Sperandio et al. (45) found the link between AI-2 and motility for the two-component regulatory system qseBC, yet they proposed that additional regulators in the cascade that mediate motility and quorum sensing need to be found and characterized. One of these links is now found, and it connects AI-2, MqsR, QseBC, and biofilm formation.
Our model (Fig. 3) is that AI-2 stimulates biofilm formation by stimulating expression of MqsR, which then directly or indirectly induces expression of QseBC, which then promotes cell motility via the master regulon flhDC, which then stimulates MotA and FliA and leads to biofilm formation. Without this stimulation of motility, biofilm formation is severely impaired (Fig. 1). We found that qseB is controlled by MqsR (Fig.5A) and that MqsR controls flhDC (Table 4) and therefore motility. We also found that MqsR induces curli expression through crl (Table 4) and possibly induces motility through csrA. Hence, MqsR controls biofilm formation by inducing motility and curli synthesis. Considering that MqsR controls 108 proteins with unknown functions (Tables 4 and 5) and that MqsR is a global AI-2-controlled regulator, there are many new proteins to investigate in regard to biofilm formation, control, and quorum sensing.
In summary, we have determined that the species-nonspecific,quorum signal AI-2 directly stimulates biofilm formation in E. coli, that the mechanism is through stimulating motility genes, and that MqsR mediates this effect prior to QseBC. These results serve to make sense of our previous microarray data and serve to give a deeper understanding of how plant biofilm inhibitors work. Hence, our results are helpful for understanding and preventing biofilm formation by the archetypal strain, as well as helpful for combating related pathogenic strains such as E. coli O157:H7 (30).
ACKNOWLEDGMENTS
A.F.G.B. was supported by a Fulbright scholarship (FB2454106-2), and this research was supported by the National Science Foundation (BES-0124401) and the U.S. Environmental Protection Agency.
We thank A. Heydorn from the Technical University of Denmark for kindly providing COMSTAT, S. Molin from the Technical University of Denmark for sending plasmid pCM18, and J. Kaper from the University of Maryland for sending plasmids pVS159, pVS176, pVS175, pVS182, and pVS183.
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ABSTRACT
The cross-species bacterial communication signal autoinducer 2 (AI-2), produced by the purified enzymes Pfs (nucleosidase) and LuxS (terminal synthase) from S-adenosylhomocysteine, directly increased Escherichia coli biofilm mass 30-fold. Continuous-flow cells coupled with confocal microscopy corroborated these results by showing the addition of AI-2 significantly increased both biofilm mass and thickness and reduced the interstitial space between microcolonies. As expected, the addition of AI-2 to cells which lack the ability to transport AI-2 (lsr null mutant) failed to stimulate biofilm formation. Since the addition of AI-2 increased cell motility through enhanced transcription of five motility genes, we propose that AI-2 stimulates biofilm formation and alters its architecture by stimulating flagellar motion and motility. It was also found that the uncharacterized protein B3022 regulates this AI-2-mediated motility and biofilm phenotype through the two-component motility regulatory system QseBC. Deletion of b3022 abolished motility, which was restored by expressing b3022 in trans. Deletion of b3022 also decreased biofilm formation significantly, relative to the wild-type strain in three media (46 to 74%) in 96-well plates, as well as decreased biomass (8-fold) and substratum coverage (19-fold) in continuous-flow cells with minimal medium (growth rate not altered and biofilm restored by expressing b3022 in trans). Deleting b3022 changed the wild-type biofilm architecture from a thick (54-μm) complex structure to one that contained only a few microcolonies. B3022 positively regulates expression of qseBC, flhD, fliA, and motA, since deleting b3022 decreased their transcription by 61-, 25-, 2.4-, and 18-fold, respectively. Transcriptome analysis also revealed that B3022 induces crl (26-fold) and flhCD (8- to 27-fold). Adding AI-2 (6.4 μM) increased biofilm formation of wild-type K-12 MG1655 but not that of the isogenic b3022, qseBC, fliA, and motA mutants. Adding AI-2 also increased motA transcription for the wild-type strain but did not stimulate motA transcription for the b3022 and qseB mutants. Together, these results indicate AI-2 induces biofilm formation in E. coli through B3022, which then regulates QseBC and motility; hence, b3022 has been renamed the motility quorum-sensing regulator gene (the mqsR gene).
INTRODUCTION
There is an explosive amount of research on biofilms with the ultimate aim of their control (24); however, little is known about the regulation of this complex process of cell attachment leading to exquisite architecture (11). Since 65% of human bacterial infections involve biofilms (31), understanding the genetic basis of biofilm formation to find effective ways to prevent biofilms is important for combating disease and for engineering applications. To this end, we have studied the whole bacterial genome with DNA microarrays by two complementary approaches: studying biofilm gene expression relative to planktonic cells (34, 35) and studying plant-derived biofilm inhibitors that do not alter the bacterial growth rate, such as ursolic acid (38) and (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone (furanone) of the alga Delisea pulchra (36, 37). We found that furanone inhibits Escherichia coli biofilm formation and that 80% of the genes that were repressed by furanone were induced by cross-species quorum-sensing signal autoinducer 2 (AI-2) (36); hence, AI-2 should stimulate biofilm formation.
Bacteria use quorum sensing to regulate some forms of gene expression by sensing their population density via small signaling compounds that are secreted into the environment (3). AI-2 is produced by LuxS, is a species-nonspecific signal used by both gram-negative and gram-positive bacteria (47), and has been found in at least 55 strains (4). Three groups have used DNA microarrays to show AI-2 controls 166 to 404 genes, including those for chemotaxis, flagellar synthesis, motility, and virulence factors in E. coli (15, 36, 44). However, the species-nonspecific signal AI-2 has not been shown directly to control biofilms.
Quorum sensing has been linked to biofilms previously, since a species-specific signal, N-(3-oxododecanoyl)-L-homoserine lactone, has been shown to influence biofilm formation in Pseudomonas aeruginosa (14). In addition, quorum sensing controls biofilm formation by controlling exopolysaccharide synthesis in Vibrio cholerae (which has homoserine lactone and AI-2 signals) (19), by controlling cell aggregation in Serratia liquefaciens (which has a homoserine lactone signal) (24), and by controlling genetic competence in Streptococcus mutans (which has a peptide signal) (25). AI-2 has been found to influence biofilm formation in a mixed-species biofilm between Streptococcus gordonii and Porphyromonas gingivalis (26) and has been shown to impact slightly the architecture of Klebsiella pneumoniae (although no effect of AI-2 on biofilm formation was found using a luxS mutant for intestinal colonization and colonization on polystyrene) (2) and to affect attachment in Helicobacter pylori (a luxS homolog has been found that negatively regulates biofilm formation) (10). For these few AI-2 results with biofilms, mutants or conditioned media were used rather than the signal itself and the role of AI-2 was not clear; indeed, a recent report indicates that LuxS has no effect on biofilm formation of Haemophilus influenzae (12). Here, we show conclusively that synthesized AI-2 directly stimulates biofilm formation in E. coli, that it controls biofilm architecture, that it controls this phenotype by stimulating bacterial motility, and that it does this through the uncharacterized protein MqsR (B3022).
Although the E. coli locus mqsR (b3022) was found to be induced eightfold in biofilms (35), there is little information about the function of MqsR. MqsR appears to be a conserved regulator protein (98 amino acids), since it has >50% homology with hypothetical proteins from Yersinia pseudotuberculosis, Yersinia pestis, Cupriavidus oxalaticus, Bordetella bronchiseptica, Pseudomonas fluorescens, and Bordetella pertussis (8, 27, 29, 43, 48). As part of the 300-gene, quorum-sensing regulon in E. coli (15, 36, 44), qseBC (b3025 and b3026) are organized in an operon in the E. coli chromosome with QseB playing a role as a response regulator and QseC playing a role as the sensor kinase (45). Flagellum expression is temporally regulated, and the operons are divided into early, middle, and late genes. QseBC regulates transcription of the master regulon flhDC and therefore expression of the middle operon (e.g., fliA encoding sigma factor 28) and late operon (e.g., fliC encoding flagellin and motA encoding the proton exchange conductor for flagellum movement) (9). Here, we determined that MqsR controls biofilm formation in E. coli by positively regulating qseBC; hence, MqsR is the mediator between AI-2 and motility.
MATERIALS AND METHODS
Bacterial strains, growth media, and toxicity testing. The strains and plasmids of this study are listed in Table 1. Luria-Bertani medium (LB) (41) was used to preculture all the E. coli cells. LB, minimal M9 medium with 0.4% (wt/vol) Casamino Acids and 0.8-g/liter sodium citrate (M9C citrate) (40), LB supplemented with 0.2% (wt/vol) glucose (LB glu), and M9 supplemented with 0.4% (wt/vol) glucose and 0.4% (wt/vol) Casamino Acids (M9C glu) were used for the 96-well biofilm experiments. For the flow cell experiments, M9C glu medium was used for MG1655 and MG1655 mqsR, and LB was used for ATCC 25404. For purification of Pfs and LuxS, E. coli DH5 carrying pTrcHis-pfs or pTrcHis-luxS from E. coli W3110 (21) was cultured in SOB medium (20 g of tryptone/liter, 5 g of yeast extract/liter, 0.5 g of sodium chloride/liter, 2.5 mM potassium chloride, and 10 mM magnesium chloride) (41).
Construction of the complementation plasmid pVLT31 mqsR+. To show that the mqsR gene is responsible for the altered motility phenotype, a complementation plasmid was constructed using the low-copy-number plasmid pVLT31 (16). The whole coding sequence of mqsR was amplified using the primer pair pA (front primer, 5'-GGTTATAACTGAATTCACAGGGAGGCGGGG-3') and pB (rear primer, 5'-GCCAGAAACCATTTCTAGATGGTGGCAAACCGG-3'). The PCR product was digested with EcoR I and XbaI and cloned into the multiple cloning site of pVLT31 that was digested with the same two enzymes to create the complementation plasmid pVLT31 mqsR+. This recombinant plasmid was confirmed through DNA sequencing upstream of the ptac promoter using primer pC (5'-GAGCGGATAACAATTTCACACAGG-3'). Since pVLT31 carries lacIq, expression of the mqsR+ gene requires isopropyl- D-thiogalactopyranoside (IPTG; Sigma, St. Louis, Mo.), which was added at 0.4 mM for complementing motility and at 0.2 mM for complementing biofilm formation of the mqsR mutant.
Synthesis of AI-2. At an optical density at 600 nm (OD600) of 0.4 to 0.6, 1 mM IPTG was added to induce expression of His-Pfs or His-LuxS. After 4 h of induction, cells were collected by centrifugation at 14,000 x g for 20 min at 4°C. The cells were stored at –80°C and lysed using BugBuster solution (Novagen) (300 μl and 2.5 ml culture pellets for His-Pfs or 1.5 ml and 50 ml culture pellets for His-LuxS) for 20 min at room temperature. Soluble cell extracts were collected by centrifugation at 14,000 x g for 20 min at 4°C, mixed with Co2+ affinity resin (BD TALON; BD Biosciences), and washed with equilibration-wash buffer (50 mM sodium phosphate, 0.3 M sodium chloride [pH 7.0]). Twenty microliters and 600 μl of Co2+ resin suspension were mixed with the soluble cell extracts from the 2.5-ml and 50-ml cultures. His-Pfs or His-LuxS was bound to the Co2+ resin, and the Co2+ resin was washed to remove nonspecifically bound proteins as described in the manufacturer's protocol. The bound proteins were eluted with 300 μl of elution buffer (125 mM imidazole in equilibration-wash buffer) containing 100 μM zinc chloride, 10 mM mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride. His-tagged protein purification was performed at 0°C, Co2+ resin was removed by centrifugation, and the supernatant was extracted twice with chloroform. Image analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels indicated Pfs (28,899 Da) and LuxS (23,962 Da) were highly purified (>99%; data not shown), and no other bands were seen. In addition, no other immunoreactive bands were detected by using anti-His immunoblots.
The purified Pfs and LuxS enzymes were used to synthesize AI-2 from 1 mM S-adenosylhomocysteine in 50 mM Tris-HCl, pH 7.8 (41), containing 100 μM zinc chloride and 1 mM phenylmethylsulfonyl fluoride. In vitro AI-2 synthesis reactions were carried out at 37°C overnight. Concentrations of His-Pfs and His-LuxS in the reaction mixtures were 8 μM and 69 μM, respectively. High-performance liquid chromatography showed complete conversion of SAH by Pfs, as well as the complete conversion of S-ribosylhomocysteine by LuxS in the AI-2 samples.
Autoinducer activity assay. Activity of the synthesized AI-2 was assayed as described previously (46). Briefly, the reporter strain Vibrio harveyi BB170 was grown in autoinducer bioassay medium (0.3 M NaCl, 0.05 M MgSO4, 0.2% Casamino Acids, 10 μM KH2PO4, 1 μM L-arginine, 20% glycerol, 0.01 μg/ml riboflavin, and 1 μg/ml thiamine) overnight and diluted 1:5,000 into the fresh AB medium, and then AI-2 was added at 0.2, 0.4, 0.8, or 1.6 μM. The time course of bioluminescence was measured with a 20/20 luminometer (Turner Design, Sunnyvale, CA) and reported as relative light units. The cell density of the V. harveyi reporter strain was measured by spreading the cells on Luria marine medium (20 g/liter NaCl, 10 g/liter Bacto tryptone, and 5 g/liter yeast extract) plates and counting the CFU after 24 h. Each experiment was conducted in duplicate. The optimum concentration of AI-2 for bioluminescence (0.8 μM) was used as the basis for evaluating the effect of AI-2 on E. coli biofilm formation (0.8, 1.6, 3.2, and 6.4 μM).
Crystal violet biofilm assay. This assay was adapted from those reported previously (32). E. coli was grown in polystyrene 96-well plates at 37°C for 2 days without shaking in LB medium, M9C glu, LB glu medium, or M9C citrate supplemented with AI-2. Each data point was averaged from four replicate wells, and the standard deviations were calculated. Plates were processed after 24 h. The experiments were conducted twice using two independent cultures with each culture evaluated in four wells (total of eight wells). Negative controls were wells containing 11 μM (each) adenine and homocysteine.
Flow cell biofilm experiments and image analysis. LB medium was supplemented with 200-μg/ml erythromycin to maintain pCM18 (20) (Table 1), which contains the constitutive green fluorescent protein (GFP) vector and which allows visualization of the biofilm. The biofilm was formed at 37°C in a continuous flow cell that consists of a standard glass microscope slide on one side and a plastic coverslip on the other side with dimensions of 47.5 mm by 12.7 mm with a 1.6-mm gap between the surfaces (BST model FC81; Biosurface Technologies Corp., Bozeman, MT). Overnight cultures in LB medium supplemented with 120- to 200-μg/ml erythromycin (to retain the GFP plasmid pCM18) were centrifuged and resuspended in LB medium with erythromycin. This diluted culture (OD600, 0.05) was used to inoculate the flow cell for 2 h at a flow rate of 10 ml/h before fresh LB or M9C glu medium flow with erythromycin was started at the same flow rate; the initial inoculum was 1.5 x 108 cells/ml. To determine the impact of AI-2, 6.4 μM was added upon inoculation and in the continuous feed or homocysteine and adenine were added (each, 6.4 μM) as the negative control. Biofilm development in the flow cell was monitored with a TCS SP2 scanning confocal laser microscope (Leica Microsystems, Heidelberg, Germany) with a 40x objective at 16 and 24 h.
Color confocal flow cell images were converted to grayscale using Image Converter (Neomesh Microsystems, Wainuiomata, Wellington, New Zealand). Biomass, substratum coverage, surface roughness, and mean thickness were determined with COMSTAT image-processing software (22) written as a script in Matlab 5.1 (The MathWorks) and equipped with the Image Processing Toolbox. Thresholding was fixed for all image stacks. At each time point, nine different positions were chosen for microscope analysis, and 225 images were processed for each time point. Values are means of data from the different positions at the same time point, and standard deviations were calculated based on these mean values for each position. Simulated three-dimensional images were obtained by using IMARIS (BITplane, Zurich, Switzerland). Twenty-five pictures were processed for each three-dimensional image.
Motility assay. LB overnight cultures were used to assay motility in plates containing 1% tryptone, 0.25% NaCl, and 0.3% agar (45). The motility halos were measured at 8 h for ATCC 25404, MG1655, and MG1655 mqsR/pVLT31 mqsR+ and at 16 h for DH5, JM109, and BW25113. Between 3 and 25 plates were used to evaluate motility in each strain. Motility agar plates containing AI-2 (0.8 or 3.2 μM) were used to test the impact of AI-2 on motility, and homocysteine and adenine (each, 0.8 or 3.2 μM) were added to the agar as a negative control. Each experiment was performed in duplicate.
Transcription reporter assays. To determine the effect of AI-2 on the expression of the motility genes, ATCC 25404 cultures with the lacZ fusion transcriptional reporters qseB::lacZ, flhD::lacZ, fliA::lacZ, fliC::lacZ, and motA::lacZ (45) were cultured overnight in LB ampicillin (100 μg/ml), diluted 1:100 in LB medium, and then grown to stationary phase to an OD600 of 3, since internalization of AI-2 takes place primarily in stationary phase (53). Once cells reached stationary phase, AI-2 was added at 6.4 μM for 2 h (adenine and homocysteine were added, each at a concentration of 6.4 μM, as a negative control). Galactosidase activity was evaluated as described previously (51). All activities were calculated based on a protein concentration of 0.24 mg of protein/ml/OD600 unit (17).
To determine the effect of mqsR and qseB on the expression of the motility genes, MG1655 and MG1655 mqsR were cultured overnight in LB ampicillin (100 μg/ml), diluted 1:100 in LB medium, and grown to exponential phase to an OD600 of 1. When the effect of AI-2 on qseB expression in MG1655 and MG1655 mqsR was tested, cells were cultured overnight in LB ampicillin (100 μg/ml), diluted 1:100 in M9C glu medium, different concentrations of AI-2 (0, 3.2, and 6.4 μM) were added, and the cells were grown to exponential phase (OD600 of 1). Homocysteine and adenine (each, 6.4 μM) were added to cultures as a negative control. The same procedure was followed for testing qseB expression in MG1655 mqsR complemented with pVLT31 mqsR+, but instead of AI-2, IPTG was added at different concentrations (0, 0.1, 0.2, 0.4, and 1 mM).
Microarray analysis. The strains were cultured in LB medium overnight (with kanamycin added to MG1655 mqsR), diluted 1:100 in LB medium, and grown to exponential phase (OD600, 1); total RNA was isolated as described previously (35). The Affymetrix E. coli GeneChip antisense genome array (catalogue no. 900381), which contains probe sets for all 4,290 open reading frames, rRNA, tRNA, and 1,350 intergenic regions, was used to study the effect of the mqsR deletion on the gene expression profile of E. coli. Briefly, the total RNA samples were first converted into cDNA through a reverse transcription reaction with poly(A) RNA controls spiked into the same reaction mixture to monitor the entire target labeling process. The cDNA was then digested with DNase I (Amersham Biosciences) to produce 50- to 200-bp fragments, which were checked by running the fragmented cDNA on a 2% agarose gel. The fragmented cDNA was labeled at the 3' termini by the Enzo BioArray Terminal Labeling kit with Biotin-ddUTP (catalogue no. 900181; Affymetrix). The biotin-labeled target was hybridized to the Affymetrix GeneChip E.coli antisense array at 45°C for 16 h at 60 rpm using the Hybridization Oven 640 (Affymetrix), and then a three-step fluorescent staining was conducted using the Fluidics Station 450 (Affymetrix) during the washing and staining procedure. This includes binding of streptavidin to the biotin-labeled cDNA in the first staining solution, binding of biotin-conjugated streptavidin antibody to the streptavidin in the second staining solution, and binding of phycoerythrin-conjugated streptavidin to the biotin-labeled antibody in the third staining solution. The microarray was scanned at 570 nm to get an image file with the GeneChip Scanner 3000 (Affymetrix). Using GeneChip Operating Software, individual strain reports for both the wild-type strain and mutant cDNA samples were obtained, as well as reports comparing the mqsR mutant to wild-type E. coli. Total cell intensity was scaled automatically in the software to an average value of 500. Since the standard deviation for the expression ratio for all the genes was 2.7, genes with a 4-fold change in intensity between the two chips and a P value of <0.05 were considered differentially expressed.
RESULTS
AI-2 stimulates E. coli biofilm formation in 96-well plates. We suspected AI-2 was involved directly in biofilm formation, since our microarray data indicated that AI-2 controls motility-related genes (e.g., cheABRWYZ, flgABCDEFGHIJKLMN, fliACDFHKLMNOPQ, and motAB) (36) and since the plant-derived furanone inhibits biofilms and represses the same AI-2-controlled genes (36). To investigate this hypothesis, we synthesized AI-2 (there is no commercial source) using two E. coli enzymes and found it was active via a 2,400-fold increase in bioluminescence in the V. harveyi BB170 reporter (0.8 μM AI-2). Then, this active AI-2 (0.2 to 11 μM) was evaluated for its effect on the biofilm formation of three E. coli wild-type strains (ATCC 25404, MG1655, and BW25113), the LuxS-deficient strains E. coli BW25113 luxS and E. coli DH5, and the well-known laboratory strain E. coli JM109. Biofilm formation was stimulated significantly by 11 μM AI-2 in LB medium at 24 h for ATCC 25404 (26 ± 2 fold), DH5 (29 ± 0.3 fold), and MG1655 (4 ± 2 fold) (Table 2). Biofilm formation was also stimulated in JM109 and BW25113 (2 ± 1 fold at 3.2 μM) and in the luxS mutant of BW25113 (1.7 ± 0.3 fold at 1.25 μM). These results with rich media were corroborated with M9C citrate, where 3.2 μM AI-2 stimulated biofilm formation after 24 h for DH5 (2.2 ± 1 fold) and JM109 (1.7 ± 0.4 fold).
Note that in the absence of AI-2, BW25113 luxS made 50% less biofilm than the isogenic wild-type strain, which indicates again that AI-2 stimulates biofilm formation in E. coli, since LuxS forms AI-2. Biofilm could be restored by adding complementing luxS in trans using plasmid pCA24N luxS+ (46% of the wild-type biofilm was formed at 0 mM IPTG and 110% of the wild-type biofilm was formed at 0.25 mM IPTG in LB medium).
To confirm that AI-2 was the cause of the increase in biofilm formation, we measured biofilm stimulation with the isogenic MG1655 lsrK mutant because this mutation dramatically impairs the AI-2 uptake compared with other mutations in the lsr system (49, 52). As expected, AI-2 was not able to induce biofilm formation of the lsrK mutant at 6.4 μM (Fig. 1A); hence, AI-2 induces biofilm formation through the LsrK transport pathway (49).
E. coli DH5, which is deficient in AI-2 synthesis (36) and which was found here to be completely nonmotile, was also studied using the continuous flow cell to see the effect of the luxS mutation on biofilm formation (Table 3) and architecture (image not shown). Although we recognize that this strain is not isogenic with ATCC 25404, we thought it might be informative to see if it responded to AI-2 and to look at its architecture. Compared with ATCC 25404 without AI-2, DH5 displayed less biomass (15 ± 4 μm3/μm2 versus 8 ± 3 μm3/μm2), less substratum coverage (57% ± 7% versus 41% ± 11%), and less thickness (25 ± 7 μm versus 10 ± 4 μm).
We also tested the effect of AI-2 (6.4 μM) on biofilm formation when strains harbored the derepressed conjugation plasmid R1drd19, which enhances biofilm formation (Fig. 1A) (18). Biofilm formation was not significantly induced with either MG1655 or BW25113 when R1drd19 was present.
AI-2 promotes E. coli biofilm formation in a continuous flow cell. To further investigate the effect of AI-2 on biofilm architecture, as well as to corroborate the 96-well plate crystal violet results, a continuous flow cell with LB medium was used to study the biofilm of ATCC 25404 (harboring the GFP plasmid pCM18). In the absence of AI-2, ATCC 25404 developed regular microcolonies covering 41 and 57% of the surface at 16 and 24 h, respectively (Table 3; Fig. 2A); previously similar structures were seen for E. coli SAR18 and MG1655 (33). The addition of 6.4 μM AI-2 also led to the formation of typical microcolonies (Fig. 2B), but the amount of attached cells was greater, since the biomass and thickness increased 5-fold ± 3-fold and 7-fold ± 2-fold at 16 h and 5-fold ± 1-fold and 4-fold ± 1-fold at 24 h, respectively. The roughness coefficient (3-fold ± 1-fold decrease) (Table 3 and Fig. 2) indicated that there was less heterogeneity when AI-2 is added, since the biofilm had fewer interstitial spaces at both times analyzed.
AI-2 increases motility in E. coli through QseB. To determine how AI-2 stimulates biofilm formation in E. coli, we investigated whether AI-2 addition affected the motility of five strains, since our microarrays (36) indicated that these genes were induced by AI-2 (determined by using a luxS mutant). The motility of both ATCC 25404 and MG1655 increased about 30% upon the addition of 0.8 μM AI-2. It was necessary to increase the AI-2 dose to 3.2 μM to see an effect with DH5 and BW25113 (80% and 43% increases in motility, respectively). JM109 did not respond significantly to AI-2 addition at 3.2 μM.
To discern the genetic basis of this increase in motility upon AI-2 addition, we probed the ability of AI-2 to induce the promoters of motility genes qseB, flhD, fliA, fliC, and motA. Upon addition of 6.4 μM AI-2, the quorum-sensing flagellum regulon qseB (45) was induced 8-fold ± 3-fold. These results corroborated the ones reported by Sperandio et al. (45), who previously found that qseB was induced 17-fold compared with the luxS mutant through DNA microarray studies with E. coli O157:H7 and its isogenic luxS mutant. The induction of qseB here led to a 4.0-fold ± 0.1-fold increase in the transcription of flhD (master controller of the flagellum regulon), a 2.6-fold ± 0.3-fold increase in fliA (sigma factor 28), a 3.6-fold ± 0.8-fold increase in fliC (flagellin), and a 6-fold ± 0.3-fold increase in motA (proton conductor for flagellum movement).
Based on this increase in motility through QseB upon AI-2 addition, we hypothesized that AI-2 induces biofilm formation by inducing motility and that this increase in motility leads to increased attachment. To test our hypothesis, we measured biofilm formation upon the addition of AI-2 to two motility-deficient strains. We added AI-2 to the paralyzed isogenic MG1655 motA mutant (9) (we confirmed that this strain is nonmotile), which has reduced biofilm formation (32), and to the isogenic MG1655 qseB mutant, which we found to have impaired motility as previously reported (45). As expected, biofilm formation (Fig. 1A) was not altered when AI-2 was added to both motility mutants, nor did it affect MG1655 fliA (reduction of motility was corroborated for this strain, too).
Deletion of mqsR decreases biofilm formation in 96-well plates and continuous flow system. Since mqsR is induced eightfold in biofilms (35) and is near qseBC (Fig. 3), we investigated its role in AI-2-controlled biofilm formation. Initially, we confirmed the impact of the mqsR deletion on biofilm formation of MG1655 by using 96-well plates after 24 h. Deletion of mqsR decreased biofilm formation in LB (74%), M9C glu (46%), and LB glu (78%) (Fig. 1B). Biofilm formation in flow cells corroborated these results (Table 3; Fig. 2C and D), since deleting mqsR at 48 h led to an 8-fold ± 14-fold reduction in biomass, a 19-fold ± 12-fold reduction in substratum coverage, and a 4-fold ± 3-fold change in thickness. Deleting mqsR changed the biofilm architecture significantly from a 54-μm-thick film with microcolonies to one with nearly no biomass (few colonies remaining). The 7.5-fold ± 4.6-fold increase in the roughness coefficient (24 h) also indicated that there were few colonies formed after deletion of mqsR. Growth was not altered after mqsR was deleted, so the changes in the biofilm were not a result of growth rate differences; the specific growth rates in LB were 1.64 ± 0.02/h versus 1.720 ± 0.008/h for MG1655 and MG1655 mqsR, respectively, while in M9C glu the specific growth rates were 0.990 ± 0.004/h versus 0.93 ± 0.03/h, respectively.
To corroborate that mqsR actually controls biofilm formation, we complemented mqsR in trans by constructing a low-copy-number plasmid (pVLT31) which expresses mqsR+ upon IPTG addition. By the addition of IPTG to MG1655mqsR/pVLT31 mqsR+ in LB medium, biofilm formation was restored from 30% of MG1655/pVLT31 at 0 mM IPTG to 84% at 0.2 mM IPTG; hence, MqsR regulates biofilm formation.
Note that the deletion of fliA, qseB, and motA also inhibited biofilm formation substantially (Fig. 1A and B). This appears to be the first report about the regulation of biofilm by QseB.
MqsR controls motility by regulating QseBC, FliA, and MotA. QseB and QseC are a two-component, quorum-sensing controlled regulator system for motility (45). We hypothesized that mqsR induces biofilm formation by regulating the two-component regulatory system qseBC, which then regulates the motility master regulon flhD (9). In agreement with this hypothesis, we found that when mqsR was deleted, motility was abolished (Fig. 4). Furthermore, this lack of motility due to the deletion of mqsR was caused by a reduction in transcription of qseB (61-fold ± 27-fold), fliA (2.4-fold ± 2-fold), and motA (18-fold ± 10- fold) (Fig. 5A) in M9C glu. Similar results were found with LB, as qseB, fliA, and motA decreased 2.3-fold ± 1.4-fold, 5-fold ± 1-fold, and 11-fold ± 11-fold, respectively (results not shown). Note that although flhD transcription was not altered substantially in these experiments, its expression was altered greatly in the DNA microarrays (Table 4). To corroborate that mqsR abolishes motility, we complemented mqsR in trans with the low-copy-number plasmid pVLT31, which carries mqsR+ (Fig. 4). Increasing the expression of mqsR by adding IPTG reestablished cell motility in a dose-dependent manner until it reached 50% of wild-type motility at 0.4 mM IPTG. Hence, MqsR regulates biofilm formation by inducing motility through QseBC.
MqsR is a global regulator in E. coli MG1655. Considering the size of MqsR (98 aa), we hypothesized it could be a global regulator in E. coli. To investigate this, differential gene expression was determined for MG1655 and MG1655 mqsR in LB liquid culture. By deleting mqsR, 41 genes were down-regulated >4-fold, while 33 genes were up-regulated >4-fold (Tables 4 and 5). Of the 246 genes that were down-regulated two- to ninefold, 14% (34 genes) were reported to be AI-2 controlled (15, 36, 44), which supplies additional evidence that MqsR is a global mediator between AI-2 and E. coli. Note that the genes that encode the master flagellum regulons flhD and flhC were down-regulated 24- and 7.5-fold, respectively, which also corroborates that MqsR regulates motility by controlling the master flagellum regulon flhDC. It was also found MqsR induced curli expression, based on its 26-fold differential expression of crl (Table 4), a transcriptional regulator of the cryptic csgAB locus for curli surface fibers (6), which has been reported to play a role in E. coli biofilm formation (7). The array results also showed that MqsR regulates motility by controlling not only QseB (Fig. 5A) but also csrA, which is down-regulated in the mqsR mutant (twofold) and which regulates motility master regulon expression in E. coli (50).
MqsR links AI-2 quorum-sensing signal and biofilm formation. We tested the effect of the mqsR deletion on the ability of AI-2 to induce biofilm formation in LB by using microtiter plates. The addition of 6.4 μM AI-2 increased MG1655 biofilm mass by 2.8-fold ± 0.5-fold, while it had no effect when mqsR was deleted (Fig. 1A). Therefore, induction of biofilm formation by increasing motility is mediated by MqsR.
Induction of motility with AI-2 is mediated by MqsR and then QseBC. To corroborate the results obtained by Sperandio et al. (45), we measured the expression of qseB in MG1655 upon the addition of our synthesized AI-2 (0.8 μM). As expected, qseB transcription increased eightfold upon the addition of AI-2 (results not shown), while Sperandio et al. found sixfold induction by using preconditioned Dulbecco's modified Eagle medium. To find if the induction of motility with AI-2 was mediated by both MqsR and QseBC, we then measured motA expression in the qseB and mqsR mutants upon the addition of AI-2 (0.8 μM) and compared it to that of wild-type MG1655 (Fig. 5B). The addition of AI-2 induced motA activity for the wild-type strain but did not induce motA in the mqsR and qseB mutants; therefore, the induction of motility was mediated by both MqsR and QseBC (Fig. 3).
Further evidence that mqsR is first in the cascade was provided by measuring the transcription of qseB with the wild-type strain and the mqsR mutant upon the addition of AI-2. If MqsR is first in the cascade and necessary for the transduction of the AI-2 signal, then the addition of AI-2 should only increase qseB transcription when mqsR is not deleted. We found that adding AI-2 at 6.4 μM induced the expression of qseB 3.2-fold in the wild-type strain in M9C glu but did not induce qseB in the mqsR mutant (Fig. 6A). As expected, the wild-type strain responded to AI-2 addition in a dose-dependent manner. To show further that MqsR is first in the cascade, the expression of qseB from pVS159 was also measured while inducing MqsR expression in trans in the mqsR mutant by adding IPTG to strains harboring pVLT31 mqsR+. As expected if MqsR was required for signal transduction to QseB, expression of qseB was induced fourfold in a dose-dependent manner in M9C glu (Fig. 6B). Hence, MqsR is first in the cascade (Fig. 3).
DISCUSSION
We focused on E. coli biofilms, since this strain is the most thoroughly studied bacterium (5), its genome is sequenced (5), microarrays are available (35, 42), the functions of many of its proteins have been elucidated (39), and many isogenic mutants are available (23). Also, our group has experience in determining the genetic basis of E. coli biofilm formation and its inhibition with natural, plant-derived antagonists (37, 38). Although E. coli is well studied, its biofilm has not received the same scrutiny, since many (but not all) K-12 strains make a poor biofilm if they lack a conjugative plasmid (18, 33).
In this study, we show that cross-species quorum-sensing signal AI-2 stimulates biofilm formation in five different E. coli hosts (ATCC 25404, MG1655, BW25113, DH5, and JM109), in two different media (M9 citrate and LB), and in both batch and continuous flow systems (which enables the biofilm to be studied under two different hydrodynamic conditions, corroborates the results, and allows the biofilm architecture to be examined); hence, stimulation of biofilm formation by AI-2 is a general phenomenon. Our results here serve to explain two results that have been found previously: that AI-2 controls chemotaxis, flagellar synthesis, and motility in E. coli (36, 44) and that the quorum-sensing antagonist furanone was effective in preventing the biofilms of E. coli by repressing these same chemotaxis, flagellar synthesis, and motility genes (36). Therefore, AI-2 stimulates biofilm formation directly, flagellar synthesis and motility are clearly involved in biofilm formation, and furanone inhibits biofilm formation by masking AI-2.
We have also shown here that AI-2 stimulates biofilm formation by increasing motility, since addition of AI-2 stimulated the motility of two strains (ATCC 25404 and MG1655), since AI-2 addition had no effect on the biofilm formation of the motility-impaired mutants motA and qseB (Fig. 1A), since the transcription of flagellar genes is induced by AI-2, and since the biomass, substratum coverage, and biofilm thickness of the luxS mutant E. coli DH5, which has abolished motility, are less than those of E. coli ATCC 25404 with AI-2 (Table 3). Furthermore, by stimulating motility, the addition of AI-2 changes the architecture of the ATCC 25404 biofilm to a flatter phenotype in flow cells (Fig. 2). Previous reports have indicated that motility plays an important role in the attachment of cells to the surface (32), but here we show that motility (stimulated by AI-2) affects the architecture, too.
Previous reports have indicated AI-2 is not necessary for mature biofilm formation when a conjugation plasmid such as R1drd19 is present in minimal AB medium with glucose (33). Here, along with one of the first applications of synthesized AI-2, we used wild-type strains that lack a conjugation plasmid and were cultured in rich medium and found AI-2 plays a surprisingly large role in biofilm formation (25-fold). The difference in results was most likely due to the lack of the conjugation plasmid, as we showed here (Fig. 1A), as well as to the difference in hosts used (that is one reason we verified our results with five familiar strains). Contrary to previous reports (18), the wild-type strain (MG1655) harboring the conjugative plasmid forms less biofilm in the presence of AI-2 than the non-plasmid-carrying strain (Fig. 1A). One explanation may be that we found that the addition of conjugation plasmids induces biofilm formation by inducing cell aggregation, not by changing motility (18a). We believe that cells harboring R1drd19 in the presence of AI-2 have induced motility, which may impede cell aggregation and thereby decrease biofilm formation. The fact that we saw the smallest stimulation of both biofilm formation and motility with AI-2 for JM109 corroborates this, since JM109 contains the F' conjugation plasmid.
We also found that AI-2 stimulates biofilm formation via the uncharacterized protein MqsR by showing that MqsR induces motility (Fig. 4) and biofilm formation in both batch and continuous systems (Fig. 1B and 2), that AI-2 stimulates motility through MotA (Fig. 5B) and biofilm formation through MqsR (Fig. 1A), and that MqsR stimulates QseB (Fig. 5A), which controls motility in E. coli. Previous reports have found relationships between quorum sensing and biofilms (28), but these reports have not found the genetic underpinnings behind the biofilm phenotype. Based on the discovery in the present work that AI-2 stimulates biofilms directly, we propose a genetic model (Fig. 3) for how AI-2 controls biofilm formation in E. coli. Sperandio et al. (45) found the link between AI-2 and motility for the two-component regulatory system qseBC, yet they proposed that additional regulators in the cascade that mediate motility and quorum sensing need to be found and characterized. One of these links is now found, and it connects AI-2, MqsR, QseBC, and biofilm formation.
Our model (Fig. 3) is that AI-2 stimulates biofilm formation by stimulating expression of MqsR, which then directly or indirectly induces expression of QseBC, which then promotes cell motility via the master regulon flhDC, which then stimulates MotA and FliA and leads to biofilm formation. Without this stimulation of motility, biofilm formation is severely impaired (Fig. 1). We found that qseB is controlled by MqsR (Fig.5A) and that MqsR controls flhDC (Table 4) and therefore motility. We also found that MqsR induces curli expression through crl (Table 4) and possibly induces motility through csrA. Hence, MqsR controls biofilm formation by inducing motility and curli synthesis. Considering that MqsR controls 108 proteins with unknown functions (Tables 4 and 5) and that MqsR is a global AI-2-controlled regulator, there are many new proteins to investigate in regard to biofilm formation, control, and quorum sensing.
In summary, we have determined that the species-nonspecific,quorum signal AI-2 directly stimulates biofilm formation in E. coli, that the mechanism is through stimulating motility genes, and that MqsR mediates this effect prior to QseBC. These results serve to make sense of our previous microarray data and serve to give a deeper understanding of how plant biofilm inhibitors work. Hence, our results are helpful for understanding and preventing biofilm formation by the archetypal strain, as well as helpful for combating related pathogenic strains such as E. coli O157:H7 (30).
ACKNOWLEDGMENTS
A.F.G.B. was supported by a Fulbright scholarship (FB2454106-2), and this research was supported by the National Science Foundation (BES-0124401) and the U.S. Environmental Protection Agency.
We thank A. Heydorn from the Technical University of Denmark for kindly providing COMSTAT, S. Molin from the Technical University of Denmark for sending plasmid pCM18, and J. Kaper from the University of Maryland for sending plasmids pVS159, pVS176, pVS175, pVS182, and pVS183.
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