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Applied and Environmental Microbiology, July 2005, p. 4022-4034, Vol. 71, No. 7
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.7.4022-4034.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Differential Gene Expression for Investigation of Escherichia coli Biofilm Inhibition by Plant Extract Ursolic Acid

Dacheng Ren,1,{dagger} Rongjun Zuo,1,{dagger} Andrés F. González Barrios,1 Laura A. Bedzyk,2 Gary R. Eldridge,3 Mark E. Pasmore,4 and Thomas K. Wood1*

Departments of Chemical Engineering and Molecular and Cell Biology, University of Connecticut, 191 Auditorium Road, Storrs, Connecticut 06269,1 Experimental Station E328/B33, DuPont Central Research and Development, Wilmington, Delaware 19880,2 Sequoia Sciences, 11199 Sorrento Valley Road, Suite H, San Diego, California 92121,3 Center for Biofilm Engineering, Montana State University, 366 EPS Building, Bozeman, Montana 597174

Received 13 September 2004/ Accepted 31 January 2005


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ABSTRACT
 
After 13,000 samples of compounds purified from plants were screened, a new biofilm inhibitor, ursolic acid, has been discovered and identified. Using both 96-well microtiter plates and a continuous flow chamber with COMSTAT analysis, 10 µg of ursolic acid/ml inhibited Escherichia coli biofilm formation 6- to 20-fold when added upon inoculation and when added to a 24-h biofilm; however, ursolic acid was not toxic to E. coli, Pseudomonas aeruginosa, Vibrio harveyi, and hepatocytes. Similarly, 10 µg of ursolic acid/ml inhibited biofilm formation by >87% for P. aeruginosa in both complex and minimal medium and by 57% for V. harveyi in minimal medium. To investigate the mechanism of this nontoxic inhibition on a global genetic basis, DNA microarrays were used to study the gene expression profiles of E. coli K-12 grown with or without ursolic acid. Ursolic acid at 10 and 30 µg/ml induced significantly (P < 0.05) 32 and 61 genes, respectively, and 19 genes were consistently induced. The consistently induced genes have functions for chemotaxis and mobility (cheA, tap, tar, and motAB), heat shock response (hslSTV and mopAB), and unknown functions (such as b1566 and yrfHI). There were 31 and 17 genes repressed by 10 and 30 µg of ursolic acid/ml, respectively, and 12 genes were consistently repressed that have functions in cysteine synthesis (cysK) and sulfur metabolism (cysD), as well as unknown functions (such as hdeAB and yhaDFG). Ursolic acid inhibited biofilms without interfering with quorum sensing, as shown with the V. harveyi AI-1 and AI-2 reporter systems. As predicted by the differential gene expression, deleting motAB counteracts ursolic acid inhibition (the paralyzed cells no longer become too motile). Based on the differential gene expression, it was also discovered that sulfur metabolism (through cysB) affects biofilm formation (in the absence of ursolic acid).


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INTRODUCTION
 
Bacterial biofilms are sessile communities with high cell density that are ubiquitous in natural, medical, and engineering environments (14, 42). Biofilms formed by pathogenic Escherichia coli strains can pose serious problems to human health such as prostatitis, biliary tract infections, and urinary catheter cystitis (8). Deleterious biofilms are also problematic in industry since they cause fouling and corrosion in systems such as heat exchangers, oil pipelines, and water systems (14). Recently, there has been a tremendous increase in biofilm research, most of it with the ultimate aims of biofilm prevention, control, or eradication (32).

Biofilm cells survive antibiotics more readily than planktonic ones and are often responsible for reoccurring symptoms and medical treatment failure (39, 42). Because bacterial cells in a biofilm are embedded in a matrix of polysaccharide, they encounter oxygen limitation and low metabolic activity, which protect them from antibiotics (66). In addition, it was found that 40% of the cell wall proteins in biofilm cells are different from those of the planktonic cells; therefore, some antibiotics may lose their targets (42). Since many cells in a mature biofilm live extended times without division, they are highly resistant to antibiotics which are primarily effective on dividing cells (42). For these reasons, biofilms are highly resistant to antibiotics (39); hence, novel antagonists with potential to remove mature biofilms are needed. By concentrating on novel antagonists that do not inhibit growth, we seek to avoid selection pressure for resistance (21).

There are few known natural compounds that inhibit biofilm formation while not affecting cell growth, but the quorum-sensing antagonist (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone (furanone) from the marine alga Delisea pulchra (12) inhibits biofilm formation in E. coli without inhibiting its growth (51). Bacteria use quorum sensing to regulate some forms of gene expression by sensing their population density via the small signaling compounds called autoinducers (AIs) that are excreted into the environment (2). As the AI concentration increases with cell density, the binding of AIs to the cellular receptors triggers genes for different phenotypes including biofilm formation (9), production of virulence factors (4), siderophore synthesis (62), bioluminescence (7), protein production (10), and plasmid conjugation (35). Different species use different quorum-sensing signals; however, AIs are mainly divided into two groups: acylated homoserine lactones (AI-1, regulated by LuxI/LuxR systems) for gram-negative bacteria and peptides for gram-positive bacteria (2). A common signal called AI-2 (produced by LuxS) has been discovered as a species nonspecific signal used by both gram-negative and gram-positive bacteria (64). Sperandio et al. used DNA microarrays to study gene expression of wild-type E. coli O157:H7 and its luxS mutant and found that AI-2 is a global regulatory signal which regulates more than 400 genes, including those for chemotaxis, flagellar synthesis, motility, and virulence factors (58). Using DNA microarrays, furanone has been shown by us to repress 44 of the 67 E. coli genes that are positively controlled by AI-2 (49), and Hentzer et al. (22) have shown that brominated furanone also inhibits 80% of the AI-1 quorum-sensing-controlled genes in Pseudomonas aeruginosa PAO1.

To search for additional natural compounds that inhibit biofilm formation without affecting growth, a library of compounds from 176 plant families was created (13). From this library, ursolic acid (3ß-hydroxy-urs-12-en-28-oic acid) from Diospyros dendo, the tree used for ebony (e.g., black piano keys) from Gabon, Africa, was identified (Fig. 1). Ursolic acid is a relatively nontoxic active ingredient of many medicinal plants such as Sambucus chinesis, Glechoma hederacea, Eribotrya japonica, and Pyrola rotundifolia and has a broad range of pharmacology effects, including protection against liver injury, antitumor activity, inhibition of mutagenesis in bacteria, anti-inflammation, and antiulcer activity (36). Here, by using 96-well plates and a flow chamber, ursolic acid is shown to inhibit biofilm formation of the reference microorganism E. coli without affecting its growth rate. To investigate the genetic basis of this biofilm inhibition, DNA microarrays, which have been used to monitor global gene expression profiles in response to different stimuli (57) including heat shock and other stresses (20, 70, 73), quorum sensing (11, 58), anaerobic metabolism (72), sporulation (15), and biofilm formation (46, 47, 55, 60, 69), were used to study the differential gene expression of E. coli K-12 with or without ursolic acid. This is the first report of using ursolic acid to inhibit biofilms and of the role of sulfur metabolism in biofilm formation.



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  		FIG. 1. Structure of ursolic acid. Carbons 19 and 20 are shown.


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MATERIALS AND METHODS
 
Bacterial strains, growth media, and toxicity testing.
The strains and plasmids used in the present study are listed in Table 1 (including two E. coli K-12 MG1655 strains for use with their respective mutants). LB medium (54) was used for routine culturing of the E. coli strains. LB medium and LB medium supplemented with 0.2% glucose, 1% sodium citrate, or 2% sodium pyruvate was also used to study biofilm formation in the 96-well plate assay. M9 medium (53) supplemented with 0.4% glucose and 0.4% Casamino Acids was used to establish the E. coli biofilm in the flow chamber and to study biofilm formation by Vibrio harveyi in the microtiter plates. Autoinducer bioassay (AB) medium (18) was used to grow V. harveyi, and LM medium (3) was used to determine its CFU. Plasmids were electroporated at 15 kV/cm, 200 {Omega}, and 25 µF by using a Gene Pulser (Bio-Rad, Richmond, CA).


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  		TABLE 1. Strains and plasmids used in this study

The specific growth rates of E. coli K-12 ATCC 25404 with or without conjugation plasmid R1drd19 in the presence of 10 or 30 µg of ursolic acid/ml in LB medium were determined by using the optical density at 600 nm (OD600) and calculated by using the linear portion of the logarithm of OD600 versus time (OD600 from 0.05 to 0.4). Similarly, the effects of the cysB and motAB mutations on growth rates were determined. Hepatocyte toxicity testing with 13.7 µg of ursolic acid/ml (30 µM) was performed by Cerep (Redmond, WA) according to the method of Nociari et al. (40) using a sensitive fluorometric assay based on AlamarBlue and the HepG2 cell line.

Plant collection and purification of ursolic acid.
To discover novel biofilm inhibitors, plant extracts were separated by high-pressure liquid chromatography (HPLC) and screened with P. aeruginosa PAO1 biofilm assays by using microtiter plates and staining with crystal violet. Fractions that inhibited biofilm formation were purified, and the component compounds were tested individually. The plants were collected from Gabon, Africa, and the Missouri Botanical Garden at St. Louis, Mo. A total of 13,000 fractions (6,699 from Missouri and 5,941 from Gabon) were screened, representing 83 families and 167 genera. The plant collection included whole plants and separated parts such as fruits, roots, leafs, and stems (13). From these fractions ursolic acid was identified. Ursolic acid (Fig. 1) was extracted by using preparative HPLC from a leaf/flower extract from D. dendo as described previously (13). This process differs from traditional biologically guided fractionation because there was only one purification step. The separation was optimized to achieve baseline resolution of ursolic acid and the other compounds in the sample mixture. The HPLC method used to obtain ursolic acid consisted of a linear gradient of acetonitrile from 75 to 95% in 28 min on a semipreparative C18 column. Ursolic acid was eluted at 21.2 min and stored as a powder at –20°C. Within 48 h prior to the experiment, ursolic acid was dissolved in 95% ethanol at 2.5 mg/ml and stored at 4°C.

Total RNA isolation for DNA microarrays.
To identify the genes controlled by ursolic acid, E. coli K-12 ATCC 25404 was grown in LB medium overnight, diluted 1:100 in fresh LB supplemented with 0, 10, or 30 µg of ursolic acid/ml. The same amount of ethanol was supplemented to eliminate solvent effects. The cultures were grown to an OD600 of 0.9. The cells were centrifuged in a microcentrifuge for 15 s at 20,000 x g in Mini-Bead Beater tubes (Biospec, Bartlesville, Oklahoma) that were cooled to –80°C before sampling. The cell pellets were flash frozen in a dry ice-ethanol bath and stored at –80°C until RNA isolation.

To lyse the cells, 1.0 ml of RLT buffer (Qiagen, Inc., Valencia, CA) and 0.2 ml of 0.1 mm zirconia-silica beads (Biospec) were added to the frozen bead beater tubes containing the cell pellets. The tubes were closed tightly and beat for 30 s at the maximum speed in a Mini-Bead Beater (catalog no. 3110BX; Biospec). The total RNA was isolated by following the protocol of the RNeasy Minikit (Qiagen), including an on-column DNase digestion with RNase-free DNase I (Qiagen). OD260 was used to quantify the RNA yield. 23S/16S rRNA values (measured by using a 2100 Bioanalyzer, Agilent Technologies, Palo Alto, CA) and OD260 and OD280 values were used to check the purity and integrity of RNA (RNeasy Mini handbook; Qiagen).

DNA microarrays.
The E. coli DNA microarrays were prepared as described previously (68). Each gene probe was synthesized by PCR and has the size of the full open reading frame (ORF; 200 to 2,000 nucleotides). The double-strand PCR products were denatured in 50% dimethyl sulfoxide and spotted onto aminosilane slides (Full Moon Biosystems, Sunnyvale, CA) as probes to hybridize with the mRNA-derived cDNA samples. It has been shown that each array can detect 4,228 of the 4290 E. coli ORFs (68). Each gene has two spots per slide.

The detailed microarray protocol has been described in our recent publication (50). Briefly, the total RNA from the E. coli K-12 ATCC 25404 samples grown with or without ursolic acid was first converted into labeled cDNA. The cDNA samples (6 µg of each) were then each labeled with both Cy3 and Cy5 dyes to remove artifacts related to different labeling efficiencies; hence, each experiment needed at least two slides. The Cy3-labeled sample without ursolic acid and the Cy5-labeled ursolic acid sample (with 10 or 30 µg of ursolic acid/ml) were hybridized on the first slide. Similarly, the Cy5-labeled sample without ursolic acid and the Cy3-labeled ursolic acid sample were hybridized on the second slide. Since each gene has two spots on a slide, the two hybridizations generated eight datum points for each gene (four points for the sample without ursolic acid and four points for the ursolic acid sample). The microarray experiments with dye-swapping were repeated for both concentrations of ursolic acid.

The labeled cDNA samples were hybridized to the DNA microarrays and scanned, and the images were analyzed as described previously (50). Genes were identified as differentially expressed if the expression ratio was >1.4 and the P value (t test) was <0.05. P values were calculated on log-transformed, normalized intensities. Including the P value criterion ensures the reliability of the induced or repressed gene list. Normalization was relative to the median total fluorescent intensity per slide per channel. The gene functions were obtained from the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov/).

Autoinducer activity assay.
Bacterial supernatants were assayed by using the method of Surette and Bassler (63), as we described previously (51). Reporter strains V. harveyi BB170 or BB886 were grown in AB medium overnight and diluted 1:5,000 into the fresh AB medium supplemented with 0, 1, 5, or 10 µg of ursolic acid/ml. The time course of bioluminescence was measured with a 20/20 luminometer (Turner Design, Sunnyvale, CA) and reported as relative light units (RLU). The cell density of the V. harveyi reporter strains was measured by spreading the cells on LM plates and counting CFU after 24 h.

Ninety-six-well plate biofilm assay.
This assay was adapted from those reported previously (33, 43). E. coli was grown in polystyrene 96-well plates at 37°C for 2 days without shaking in LB medium (or in LB medium supplemented with 0.2% glucose or other carbon sources according to the requirements of the individual strains) supplemented with 5, 10, or 30 µg of ursolic acid/ml as indicated. The appropriate amount of 95% ethanol was added to all samples to eliminate solvent effects (each well contained 1.2% [vol/vol] ethanol). Before the biofilm mass in each well was measured, the OD reading at 620 nm was taken to quantify the growth of the cells with or without ursolic acid. To quantify the total biofilm mass, the suspension cultures were decanted, the plates were washed three times with water, and the biofilms were stained with 0.1% crystal violet (Fisher, Hanover Park, IL) for 20 min. The extra dye was removed by three washes with water. All of the dye associated with the attached biofilm (air-liquid interface biofilm as well as bottom liquid-solid biofilm) was dissolved with 300 µl of 95% ethanol, and an OD reading at 540 nm was used to quantify the total biofilm mass. Each datum point was averaged from four replicate wells and the standard deviations were calculated (Fig. 2; see also Fig. 4). To study the overall effect of ursolic acid, it was added upon inoculating E. coli, and a time course of biofilm mass was measured. To study whether ursolic acid can remove mature biofilms, it was also added 24 h after inoculation. One plate was processed every 8 to 24 h so that a total of four plates were prepared for each condition to give a time course, and the experiments were conducted twice (eight plates total); these experimental conditions apply for strains E. coli K-12 ATCC 25404 [R1drd19], K-12 MG1655 with or without R1drd19, K-12 MG1655 motAB with or without R1drd19, K-12 MG1655 cysB [R1drd19], JM109, EJ500, MC4100 [R1drd19], MC4100 cysB [R1drd19], C600 [R1drd19], and C600 cysB [R1drd19]. For P. aeruginosa PAO1, 1% sodium citrate was added into LB medium to promote biofilm formation since P. aeruginosa PAO1 made a very poor biofilm in LB medium (biofilm increased fourfold upon addition of citrate); similarly, 1% sodium citrate was used with M9 minimal medium to promote biofilm formation (only carbon and energy source). For V. harveyi BB120, the medium was M9 minimal medium supplemented with 0.4% glucose, 0.4% Casamino Acids, and 10 µg of thiamine/ml since V. harveyi BB120 made a very poor biofilm in LB medium.



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  		FIG. 2. Inhibition of biofilm formation by E. coli K-12 ATCC 25404 [R1drd19], E. coli JM109, P. aeruginosa PAO1, and V. harveyi BB120 by the addition of 10 µg of ursolic acid/ml. For E. coli K-12 ATCC 25404 [R1drd19], data were collected 16 h after the addition of ursolic acid in LB medium; for E. coli JM109, data were collected 24 h after the addition of ursolic acid in LB medium supplemented with 0.2% glucose; for P. aeruginosa PAO1, data were collected 18 h after the addition of ursolic acid in LB medium plus 1% sodium citrate; and for V. harveyi BB120, data were collected 18 h after the addition of ursolic acid in M9 medium. All biofilm mass readings at OD540 were normalized based on the reading of wild type without ursolic acid, which was normalized to 1. One standard deviation shown.



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  		FIG. 4. Effect of the E. coli motAB mutation on ursolic acid biofilm inhibition (E. coli K-12 MG1655 [R1drd19] versus E. coli K-12 MG1655 motAB [R1drd19], data collected 24 h after addition of 30 µg of ursolic acid/ml in LB medium), and the effect of the E. coli cysB mutation on biofilm formation (E. coli K-12 MG1655 [R1drd19] versus E. coli K-12 MG1655 cysB [R1drd19], data collected 24 h after inoculation in LB, LB supplemented with 0.2% glucose, and M9 minimal medium supplemented with 0.4% glucose and 0.4% Casamino Acids). Biofilm mass was normalized based on the reading of wild type without ursolic acid. One standard deviation shown.

Flow chamber biofilm experiments and image analysis.
M9-0.4% glucose-0.4% Casamino Acids medium supplemented with 30 µg of chloramphenicol/ml and 150 µg of erythromycin/ml to maintain R1drd19 and pCM18, respectively, was used to grow biofilm at 37°C in a continuous flow chamber (BST model FC81; Biosurface Technologies, corp., Bozeman, MT) with chamber dimensions of 2.54 by 12.7 by 50.8 mm. The flow cell contains a standard glass microscope slide on one side and a glass coverslip on the other side. An overnight culture of E. coli K-12 ATCC 25404 [R1drd19, pCM18] in LB medium supplemented with 30 µg of chloramphenicol/ml and 150 µg of erythromycin/ml was centrifuged and resuspended in the M9 medium. This diluted culture (OD600 = 0.05) was allowed to flow into the flow chamber for 2 h at a flow rate of 10 ml/h (in laminar flow) before the fresh M9 medium (supplemented with chloramphenicol and erythromycin) flow was started at the same flow rate. Ursolic acid was added at both inoculation and in the fresh medium at 10 µg/ml to investigate its effect on biofilm formation. The biofilm development in flow chamber was monitored by using a Leica TCS SP2 scanning confocal laser microscope (Leica Microsystems, Heidelberg, Germany) with a 40x objective lens.

Color confocal flow chamber images were converted to gray scale by using Image Converter (Neomesh Microsystems, Wainuiomata, Wellington, New Zealand). Biomass, substratum coverage, surface roughness, and mean thickness were determined by using COMSTAT image-processing software (23) written as a script in Matlab 5.1 (The MathWorks) equipped with the Image Processing Toolbox. Thresholding was fixed for all images stacks. At each time point, 3 to 15 different positions were randomly chosen for microscope analysis and 30 to 100 images were processed for each position; in all, 26 positions with 1,597 images were obtained for the biofilm without ursolic acid, and 58 positions with 2,740 images were obtained for the biofilm with ursolic acid. 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). Fifty pictures were processed for each three-dimensional image.

Chemotaxis.
To investigate whether ursolic acid is a chemoattractant or a chemorepellent for E. coli, a chemotaxis experiment was performed based on the protocol of Adler (1). E. coli K-12 ATCC 25404 was grown overnight in chemotaxis growth medium containing 5% glycerol as a carbon source with shaking at 30°C (1). The overnight culture was diluted in the same fresh medium to an OD600 of 0.05 and incubated at 30°C with shaking. The culture was harvested at an OD600 of 0.38 and washed three times with chemotaxis buffer. To prepare the agar plug, 100 mg of Seakem Gold agarose (FMC BioProducts, Rockland, ME) was added to 5 ml of chemotaxis buffer and mixed. Then, 0.5 ml of the agarose solution and ursolic acid (30 or 100 µg/ml) were added to a microcentrifuge tube, followed by incubation at 70°C (this is the agarose plug mixture). Next, 10 µl of the above agarose plug mixture was placed between two coverslips on a microscope slide (warmed at 30°C first) and immediately covered with a third coverslip to make an agarose plug. After 4 min, 100 µl of cells was added to the agar plug, followed by incubation at 30°C for 0.5 to 1 h. The plug was examined under the microscope (Zeiss Axioskop; Zeiss, Oberkochen, Germany).

RESULTS
Ursolic acid does not inhibit the growth of E. coli, P. aeruginosa, V. harveyi, or hepatocytes.
Ursolic acid was tested for its impact on the growth of E. coli K-12 ATCC 25404 in LB medium. The specific growth rate was 1.08 ± 0.02 h–1 without ursolic acid, 1.11 ± 0.07 h–1 with ursolic acid at 10 µg/ml, and 1.04 ± 0.13 h–1 with ursolic acid at 30 µg/ml; hence, ursolic acid is not toxic to E. coli. Ursolic acid also did not affect the growth rate of E. coli K-12 ATCC 25404 [R1drd19] (e.g., the specific growth rate was 1.29 ± 0.12 h–1 without ursolic acid, 1.36 ± 0.07 h–1 with ursolic acid at 10 µg/ml, and 1.16 ± 0.08 h–1 with ursolic acid at 30 µg/ml). Further, ursolic acid also did not affect the growth yields of JM109 in both LB medium and LB medium supplemented with 0.2% glucose in 96-well plates (5 and 10 µg/ml) and did not affect the growth yield of both wild-type V. harveyi (5 and 10 µg/ml in AB minimal medium and in 96-well plates) and wild-type P. aeruginosa PAO1 (5 and 10 µg/ml in LB supplemented with 1% sodium citrate in 96-well plates). Ursolic acid at 13.7 µg/ml was also not toxic to hepatocytes since it inhibited HepG2 cell viability by only 1%. These results are similar to those found with oleanolic acid, which is also a triterpenoid that differs from ursolic acid by a methyl group at positions 19 and 20 (Fig. 1) (36). Hence, ursolic acid was not toxic at least at 10 µg/ml for all of the tested organisms. However, it significantly inhibited bacterial biofilm formation at 10 µg/ml.

Ursolic acid inhibited E. coli biofilm formation in 96-well plates.
To study the effects of ursolic acid on E. coli biofilm formation, plasmid R1drd19 was electroporated into E. coli K-12 ATCC 25404 to promote biofilm formation since this strain makes a better biofilm in the presence of conjugation pili (16, 45). Biofilm formation was inhibited clearly in LB medium when ursolic acid was added to a 24-h biofilm (Fig. 2). The biofilm without ursolic acid increased with time until 40 h after inoculation and then became stable. For the wells that contained 10 µg of ursolic acid/ml, however, the biofilm decreased by 79% 16 h after addition of the ursolic acid (Fig. 2). Also, 30 µg of ursolic acid/ml decreased biofilm formation by 40% in M9 medium supplemented with glucose. Similarly, biofilm formation was inhibited by 87% for P. aeruginosa PAO1 in complex medium and 95% in minimal medium and by 57% for V. harveyi in minimal medium (Fig. 2).

Other E. coli strains and media were also tested to investigate the effects of ursolic acid on biofilm formation, including its effect on a relatively good biofilm-forming strain, JM109. Ursolic acid was found to inhibit the biofilm formation of E. coli K-12 ATCC 25404 [R1drd19] in LB supplemented with 0.2% glucose (51% reduction observed 39 h after the addition of 10 µg/ml), of E. coli JM109 in LB (62% reduction observed 24 h after the addition of 10 µg/ml) and LB supplemented with 0.2% glucose (72% reduction at 24 h after the addition of 10 µg/ml, Fig. 2), and of E. coli EJ500 in LB and LB supplemented with 0.2% glucose (92% reduction at 23 h after the addition of 10 µg/ml to both media), as well as to inhibit the biofilm of E. coli JCB495 in LB supplemented with 2% sodium pyruvate to promote biofilm formation (60% reduction at 25 h after the addition of 10 µg/ml). Hence, for the bacteria tested, the inhibition of biofilm formation was not species, strain, or medium specific, and there were no signs of toxicity.

Ursolic acid inhibited E. coli biofilm formation in a continuous flow chamber.
To further investigate the effect of ursolic acid on biofilm architecture, as well as to corroborate the 96-well plate crystal violet results, a continuous flow chamber was used to establish the biofilm of E. coli K-12 ATCC 25404 [R1drd19, pCM18]. Since E. coli biofilm formation with LB is too robust (data not shown) and it is difficult to see distinct biofilm structure, minimal medium was used to monitor the effect of ursolic acid. The green fluorescent protein plasmid pCM18 was found not to affect the biofilm formation of E. coli K-12 ATCC 25404 carrying R1drd19 for up to 31 h in LB or M9 medium in a 96-well plate biofilm assay (e.g., at 31 h, the OD reading at 540 nm for total biofilm after crystal violet staining was 1.72 for E. coli K-12 ATCC 25404 [R1drd19, pCM18] and 1.87 for E. coli K-12 ATCC 25404 [R1drd19]).

Over 65 h, the biofilm development and architecture were monitored in the flow chamber. In the absence of ursolic acid, cells attached to the glass surface and began to make cell clusters within 15 h (data not shown). These cell clusters matured to larger organized microcolonies within 22 h and formed a biofilm 50 µm thick (Fig. 3A). In contrast, when 10 µg of ursolic acid/ml was added, very few microcolonies were seen within 22 h; instead, only single cells or some small dispersed cell clusters were seen (Fig. 3C). Within 65 h, no mature biofilm structure was observed in the presence of ursolic acid, and the biofilm thickness was much less than that without ursolic acid (Fig. 3B and D). COMSTAT analysis of biofilm structure at 22 and 65 h after inoculation confirmed that biofilm development was inhibited by the presence of ursolic acid (Table 2) with significant decreases in biomass, surface coverage, and mean thickness, as well as an increase in the roughness coefficient.



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  		FIG. 3. Inhibition of E. coli K-12 ATCC 25404 [R1drd19, pCM18] biofilm development in flow cells upon the addition of 10 µg of ursolic acid/ml at inoculation to M9 medium (supplemented with 0.4% glucose and 0.4% Casamino Acids). (A) No ursolic acid, 22 h; (B) no ursolic acid, 65 h; (C) 10 µg ursolic acid/ml, 22 h; (D) 10 µg ursolic acid/ml, 65 h. Scale bar, 10 µm.


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  		TABLE 2. Inhibition of E. coli K-12 ATCC 25404 [R1drd19, pCM18] biofilms by the addition of ursolic acid in flow chambers

Ursolic acid induces genes for chemotaxis, heat shock, and unknown functions.
To investigate the inhibition of biofilm by ursolic acid on a global genetic basis and to compare the findings with our earlier study of another natural biofilm inhibitor furanone (49), DNA microarrays were used here to study the gene expression profiles of E. coli K-12 ATCC 25404 grown with or without ursolic acid. These experiments were done in the absence of the conjugative plasmid R1drd19 so that the results are comparable to the earlier furanone studies and so that the artifact of the roughly 100 genes of R1drd19 are not introduced. Also, ursolic acid was used with suspension cells since it was not practical to get a reasonable biofilm sample due to the inhibition of biofilm by ursolic acid.

The effects of both 10 and 30 µg of ursolic acid/ml were studied. Each experiment was conducted in duplicate (therefore, there were four sets of DNA microarrays for each concentration of ursolic acid), and a number of genes were found to be induced or repressed consistently (Tables 3 and 4). For the genes affected by ursolic acid, the DNA microarray data of E. coli K-12 ATCC 25404 differential gene expression due to furanone and due to AI-2 quorum sensing (49) are also listed in Tables 3 and 4 for comparison. There were 32 genes and 61 genes induced by 10 and 30 µg of ursolic acid/ml, respectively, and 19 genes were consistently induced by both 10 and 30 µg of ursolic acid/ml (Table 3). There was even better consistency in that 11 of the other 13 genes induced by 10 µg of ursolic acid/ml were also upregulated by 30 µg of ursolic acid/ml; they are not listed as induced due to either their high P values or because the induction ratios are lower than 1.4 (Table 3). Hence, the upregulation of these genes was consistent, and there were few differences related to the concentration of ursolic acid used with the microarrays.


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  		TABLE 3. E. coli K-12 genes induced by ursolic acida


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  		TABLE 4. E. coli K-12 genes repressed by ursolic acida

The induced genes with known functions could be divided into five groups. The first has functions for chemotaxis and motility, such as cheA, motAB, tap, and tsr. Although only cheA in the che operon was consistently induced by 10 and 30 µg of ursolic acid/ml, the other genes in the che operon, cheBWYZ, were also induced by 10 µg of ursolic acid/ml (Table 3) and upregulated by 30 µg of ursolic acid/ml (all ~1.4-fold, but the P values were higher than 0.05; Table 3). The induction of the chemotaxis and motility genes indicated that ursolic acid promotes cell movement. However, ursolic acid appeared to be neither a chemoattractant nor a chemorepellent for E. coli based on our chemotaxis assay (data not shown).

The second group of induced genes are involved in a heat shock and stress response such as hslSTV, htpG, and mopB. hslT (alternate name ibpA) and hslS (alternate name ibpB) encode small proteins for responding to heat shock and superoxide stresses (30). Hence, the induction of these heat shock genes suggest the cells were under some kind of stress. The third group of induced genes are involved in transport, such as dcuA, emrK, and malE (Table 3). There were also a number of genes for synthesis and metabolism, as well as phage-related genes that were induced by ursolic acid. Nineteen genes with unknown functions were also induced with 10 and/or 30 µg of ursolic acid/ml (Table 3), such as b1566, b1760, b2674, yjjS, yrfH, and yrfI. b1566 is also known as flxA, and its expression is dependent on the sigma factor for the class 3 flagellar operons; however, the flxA mutant is not deficient in motility (27). b1566 was also found induced by AI-2 and repressed by furanone in our recent study (49). In addition, we also found recently using DNA microarrays that b1566 is induced 8.3-fold during E. coli K-12 biofilm formation on mild steel plates compared to suspension cells (47). Therefore, this gene may be involved in cell movement and biofilm formation.

Genes repressed by ursolic acid.
Based on the microarray results, it was found that 10 and 30 µg of ursolic acid/ml repressed 31 and 17 genes, respectively, and 12 genes were consistently repressed by both 10 and 30 µg of ursolic acid/ml, including cysDJK (Table 4) belonging to the cysteine regulon and regulated by CysB (25, 37). Interestingly, narH for anaerobic respiration was repressed (Table 4). Since biofilm cells are encountering oxygen limitations (44), the repression of narH may help explain biofilm removal by ursolic acid.

Ursolic acid has no effect on the AI-1 and AI-2 quorum-sensing systems.
In a recent study, we found that genes for flagellar synthesis, chemotaxis, and motility were induced by AI-2 and repressed by furanone (49) (the expression ratios affected by AI-2 and furanone for the genes induced/repressed by ursolic acid are listed in Tables 3 and 4). Therefore, the induction of chemotaxis genes in the present study suggests a possible interaction between ursolic acid and AI-2 quorum sensing. To test whether ursolic acid stimulates AI-2 quorum sensing via production of AI-2, ursolic acid was added at different concentrations (0, 1, 5, and 10 µg/ml) to cultures of V. harveyi BB170 (which senses AI-2), and AI-2 activity was measured. The experiment was conducted in duplicate. No apparent induction of AI-2 activity was observed (relative AI-2 activities of 0.48 ± 0.4 x 10–5 RLU without ursolic acid, 1.0 ± 0.2 x 10–5 RLU with 5 µg of ursolic acid/ml, and 0.6 ± 0.005 x 10–5 RLU with 10 µg of ursolic acid/ml). In contrast, the positive control showed an induction of 3,400-fold.

To understand whether ursolic acid inhibits AI-2 quorum sensing, cell-free supernatants from V. harveyi BB120 containing AI-2 were added to V. harveyi BB170 cultures as 10% (vol/vol), and the effect of ursolic acid at 0, 1, 5, 10, and 30 µg/ml was studied (an individual control sample was used for each ursolic acid concentration which had the same amount of ethanol as the corresponding ursolic acid sample). No apparent effect on AI-2 activity was found (e.g., relative AI-2 activity was 0.0034 RLU without ursolic acid and 0.0040 RLU with 10 µg of ursolic acid/ml). In comparison, we showed previously that E. coli AI-2 was repressed by 26,600-fold by furanone (51). Therefore, ursolic acid neither induces nor represses AI-2 quorum sensing.

Similarly, the effect of ursolic acid on V. harveyi AI-1 quorum sensing was also studied by using V. harveyi BB886 as a reporter. This experiment was conducted in duplicate. Again, ursolic acid did not induce (relative AI-1 activities of 4.5 ± 3.0 x 10–4 RLU without ursolic acid and 4.2 ± 3.2 x 10–4 RLU with 10 µg of ursolic acid/ml) or repress (relative AI-1 activities 6.8 ± 0.1 x 10–5 without ursolic acid and 7.8 ± 2.0 x 10–3 with 10 µg of ursolic acid/ml) the AI-1 activity. In contrast, the positive control showed an induction of 17-fold. Hence, the induction of chemotaxis and motility genes by ursolic acid was not through the interaction with either the AI-1 or the AI-2 quorum-sensing system. Note the ursolic acid did not inhibit the growth of the Vibrio strains in LB medium.

motAB and cysB affect biofilm formation.
Since the DNA microarrays identified that motAB was induced by ursolic acid (Table 3), we tested strains with mutations in these genes in the presence of ursolic acid to corroborate these results. Note that the motAB mutation causes cells to lack flagellar motion but not synthesis (5, 43). Without the conjugation plasmid R1drd19, deletion of motAB caused the cells to decrease biofilm formation 1.6-fold relative to the isogenic motA+B+ strain as originally reported (43); however, in the presence of the conjugation plasmid R1drd19, the strain with the motAB deletion made 2.7-fold more biofilm than its parent strain (Fig. 4), suggesting that low motility is favorable for conjugation which promotes biofilm development (16, 45). The results also showed that, compared to its isogenic motA+B+ strain, the motAB mutant is much less sensitive to ursolic acid (in terms of biofilm inhibition), i.e., in the presence of R1drd19 there was no biofilm inhibition with 30 µg of ursolic acid/ml for the motAB mutant versus 84% biofilm reduction for motA+B+ strain (Fig. 4). This is expected according to the DNA microarray data because ursolic acid decreases biofilm formation by inducing expression of motAB, making cells too motile to stay in the biofilm community stably, whereas removing motAB simply counteracts the ability of ursolic acid to inhibit biofilms since the cells are paralyzed. There was no effect of the motAB mutation on the growth of E. coli K-12 MG1655 in LB medium.

Since the microarray data indicated that the cysB-regulated operon cysDJK was repressed by ursolic acid (Table 4), we also studied biofilm formation by a cysB mutant. Interestingly, we found that the cysB mutation increased biofilm formation by 2- to 10-fold compared to its isogenic cysB+ strain in all three media tested (Fig. 4). The observation that the cysB mutation affects biofilm formation was corroborated by two other cysB mutants versus their respective cysB+ hosts (E. coli MC4100 cysB [R1drd19] in both LB medium and LB plus 0.2% glucose and E. coli C600 cysB [R1drd19] in LB medium). Although it is not explained by the microarray data, the result that the cysB mutation increases biofilm formation suggests that CysB controls biofilm formation in a way different from ursolic acid. We noted that cysB mutation decreased specific growth rates of E. coli K-12 MG1655 by 30% in LB and LB containing 0.2% glucose but not in M9 medium supplemented with 0.4% glucose and 0.4% Casamino Acids. This defect in growth by cysB mutation is not a concern for biofilm formation since biofilm is actually increased by the cysB mutant (in the absence of ursolic acid).


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DISCUSSION
 
We focused on E. coli biofilms since this strain is the most thoroughly studied bacterium (6), its genome is sequenced (6), microarrays are available (47, 56), the function of many of its proteins have been elucidated (52), and many isogenic mutants are available (17). Also, our group has experience in determining the genetic basis of E. coli biofilm formation and its inhibition with a natural plant-derived antagonist (47, 51). Furthermore, although E. coli is well studied, its biofilm has not received the same scrutiny since strain K-12 makes a poor biofilm if it lacks a conjugative plasmid (16, 45). Hence, the results of our study are helpful for understanding and preventing the biofilm of the archetypal strain, as well as helpful for combating pathogenic strains such as E. coli O157:H7 (41).

In the present study, we identify a new chemical class of biofilm inhibitor, ursolic acid, and show it is completely nontoxic to E. coli, P. aeruginosa PAO1, V. harveyi, and hepatocytes. Moreover, biofilm inhibition was seen in five different E. coli hosts (K-12, JM109, C600, EJ500, and JCB495) and in four different media (M9, LB, and LB supplemented with either 0.2% glucose or 2% sodium pyruvate); hence, the biofilm inhibition by ursolic acid does not seem to be restricted to specific species, strains, or growth conditions.

By studying the complete transcriptome, it was also shown that ursolic acid induces chemotaxis, motility, and heat shock genes in E. coli and represses genes related to sulfur metabolism. Also, ursolic acid inhibits the development of mature biofilms both in LB medium grown in the 96-well plate assay and in M9 medium (0.4% glucose and 0.4% Casamino Acids) in a continuous flow chamber. The exact mechanism of inhibition is not clear, but the induction of chemotaxis and motility genes suggests that ursolic acid may function as a signal that tells cells to remain too motile for adequate biofilm formation. It has been reported that motility is important for initial cell attachment in E. coli biofilm formation (43). For biofilm maturation, however, the cells need to stay attached to the surface and produce the polysaccharide matrix (67); hence, the motility genes may have to be coordinately regulated since regulation has been shown to be important for other biofilm-related genes (28, 61). Therefore, the induction of chemotaxis and motility genes during the wrong stage of biofilm development may destabilize the biofilm and prevent the formation of a mature biofilm. However, ursolic acid itself does not appear to be a chemoattractant or a chemorepellant for E. coli, and a direct relationship between ursolic acid and motility was not found in that both a motility assay with 30 µg of ursolic acid/ml present in soft agar plates (59) and ß-galactosidase reporter assays with 30 µg of ursolic acid/ml to check induction of the motility-related promoters qseB, fliC, motA, flhD, and fliAeh (59) did not show an impact by ursolic acid. Since mopA and mopB are induced by ursolic acid (Table 3) and since MopA and MopB are chaperons used to refold damaged proteins (38), ursolic acid may disrupt a negative regulator of motility.

Here, biofilm formation was not affected by ursolic acid with a strain that is paralyzed with the motAB mutation, which indicates the importance of torque generation for biofilm inhibition by ursolic acid. These results (Fig. 4) also indicate again that motility is not necessary for normal biofilm formation in E. coli in the presence of a conjugation plasmid (45), although it has been found to alter the biofilm structure in P. aeruginosa PAO1 (31). Note that there was less biofilm with the motAB mutation than with the wild-type strain (without the conjugation plasmid R1drd19) in the absence of ursolic acid as expected (43).

Previously, we have shown that furanone inhibited genes related to AI-2 quorum sensing (49), that it inhibited biofilm formation of E. coli (51), and that it negatively regulated chemotaxis genes (49) (see Tables 3 and 4 for the gene expression ratios for genes in common with those affected by ursolic acid). Hence, it appears that ursolic acid and furanone both inhibit biofilm formation, but by controlling the chemotaxis genes in an opposite manner and therefore affecting different stages of biofilm formation.

It is not clear whether the plants produce ursolic acid constitutively or only after infection by a pathogen. Given that ursolic acid induced chemotaxis genes, ursolic acid may be a defense response of the host after the infection of the pathogen. Further study of the surface concentration of ursolic acid on plants and the effect of environmental conditions on its production may be informative.

Although the microarray data in the present study are statistically significant since the cutoff ratio was based on a standard deviation higher than 2.5 and the P values were <0.05 for the induced and repressed genes, the induction and repression ratios of microarray results in the present study (1.5- to 2-fold; see Tables 3 and 4) are modest compared to our earlier microarray experiments. For example, our previous study showed that 5 µg of furanone/ml induced 94 genes of Bacillus subtilis >5-fold as it acted as a novel antimicrobial for this gram-positive species (48). Another recent study of ours in which furanone acts as a nontoxic biofilm inhibitor for gram-negative strains (49) showed that 60 µg/ml repressed the chemotaxis and motility genes 7- to 300-fold. Although modest, these ursolic acid microarray results are robust in that they provided important insights, as evidenced by the discovery that deleting motAB counteracts biofilm inhibition by ursolic acid and the discovery that sulfur metabolism (through cysB) affects biofilm formation.

Like furanone, the ursolic acid was effective at low concentrations since 10 µg/ml removed 72% of the E. coli JM109 biofilm. Hence, the addition of ursolic acid to destabilize biofilms is a promising approach. The structure of ursolic acid may also be altered to improve efficacy by using combinatorial chemistry and screening with the biofilm assay to identify even better biofilm inhibitors. It is also clear that by studying the molecular basis of the inhibition of biofilms with plant-derived antagonists, important discoveries may be made about the nature of biofilm formation. Here, it was found for the first time that sulfur metabolism is important for biofilms.


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ACKNOWLEDGMENTS
 
D.R. and R.Z. were supported in part by a grant from Sequoia Sciences, Inc. The discovery and isolation of ursolic acid was supported by NIH STTR grant 2R42RR016363-02.

We are grateful for the strains sent by Roberto Kolter, Goran Jovanovic, Tim Mcdermott, Bonnie L. Bassler, Søren Molin, Kazuhiro Kutsukake, and FranÇois Baneyx, as well as for the cooperation and support of the government of Gabon and IPHAMETRA/CENAREST. We thank Jim Miller, John Stone, Adam Bradley, and Gretchen Walters from the Missouri Botanical Garden for the plant collections and identification.


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FOOTNOTES
 
* Corresponding author. Mailing address: Departments of Chemical Engineering and Molecular and Cell Biology, University of Connecticut, 191 Auditorium Rd., Storrs, CT 06269-3222. Phone: (860) 486-2483. Fax: (860) 486-2959. E-mail: twood{at}engr.uconn.edu. Back

{dagger} D.R. and R.Z. contributed equally to this study. Back


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Applied and Environmental Microbiology, July 2005, p. 4022-4034, Vol. 71, No. 7
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.7.4022-4034.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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