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Journal of Bacteriology, November 2006, p. 7335-7343, Vol. 188, No. 21
0021-9193/06/$08.00+0     doi:10.1128/JB.00599-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

BdlA, a Chemotaxis Regulator Essential for Biofilm Dispersion in Pseudomonas aeruginosa

Ryan Morgan,1 Steven Kohn,1 Sung-Hei Hwang,2 Daniel J. Hassett,2 and Karin Sauer1*

Department of Biological Sciences, Binghamton University, SUNY at Binghamton, Binghamton, New York 13902,1 Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-05242

Received 27 April 2006/ Accepted 28 July 2006


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ABSTRACT
 
Multiple environmental cues have been shown to trigger biofilm detachment, the transition from surface-attached, highly organized communities known as biofilms to the motile lifestyle. The goal of this study was to identify a gene product involved in sensing environmental cues that trigger biofilm dispersion in Pseudomonas aeruginosa. To do so, we focused on novel putative chemotaxis transducer proteins that could potentially be involved in environmental sensing. We identified a locus encoding such a protein that played a role in detachment, as indicated by the observation that an isogenic mutant biofilm could not disperse in response to a variety of environmental cues. The locus was termed bdlA for biofilm dispersion locus. The BdlA protein harbors an MCP (methyl-accepting chemotaxis protein) domain and two PAS (Per-Arnt-Sint) domains that have been shown to be essential for responding to environmental signals in other proteins. The dispersion-deficient phenotype of the bdlA mutant was confirmed by treatment with the biocide H2O2 and by microscopic observations. The dispersion response was independent of motility. bdlA mutant biofilms were found to have increased adherent properties and increased intracellular levels of cyclic di-GMP (c-di-GMP). Our findings suggest that BdlA may be a link between sensing environmental cues, c-di-GMP levels, and detachment. Based on our findings, a possible involvement of BdlA in a signaling cascade resulting in biofilm dispersion is discussed.


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INTRODUCTION
 
Biofilms are highly organized, surface-attached microbial communities that can be found at almost any solid-liquid interface in industrial, environmental, and clinical settings (8, 10, 13, 16, 28-29, 57). There is compelling evidence that the biofilm lifestyle is an efficient means for microorganisms to define and maintain a protected niche provided that growth conditions are favorable. However, to spawn novel communities in new locations, microorganisms must be able to successfully transit from the biofilm to the planktonic growth state. The transition is typically induced upon sensing environmental cues. Changes in oxygen or carbon substrate concentration, pH, or other chemical parameters have been reported to induce dispersion of mature biofilms in various organisms, including Pseudomonas aeruginosa, Pseudomonas putida, and Shewanella oneidensis (3, 17, 26, 43, 49, 52).

Although the genes involved in biofilm dispersion are not as clearly defined as those required for biofilm formation, some recent progress has been made concerning the molecular basis underlying this phenomenon. Recent evidence suggests that global regulation of the central carbon flux by the RNA-binding protein CsrA plays a role in biofilm formation and dispersion of Escherichia coli (24-25). The effects of CsrA are mediated largely through the collective regulation of intracellular glycogen biosynthesis and central carbon metabolism, activation of flagellum biosynthesis, and motility (4, 24-25, 36, 58). Recent evidence also suggests that CsrA may play a role in the production of an adhesin. Wang and coworkers demonstrated that csrA disruption causes an ~3-fold increase in the production of the polysaccharide adhesin PGA (poly-ß-1,6-N-acetyl-D-glucosamine) as well as an increase in pga expression, indicating that CsrA destabilizes the pgaA transcript in vivo (54). There is no indication as to a similar role for CsrA homologues in other bacteria.

However, as detailed studies emerge, it is becoming clear that bacterial dispersion from mature biofilms triggered by environmental cues coincides with alteration of matrix components. Vats and Lee showed that a surface protein-releasing enzyme (SPRE) in Streptococcus mutans is actively involved in the degradation of attachment polymers on tooth surfaces, releasing bacteria into the bulk liquid (53). Addition of SPRE results in a 20% increase in the biofilm dispersion rate compared to that for control samples. However, until recently, it was unclear how modulation of matrix components was induced and how the signal was transduced within the cells to degrade polymers, reduce adhesiveness, and initiate biofilm dispersion. It is now widely accepted that cyclic di-GMP (c-di-GMP) signaling, first described to control extracellular cellulose biosynthesis in Gluconacetobacter xylinus (39-40), is involved in the modulation of matrix components, control of autoaggregation of planktonic cells, and biofilm formation in several microorganisms (30, 46, 51). In P. putida, two genes, PP0164 and PP0165, encoding a putative periplasmic protein and a putative transmembrane protein containing GGDEF and EAL domains, respectively, were found to be required for formation and starvation-induced dispersion of P. putida biofilms (17). These two proteins were found to regulate adhesion, probably via the adhesiveness of bacterial cells through c-di-GMP signaling (17) in a phosphorelay-mediated signaling event (43), as indicated by the finding that PP0164 mutant bacteria were unable to reduce their adhesiveness and disperse from biofilms in response to carbon starvation, while PP0165 mutant bacteria were unable to increase their adhesiveness and form biofilms (17). In S. oneidensis, a rapid cellular detachment from the biofilm occurred upon activation of yhjH, encoding an enzyme having phosphodiesterase activity which degrades c-di-GMP (50). In contrast, matrix attachment was shown to be dependent on mxdA, which encodes a cyclic bis(3',5')guanylic acid (c-di-GMP)-forming enzyme involved in c-di-GMP synthesis. The findings indicated that attachment might be controlled by exopolysaccharide (EPS) biosynthesis and increased adhesiveness via increased c-di-GMP levels while detachment is a result of reduced adhesiveness as well as controlled cessation or reduction of such activity due to reduced levels of c-di-GMP. However, it is unclear how environmental cues are sensed, resulting in the modulation of the enzymatic activities of a c-di-GMP-forming diguanylate cyclase(s) and of a c-di-GMP-hydrolyzing phosphodiesterase(s). Thormann et al. (50) proposed a mechanism in which environmental cues are sensed by a sensor protein(s), which modulates the enzymatic activities and, thus, c-di-GMP levels. However, no sensor protein(s) was identified in that study.

Here, we report the identification of a new, putative chemotaxis transducer protein that is involved in biofilm dispersion triggered by environmental cues that may link detachment and environmental cues to c-di-GMP levels. The protein encoded by PA1423 (herein termed bdlA for biofilm dispersion locus) has two PAS (Per-Arnt-Sint) domains that are essential for responding to environmental signals. Deletion of the gene resulted in the formation of biofilms that were not responsive to multiple environmental cues, such as succinate, glutamate, and various heavy metals that triggered dispersion of wild-type P. aeruginosa biofilms. Furthermore, deletion of bdlA resulted in biofilms having a higher relative hydrophobicity and higher c-di-GMP levels than wild-type biofilms. To our knowledge, this is the first study describing a locus in P. aeruginosa that is critical for sensing environmental cues that trigger biofilm dispersion. We propose a mechanism in which BdlA modulates intracellular c-di-GMP levels that affect the aforementioned processes.


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MATERIALS AND METHODS
 
Bacterial strains, media, and planktonic growth conditions. P. aeruginosa strains used in this study are listed in Table 1. All strains were grown aerobically in minimal medium (42) at 22°C in shake flasks at 250 rpm. Glutamate (1.8 mM) was used as the sole carbon source unless otherwise indicated. Biofilms were grown as described below at 22°C in minimal medium containing 1.8 mM glutamate as the carbon source.


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  		TABLE 1. Strains and plasmids used in this study

Isogenic mutants were constructed by allelic replacement using sucrose counterselection, as previously described (45), as follows. A region of genomic DNA containing genes PA1423 (bdlA), PA1561, PA1608, and/or PA1930 was amplified and the PCR products ligated to the suicide vector pEX100T. The respective genes were then replaced by a blunt-ended SacI fragment containing the gentamicin resistance cassette from pPS858 (20). The resulting plasmids were mated into PAO1, and mutants were selected on Pseudomonas isolation agar containing 200 µg/ml gentamicin. Double recombinant mutants were selected on LB plates containing 5% sucrose and confirmed by PCR. For the construction of double mutants, a kanamycin resistance cassette was used. Complementation of the bdlA mutant was accomplished using a mini-CTX system, which allows for single-copy complementation within the unique and nonessential attB site on the P. aeruginosa genome.

Biofilm formation. (i) Continuous-flow tube reactor. Biofilms were grown as described previously (43). Briefly, the interior surfaces of size 13 silicone tubing (Masterflex; Cole Parmer, Inc.) of a once-through continuous-flow tube reactor system were used to cultivate biofilms at 22°C for 5 days.

(ii) Flow cell biofilms. Biofilm architecture and development were studied by using flow cells as described previously (41, 47). Images of biofilms grown in once-through flow cells were viewed by confocal scanning laser microscopy using an LSM 510 Meta inverted microscope (Zeiss, Heidelberg, Germany). Images were obtained with an LD-Apochrome 40x/0.6 lens and with LSM 510 Meta software (Zeiss) and visualized using a live/dead BacLight stain from Invitrogen (Eugene, OR). Quantitative analysis of epifluorescence microscopic images obtained from flow cell-grown biofilms at the 5-day time point was performed with COMSTAT image analysis software (19).

(iii) Ninety-six-well microtiter dish assay. Initial biofilm formation was measured by using a 96-well microtiter dish assay system, as described previously (47).

Induction of biofilm dispersion. Biofilms were pregrown for 5 days in minimal medium containing 1.8 mM glutamate as the carbon source. After 5 days of biofilm growth, biofilm dispersion was induced by the addition of succinate (20 mM), Na3AsO3 (2 mM), AgNO3 (2 mM), or HgCl2 (2 mM) to the growth medium. Dispersion was indicated by an increase in turbidity at 600 nm in the effluent from the silicone tubing. The total numbers of viable bacteria in the effluent and the remaining biofilm were determined by serial plate counts on LB agar at 37°C.

Motility assays. Swimming and twitching motilities were assessed as described previously (47).

Microbial adhesion to hydrocarbon (MATH) test. To compare the relative hydrophobicities of P. aeruginosa wild-type and bdlA::Gm mutant strains, cell adherence to hexadecane was determined as described by Déziel et al. (13), except that 5-day-old biofilms were used.

One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane protein fractions and protein identification by mass spectrometry. Preparation of cell extracts and protein determinations were conducted as described previously (41, 43, 47). The membrane protein fraction was obtained by first removing broken cell debris and insoluble material by centrifugation at 36,000 x g for 30 min at 4°C, followed by ultracentrifugation at 150,000 x g for 60 min at 4°C. The membrane pellet was dissolved in Tris-EDTA buffer containing 1% CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate} and subsequently analyzed by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein bands of interest were excised from the gel and digested in situ with trypsin by use of a ProGest workstation (Genomics Solutions, Inc., Michigan). The resulting tryptic peptides were analyzed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry using an Ettan MALDI-TOF Pro mass spectrometer (GE Healthcare, Piscataway, NJ) as described in detail previously (1). All proteins were identified with significant certainty (probability score of <0.02). Proteins were identified with 3 to 15 matched peptides and a minimum of 5% sequence coverage.

Extraction and quantification of c-di-GMP from P. aeruginosa wild-type and bdlA mutant biofilms. c-di-GMP was extracted from biofilms grown in tube reactors as described above. Biofilm cells were harvested after 5 days, centrifuged, and washed with phosphate-buffered saline. The pellets were then resuspended in phosphate-buffered saline. c-di-GMP was extracted by heat and ethanol in triplicate (2, 46) as follows. Cells were heated at 100°C for 5 min, and then ethanol was added to a final concentration of 65%. Samples were centrifuged, the supernatant was retained, and the extraction was repeated. Combined supernatants were dried using a SpeedVac. Dried samples were resuspended in 50 µl of 20 mM ammonium bicarbonate buffer, vortexed, ultrasonicated, and centrifuged, and the supernatant was retained. This was repeated, and the supernatants were combined. Samples were analyzed by liquid chromatography-mass spectrometry and tandem mass spectrometry using a QStar quadrupole time-of-flight mass spectrometer (Applied Biosystems) equipped with a nanospray ion source (Proxeon). c-di-GMP was detected via its fragmentation patterns as described by Thormann et al. (50). The extraction also contained GTP. Since the GTP concentration was shown to be 100-fold higher than the c-di-GMP concentration and was not affected by variation in the c-di-GMP concentration (38, 51), c-di-GMP was quantified by comparing the peak intensity of c-di-GMP to that of GTP. GTP (Sigma) was used as a control.


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RESULTS
 
Identification of a P. aeruginosa chemotaxis gene that is essential for nutrient-induced biofilm dispersion. We previously described that sudden changes in the nutrient concentration induced biofilm dispersion (43). This finding indicated a potential role of chemotaxis in the dispersion response by sensing chemical gradients or changes in the availability of nutrients. To elucidate the connection between detachment and sensing of environmental cues, we focused on novel, putative chemotaxis transducer proteins that could potentially be involved in environmental sensing. Based on the P. aeruginosa genome (www.pseudomonas.com), we selected four putative chemotaxis transducer (also known as methyl-accepting chemotaxis) genes for further analysis (PA1608, PA1561 [also known as the aer, or aerotaxis, gene], PA1930, and PA1423). Our selection was based in part on the presence or absence of PAS domains that are essential for responding to environmental signals. Isogenic mutants were generated and subsequently tested for whether their biofilms were incapable of the nutrient-induced dispersion response. Biofilm dispersion was induced by a sudden 10-fold increase in the medium glutamate concentration. Overall, eight P. aeruginosa mutants (harboring mutations in either one or two of the respective genes [Table 1]) were tested, but for clarity only the results from two P. aeruginosa mutants are shown in Fig. 1. All mutants, including the aer mutant, dispersed from the biofilms, except bacteria harboring a deletion in the PA1423 gene (bdlA) (Fig. 1).


Figure 1
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  		FIG. 1. Induction of biofilm dispersion in isogenic PA1608::Gm, bdlA::Gm, and complemented bdlA::Gm mutant biofilms and P. aeruginosa wild-type (WT) biofilms. Biofilms were grown in a tube reactor, composed of size 13 tubing with an internal volume of 0.5 ml (tubing length, 1 m; flow rate, 0.05 ml/min) for 4 days in minimal medium containing 1.8 mM glutamate as the sole carbon source. The retention time of the biofilm tube reactor was 10 min. After 5 days of P. aeruginosa biofilm growth, biofilm dispersion was induced by a sudden 10-fold increase of the medium glutamate concentration. The effluent of untreated P. aeruginosa wild-type biofilms was used as a control.

Interestingly, BdlA was the only putative chemotaxis transducer protein among the putative chemotaxis transducer proteins analyzed harboring an MCP (methyl-accepting chemotaxis protein) domain and two signature motifs known as PAS (Per-Arnt-Sint) domains (www.pseudomonas.com) (22, 59). The closest known homologue of BdlA was found to be Aer (PA1561, 30% identity), the signal transducer for aerotaxis in E. coli (5). The Aer redox sensor has been shown to harbor PAS domains involved in signal transduction (35). Complementation of the bdlA mutant strain restored the rate of biofilm dispersion compared to that of wild-type bacteria (Fig. 1).

Differences in dispersion are not related to fitness or colony morphology variance. We tested whether the lack of dispersion of bdlA mutant biofilms was due to altered growth rates or initial attachment. Compared to the P. aeruginosa wild type, the bdlA mutant strain showed no difference in doubling times in liquid culture (140 and 125 min, respectively) or in initial attachment (not shown). Furthermore, previous reports have linked biofilm dispersion in bacteria to the emergence of colony morphology variants (7, 31, 56). However, we did not observe differences in the colony morphologies of wild-type and bdlA mutant strains (data not shown).

BdlA is essential for biofilm dispersion induced by various environmental cues. To further elucidate the dispersion response, we determined whether the lack in biofilm dispersion of bdlA mutant biofilms was limited to sudden increases in the medium glutamate concentration or whether the lack in dispersion response was of a more general nature. Therefore, the effects of various other compounds known to act as chemoattractants and chemorepellents (9, 14, 33), including succinate as well as salts of Hg2+, Ag+, and As3+, were tested. The process of biofilm dispersion was determined by viable cell counts. As shown in Fig. 2B, less than 20% of biofilm cells were detected in the effluent of the bdlA::Gm mutant biofilms. This loss of biomass was similar to that for untreated bdlA::Gm biofilms used as controls. The highest viable counts compared to those for untreated bdlA mutant biofilms were detected upon treatment with succinate; however, the difference was not statistically significant (P > 0.6). In contrast, dispersion was induced in the P. aeruginosa wild-type biofilm cells independent of the environmental cue tested (Fig. 2A). On average, up to 80% of wild-type biofilm bacteria dispersed in the presence of compounds known to act as chemoattractants and chemorepellents in other systems (Fig. 2A). Similar results were observed when the effect of biofilm dispersion was determined by protein content (data not shown). Our findings indicate that reduction in viable cells per biofilm was not a result of killing or cell lysis but biofilm dispersion. Furthermore, our findings indicate that BdlA is essential for the dispersion response to environmental cues.


Figure 2
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  		FIG. 2. Biomass distribution after induced dispersion in P. aeruginosa PAO1 (A) and the bdlA::Gm mutant (B). Biofilm dispersion was induced by the addition of 2 mM silver nitrate (AgNO3), mercury chloride (HgCl2), or sodium arsenate (NaAsO2) or by the addition of 20 mM succinate to the growth medium, and dispersed cells were collected. The remaining biofilm cells were harvested upon completion of the dispersion event. The biomass distribution was determined by calculating the ratio of the numbers of viable cells (CFU/ml) of the remaining biofilm population and of the dispersed biofilm population. Experiments were done in triplicate. Black bars indicate the biomasses (in percentages) of remaining biofilms postdispersion, and gray bars indicate the dispersed biofilm populations.

bdlA::Gm mutant biofilms remain resistant to biocides after induced dispersion. To confirm that the bdlA mutant biofilm architecture was indeed still intact upon nutrient-induced dispersion, we tested for resistance to the biocide H2O2. To do so, P. aeruginosa wild-type and bdlA mutant cells were grown as biofilms for 5 days, after which time biofilm dispersion was induced by a sudden 10-fold increase of the medium glutamate concentration. Upon completion of the dispersion response, the remaining biofilms were treated with 1% H2O2 for 30 min. Then, the remaining biofilms were harvested and viable cell counts determined. Dispersed, H2O2-untreated biofilm cells were used as controls. Interestingly, H2O2 treatment of P. aeruginosa wild-type biofilms resulted in a 2-log reduction in viable cell counts whereas no change in CFU was detected for bdlA mutant biofilms. Similar results were obtained by protein determination (not shown). The results confirm that bdlA mutant biofilms are deficient in biofilm dispersion. Furthermore, our data indicate that the lack of biofilm dispersion in the bdlA mutant correlated with resistance to biocides. The decreased killing of the bdlA mutant biofilm may in part be due to the difference in population (~10-fold more bacteria in the bdlA mutant biofilm than in the wild-type biofilm) or reflect an intact biofilm structure/architecture conferring resistance.

Phenotypic characterization of the dispersion response. To further investigate the biofilm architecture, biofilm formation was analyzed after extended growth by using a flow cell biofilm reactor system. The bdlA mutant was found to form biofilms with a distinct three-dimensional architecture. However, while the wild-type biofilm was composed of large, irregularly shaped microcolonies (Fig. 3A), the bdlA::Gm mutant biofilm displayed smaller, densely packed, and symmetrical microcolonies (Fig. 3B). Quantitative analysis of biofilm architecture using COMSTAT confirmed our observations, indicating that the bdlA mutant biofilm differed from the wild-type biofilm with respect to average and maximum thickness as well as total biomass (Table 2). However, when the total biomass and the total CFU/biofilm for the wild-type and bdlA mutant biofilms were determined, no difference was detected. Both wild-type and mutant biofilms generated similar biomasses (~2.25 mg) and CFU/biofilm (~8.1 x 1010), indicating that the distinct biofilm architecture of the bdlA::Gm mutant was not a result of a defect in biofilm formation.


Figure 3
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  		FIG. 3. Microscopic characterization of the dispersion response. Confocal scanning laser microscopy images of biofilms, at a magnification of x400, of P. aeruginosa PAO1, the bdlA::Gm mutant, and the P. aeruginosa bdlA::Gm mutant complemented with an intact bdlA gene after 5 days of growth, showing the xy and xz planes. The microscopic images in panels A and C show the same P. aeruginosa wild-type biofilm before (A) and after (C) a nutrient-induced dispersion event, a sudden 10-fold increase of the glutamate. The microscopic images in panels B and D show the same P. aeruginosa bdlA::Gm mutant biofilm before (B) and after (D) a nutrient-induced dispersion event. The enlarged microscopic images shown in panel E show microcolonies of bdlA::Gm mutant biofilms complemented with an intact bdlA gene as well as {Delta}pilA and {Delta}flgB mutant biofilms.


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  		TABLE 2. COMSTAT quantitative analysis of biofilm structures of P. aeruginosa wild-type and bdlA::Gm mutant strains before and after nutrient-induced dispersiona

Upon biofilm dispersion induced by treatment with a 10-fold increase in the medium glutamate concentration, the wild-type biofilms consisted of only a few attached cells and some dispersed small clusters (Fig. 3C). The average biomass decreased from about 20 µm3/µm2 to less than 1.5 µm3/µm2 following dispersion (Table 2). At the same time, the maximum thickness of the biofilms formed by the wild type decreased from approximately 70 µm to less than 20 µm whereas the average thickness decreased to less than 1.5 µm (Table 2). Similar results were obtained for the complemented mutant biofilm strain (Table 2). However, the bdlA mutant biofilm appeared unchanged in structure after treatment (Fig. 3D). Our visual observations were confirmed using the COMSTAT image analysis program. Total biomass and roughness coefficient as well as average and maximum thickness of the bdlA mutant biofilm before and after nutrient-induced dispersion were similar (Table 2). Complementation of the bdlA mutant restored both the wild-type three-dimensional biofilm architecture and the dispersion response (Fig. 3E; Table 2).

Swimming and twitching motility does not affect the biofilm dispersion response. The mutant strain showed impaired twitching motility compared to that of the wild type (not shown), indicating that the altered bdlA mutant biofilm architecture may be a result of impaired twitching motility. This was confirmed by microscopy. The bdlA mutant biofilm architecture appeared similar to that of a twitching-motility-deficient P. aeruginosa mutant, which consisted of distinct microcolonies that were regularly spaced and nearly uniform in size (Fig. 3E) (18). Complementation of the bdlA mutant restored the wild-type twitching phenotype (not shown).

We previously demonstrated that biofilm dispersion in P. aeruginosa, triggered by carbon substrate availability, was associated with decreased expression of pilus (e.g., pilA) genes in dispersed cells (43). Since the bdlA mutant was impaired in twitching motility, we further elucidated the role of twitching in biofilm dispersion. To do so, we examined the three-dimensional structure of pilA mutant biofilms before and after nutrient-induced dispersion. As shown in Table 2, the pilA mutant biofilm dispersed in response to an increase in the nutrient concentration, indicating that the dispersion-deficient phenotype of bdlA was not related to twitching motility.

A proteomic analysis combined with peptide mass fingerprinting of membrane protein profiles of both wild-type and bdlA mutant strain biofilms indicated ~6-fold-increased expression of the flagellar filament protein FliC (type B) in wild-type biofilms compared to bdlA mutant biofilms. The finding suggested reduced swimming motility of the bdlA mutant under biofilm growth conditions. However, no difference in swimming motility was detected under planktonic growth conditions by use of a swimming motility assay (not shown). Furthermore, we previously demonstrated that biofilm dispersion in P. aeruginosa, triggered by carbon substrate availability, was associated with increased expression of flagella (fliC) (43), suggesting an involvement of swimming motility in the dispersion response. We therefore determined whether the presence of flagellar and/or swimming motility is essential for the nutrient-induced dispersion response by using a nonmotile P. aeruginosa mutant strain ({Delta}flgB) and examining the three-dimensional biofilm structure before and after nutrient-induced dispersion. Interestingly, no difference in dispersion response was detected between the P. aeruginosa wild type and the flgB mutant. As shown in Table 2, the flgB mutant biofilm dispersed in response to an increase in the nutrient concentration.

We also determined whether flagellar rotation is essential for the nutrient-induced dispersion response. To do so, we tested whether the upper-tier chemotaxis regulator CheB controls the nutrient-induced dispersion response by use of an isogenic cheB mutant. This was based on the findings of several studies implicating the involvement of the phosphorelay regulators CheY and CheB in regulating the direction of flagellar rotation. We made use of a cheB mutant that was shown by Kato et al. (27) to change its swimming direction much more frequently than wild-type P. aeruginosa PAO1. No difference in the nutrient-induced dispersion response of the cheB mutant compared to that of the P. aeruginosa wild type was detected (Table 2). Overall, our data suggest that nutrient-induced biofilm dispersion may be mediated by a mechanism independent of flagellar rotation and flagellum-mediated motility.

The dispersion-deficient BdlA phenotype correlates with increased relative hydrophobicity and increased c-di-GMP levels. Bacterial dispersion of biofilms in response to environmental cues was shown to coincide with a reduction in the adhesiveness of biofilm bacteria (17). To determine the adhesiveness of biofilm bacteria, we determined the relative hydrophobicities of both wild-type and bdlA mutant biofilms by using the MATH test as a measure of adhesiveness of bacterial cells. Biofilms were grown for 5 days, harvested, and immediately tested using the MATH assay. Overall, the bdlA mutant biofilm cells were found to be more hydrophobic (~38% ± 1.5% [standard deviation]) than the wild-type biofilm cells (~24% ± 1.9%) (P < 0.05). This finding suggested that the dispersion deficiency of the bdlA mutant might coincide with increased hydrophobicity and, thus, adhesiveness. We speculated that increased adhesiveness might be a result of increased c-di-GMP levels. To test this hypothesis, we examined the intracellular levels of c-di-GMP in both wild-type and bdlA mutant biofilms. c-di-GMP was extracted and quantified by liquid chromatography-mass spectrometry analysis as described in Materials and Methods. The same extraction also allowed us to simultaneously detect GTP. Since the GTP concentration was shown to be 100-fold higher than the c-di-GMP concentration and was not affected by variation in the c-di-GMP concentration (38, 51), c-di-GMP was quantified by comparing the peak intensity of c-di-GMP to that of GTP. Mass spectrometry analysis revealed that the peak intensity ratio between GTP and intracellular c-di-GMP was 18.2 (±2.5):1 in P. aeruginosa wild-type biofilms and 3.2 (±0.74):1 in bdlA mutant biofilms, indicating that the c-di-GMP level was much lower in wild-type than in bdlA mutant biofilm cells. Thus, by comparing the intracellular pools of GTP and c-di-GMP, we determined that bdlA mutant biofilms contained intracellular c-di-GMP levels that were about five- to sixfold higher than those in P. aeruginosa wild-type biofilms.


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DISCUSSION
 
In this study, we present compelling evidence that the bdlA gene, encoding a putative chemotaxis transducer, plays a role in mediating biofilm dispersion in response to environmental cues, such as sudden changes in the concentrations of classical attractants and repellents. The dispersion-deficient phenotype of the bdlA mutant strain resulted in biofilms that were resistant to the biocide H2O2, indicating that the overall biofilm architecture remained intact upon dispersion. Microscopic observations combined with COMSTAT analyses confirmed this finding (Fig. 3; Table 2). Several reports have described P. aeruginosa colony morphology variants with hyperdetaching phenotypes. Webb et al. (55-56) reported the emergence during biofilm development of a small-colony variant that exhibited enhanced attachment and accelerated both biofilm development and detachment. The emergence of the small-colony variant was linked to the activity of the Pf4 phage in P. aeruginosa biofilms. Boles et al. discovered two variants, termed "mini" and "wrinkle," that arose spontaneously from biofilms at a high frequency (7). The wrinkle variant exhibited reduced detachment rates but increased attachment and cell cluster formation rates, while the mini variant exhibited a hyperdetaching phenotype by a mechanism requiring the biosurfactant rhamnolipid (7-8). Here, we demonstrate that the dispersion-deficient phenotype of the bdlA mutant strain was found to be independent of colony variance. Furthermore, the bdlA mutant strain did not exhibit many of the phenotypic traits associated with these variants, including accelerated attachment, biofilm development, or lack of rhamnolipid production. Consistent with these observations, we observed the formation of pronounced water channels (Fig. 3) that are maintained via the production of rhamnolipid (12). However, our proteomic analyses indicated the potential involvement of phage in the dispersion response since the production of Pf1 phage proteins was increased in the bdlA mutant biofilm (not shown).

Cellular c-di-GMP levels have been implicated in bacterial biofilm formation, in most cases, via control of EPS production (23, 30, 37, 46, 51). Furthermore, it has been speculated that c-di-GMP signaling controls the transitioning between sessile and motile lifestyles. High c-di-GMP concentrations have been shown in Salmonella enterica serovar Typhimurium to stimulate biofilm formation and EPS production (and thus, adhesiveness) but to suppress motility, while low concentrations inhibited biofilm formation, repressed the production of EPS, and stimulated swimming and swarming motilities (32, 38, 46). Recent evidence further supports the role of c-di-GMP signaling in regulating the transitioning between sessile and motile lifestyles. In P. putida, two genes have been shown to be involved in regulating bacterium:surface adhesion and, thus, starvation/oxygen-induced biofilm dispersion. These genes encode a putative transmembrane protein containing a GGDEF/EAL domain and a putative periplasmic protein (17). The intracellular level of cyclic-di-GMP is regulated through cyclases and phosphodiesterases harboring GGDEF/EAL domains. The mechanism by which these two proteins participate in biofilm dispersion is probably cyclic-di-GMP signaling (11, 15, 17, 46). Furthermore, a molecular analysis of S. oneidensis revealed two genes involved in shifting the state of a biofilm cell between attachment and detachment in a concentration-dependent manner (50). mxdA, encoding a cyclic bis(3',5')guanylic acid (c-di-GMP)-forming enzyme with an unusual GGDEF motif, was found to be essential for matrix attachment. In contrast, rapid cellular detachment from the biofilm occurred upon induction of yhjH, a gene encoding an enzyme possessing phosphodiesterase activity. Taken together, the findings from previous reports indicate that biofilm detachment coincides with low levels of c-di-GMP levels, reduced adhesiveness, and increased motility (6, 50).

Interestingly, bdlA mutant biofilms were characterized by high levels of c-di-GMP compared to levels from wild-type biofilms, enhanced adhesiveness as indicated by the MATH assay, and reduced swimming motility as indicated by the reduced production of the FliC protein. However, a direct role of BdlA in regulating the c-di-GMP level and thus biofilm dispersion in P. aeruginosa can be excluded since none of the amino acids found in the GGDEF/EAL domains are within BdlA. However, our data clearly indicate a role of BdlA in dispersion. While BdlA does not directly transduce biofilm dispersion in P. aeruginosa, our data suggest that BdlA may be involved indirectly in a signaling cascade within the cells that results in the modulation of bacterium:surface adhesion to initiate biofilm dispersion. Thus, we propose that BdlA may regulate or transduce biofilm dispersion and, as such, the adhesiveness indirectly via cyclic-di-GMP levels by acting as a sensor protein. The potential involvement of BdlA in a signaling cascade is based on the finding that the protein possesses similarities to chemotaxis transducer proteins (www.pseudomonas.com) and, although direct evidence for this notion is lacking, based on its cytoplasmic location and the signature MCP and two PAS redox-signaling domains. Proteins harboring PAS domains are all located in the cytosol and are important signaling modules that sense environmental factors that cross the cell membrane and/or affect cell metabolism, such as chemoattractants or chemorepellents, and monitor changes in light, redox potential, oxygen, and overall cellular energy status (5, 35, 48). Furthermore, the closest known homologue of BdlA was found to be Aer (PA1561). In E. coli, the PAS domain-containing Aer protein regulates the motile behavior of bacteria in gradients of oxygen, redox potential, and certain nutrients (5, 35). We therefore propose that BdlA is involved in sensing environmental cues, thus functioning as a sensor protein, which in turn modulates the enzymatic activity of a c-di-GMP-forming diguanylate cyclase(s) and/or a c-di-GMP-hydrolyzing phosphodiesterase(s), such as an EAL domain-containing protein(s). Figure 4 summarizes the involvement of BdlA as well as other key components and their mode of interaction in controlling the transitioning of biofilm cells between attachment and detachment. The model of detachment proposed here resembles that of Gjermansen et al. (17) and that of Thormann et al. (50).


Figure 4
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  		FIG. 4. Model for role of BdlA as a sensor protein in controlling biofilm formation and biofilm dispersion/detachment by c-di-GMP in P. aeruginosa. Environmental cues, such as sudden changes in the succinate concentration, are sensed by BdlA, which modulates the enzymatic activity of a c-di-GMP-forming diguanylate cyclase(s) harboring GGDEF domains and/or of a c-di-GMP-hydrolyzing phosphodiesterase(s), such as an EAL domain-containing protein(s). At this point, it is unclear how BdlA modulates the activities of GGDEF- and/or EAL-harboring enzymes that result in alteration of the c-di-GMP levels. High intracellular c-di-GMP levels coincide with biofilm formation, while low levels coincide with biofilm dispersion/detachment. The model proposed here resembles that of Gjermansen et al. (17) and that of Thormann et al. (50).

As mentioned above, low c-di-GMP concentrations stimulate not only biofilm detachment but also swimming and swarming motilities. We previously reported that biofilm dispersion triggered by carbon substrate availability was associated with increased expression of flagella (fliC) and correspondingly decreased expression of pilus (pilA) genes in dispersed bacteria (43). However, here we present evidence that motility is not required for biofilm dispersion, as corroborated by the observation that flagellum- and pilus-dependent motility is not required for detachment in P. aeruginosa (Table 2). Similar observations were made for the starvation-induced dispersion response in P. putida and in S. oneidensis, in which sudden depletion of molecular oxygen triggered biofilm detachment (17, 50). Furthermore, biofilm detachment in S. oneidensis coincided with the release of individual cells and small cell clusters independent of swimming motility. The finding suggests that c-di-GMP-controlled biofilm detachment is probably a mixture of both (i) the result of cells actively escaping from the biofilm (termed dispersion) in a process requiring swimming motility and (ii) the result of a passive process in which individual cells and cell clusters detach/slough from the biofilm, requiring the reduction of EPS biosynthesis and decreased adhesiveness.

Overall, our findings suggest that biofilm dispersion, like any other stage of biofilm development, is a dynamic, highly regulated process, controlled by a yet-uncharacterized hierarchical set of genes and triggered by particular environmental cues. Here, we demonstrated that only one gene product, BdlA, was essential to mediate the dispersion response to a variety of environmental signals, such as glutamate, succinate, and salts of silver, arsenite, and mercury. The dispersion-deficient phenotype was independent of colony variants and genes and proteins that have been reported to be involved in attachment and biofilm development. Furthermore, based on the similarity to Aer, BdlA may be involved in sensing and transducing signals within cells, resulting in the modulation of c-di-GMP levels, swimming motility, and adhesiveness of the bacterial cell surface.


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ACKNOWLEDGMENTS
 
This work was supported by grants from the National Institutes of Health (AI-40541, GM-69845, and HL073835-01), the National Science Foundation (DBI-0321046 and 0311307), and the Cystic Fibrosis Foundation (HASSETT03P0) and by a Binghamton University undergraduate student research award (awarded to R. Morgan and S. Kohn).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biological Sciences, Binghamton University, SUNY at Binghamton, 104 Science III, Binghamton, NY 13902. Phone: (607) 777-3157. Fax: (607) 777-6521. E-mail: ksauer{at}binghamton.edu. Back


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Journal of Bacteriology, November 2006, p. 7335-7343, Vol. 188, No. 21
0021-9193/06/$08.00+0     doi:10.1128/JB.00599-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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