Escherichia coli BdcA controls biofilm dispersal in Pseudomonas aeruginosa and Rhizobium meliloti
© Wood et al; licensee BioMed Central Ltd. 2011
Received: 23 June 2011
Accepted: 26 October 2011
Published: 26 October 2011
Previously we showed that BdcA controls Escherichia coli biofilm dispersal by binding the ubiquitous bacterial signal cyclic diguanylate (c-di-GMP); upon reducing the concentration of c-di-GMP, the cell shifts to the planktonic state by increasing motility, decreasing aggregation, and decreasing production of biofilm adhesins.
Here we report that BdcA also increases biofilm dispersal in other Gram-negative bacteria including Pseudomonas aeruginosa, Pseudomonas fluorescens, and Rhizobium meliloti. BdcA binds c-di-GMP in these strains and thereby reduces the effective c-di-GMP concentrations as demonstrated by increases in swimming motility and swarming motility as well as by a reduction in extracellular polysaccharide production. We also develop a method to displace existing biofilms by adding BdcA via conjugation from E. coli in mixed-species biofilms.
Since BdcA shows the ability to control biofilm dispersal in diverse bacteria, BdcA has the potential to be used as a tool to disperse biofilms for engineering and medical applications.
Bacteria prefer to grow as biofilms ; i.e., as aggregates of bacteria that form usually at interfaces. Cells in biofilms are protected and held together by the extracellular matrix that includes polysaccharide, lipids, protein, and DNA . Biofilms are notoriously difficult to remove [3, 4] and are related to 80% of all human infections . In addition, biofilm formation leads to significant energy losses in heat exchange equipment  and causes operational problems in the oil industry via metal corrosion, flow reduction, contamination, and reservoir plugging . Conversely, biofilms are becoming increasingly important for chemical transformations; for example, biofilms lead to better productivity for biofuels such as ethanol , and biofilm communities form on electrodes surfaces to produce power for microbial fuel cells . Also, for biorefineries, it will be important to be able to remove biofilms from fixed film reactors so that diverse products may be synthesized . Hence, effective strategies to remove biofilms for medical applications as well as to control biofilm formation for engineering applications are important. Biofilm development is a dynamic process that includes movement to the surface , initial reversible attachment, irreversible attachment, formation of small aggregates, maturation of the biofilm, and biofilm dispersal . Biofilm dispersal is controlled by various factors, including external forces and the physiological changes of the bacteria themselves .
Previously we discovered the biofilm dispersal protein BdcA in Escherichia coli, which works by binding to the intracellular signal cyclic diguanylate (c-di-GMP) but does not act as a phosphodiesterase . Reducing c-di-GMP concentrations increases motility and extracellular DNA production and decreases extracellular polysaccharide production (EPS), cell length, and aggregation which leads to biofilm dispersal . We also performed protein engineering on this protein and successfully abolished biofilm formation via single amino acid replacement at E50Q . Hence biofilm dispersal can be achieved by simply by producing BdcA in E. coli. However, it has not been demonstrated that this protein is functional and can cause dispersal of biofilms in other bacteria.
Since BdcA works through c-di-GMP , and c-di-GMP exists in almost all bacteria  and regulates biofilm formation by controlling cell motility in Gram-negative bacteria , we hypothesized that BdcA may also work in most Gram-negative bacteria. Hence, here we constructed a broad-host plasmid to produce BdcA (pMMB206-BdcA) and tested the effect of BdcA production on biofilm dispersal for the opportunistic pathogen Pseudomonas aeruginosa (responsible for chronic cystic fibrosis infections [15, 16]), the biocontrol agent Pseudomonas fluorescens (utilized for protecting plants from fungi infection [17, 18]), as well as the nitrogen-fixing bacterium Rhizobium meliloti [19, 20]. For all three bacteria, we find that BdcA is an effective tool for dispersing biofilms.
Bacterial strains, media, and growth conditions
Strains and plasmids used in this study
E. coli BW25113
lacI q rrnB T14 ΔlacZ WJ16 hsdR514 ΔaraBAD AH33 ΔrhaBAD LD78
E. coli DH5α
luxS supE44 ΔlacU169(Φ80 lacZΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1
E. coli DH5α/ pMMB206/pRK2013
E. coli harboring pMMB206 and pRK2013
E. coli DH5α/ pMMB206-BdcA/pRK2013
E. coli harboring pMMB206-BdcA and pRK2013
P. aeruginosa PA14
P. aeruginosa wild-type strain
P. fluorescens wild-type strain
R. meliloti 102F34
R. meliloti wild-type strain
P. aeruginosa wild-type harboring pMMB206
P. aeruginosa wild-type harboring pMMB206-BdcA
P. aeruginosa wild-type harboring pMMB206 and pP25-gfp
P. aeruginosa wild-type harboring pMMB206-BdcA and pP25-gfp
P. fluorescens wild-type harboring pMMB206
P. fluorescens wild-type harboring pMMB206-BdcA
R. meliloti 102F34/pMMB206
R. meliloti wild-type harboring pMMB206
R. meliloti 102F34/pMMB206-BdcA
R. meliloti wild-type harboring pMMB206-BdcA
R. meliloti 102F34/pMMB206/pP25-gfp
R. meliloti wild-type harboring pMMB206 and pP25-gfp
R. meliloti 102F34/pMMB206-BdcA/pP25-gfp
R. meliloti wild-type harboring the pMMB206-BdcA and pP25-gfp
Cmr, Mob+, lacI q , broad-host range plasmid
Cmr; pMMB206 with P tac-lacUV5 ::bdcA
Kmr; Tra+ Mob+ (RK2) Km::Tn7 ColEl origin, helper plasmid for mobilization
M. Bagdasarian, 
Carr; constitutive green fluorescent protein production
Static biofilm assay using crystal violet
Biofilm formation for the individual single strains was assayed in 96-well polystyrene plates using 0.1% crystal violet staining (Corning Costar, Cambridge, MA) as described previously  with small modifications. Briefly, each well was inoculated with overnight cultures at an initial turbidity at 600 nm of 0.05 and grown without shaking for 24 h with chloramphenicol, and then 0.1 mM IPTG was added to the culture to induce bdcA. For P. aeruginosa, the mixture was shaken at 150 rpm for 1 min and then incubated for 2 h (26 h total) to establish the extent of mature biofilm formation. Biofilm dispersal was determined after 19 h of IPTG induction (43 h total). Comparison of these two values gave the percentage of biofilm dispersal. Similarly, for P. fluorescens and R. meliloti, biofilm formation was determined and compared after 2 h and 21.5 h of IPTG induction. At least two independent cultures were used for each strain. To remove growth effects, biofilm formation was normalized by dividing total biofilm by bacterial growth in the bulk liquid.
For BdcA dispersal in dual-species biofilms of E. coli/R. meliloti and E. coli/P. aeruginosa formed in 96-well plates, pMMB206-BdcA was mobilized using E. coli DH5α/ pMMB206-BdcA/pRK2013 by adding 300 μL of turbidity 0.05 of these washed cells to the R. meliloti and P. aeruginosa biofilms formed for 24 h. After 4 h, 0.1 mM IPTG was added to produce BdcA, and cells were grown for an additional 26 h. To approximate the conjugation efficiency, biofilm cells were taken from the air/liquid biofilm (top) of each well in the 96-well plates at the end of the experiment and plated on LB, LB + rifampicin, and LB + rifampicin + chloramphenicol selective plates. For rifampicin, 20 μg/mL inhibits E. coli, R. meliloti is resistant to 20 μg/mL, and P. aeruginosa is resistant to 100 μg/mL; chloramphenicol was used to select for cells harboring pMMB206 or pMMB206-BdcA. The conjugation efficiency of R. meliloti and P. aeruginosa was calculated by comparing the number of recipient cells harboring pMMB206 or pMMB206-BdcA with the total number of recipient cells for each species. In addition, five random colonies were selected from LB plates with 100 μg/mL rifampicin and 250 μg/mL chloramphenicol (selective plates for P. aeruginosa/pMMB206-BdcA) and another five colonies were selected from LB plates with 20 μg/mL rifampicin and 50 μg/mL chloramphenicol (selective plates for R. meliloti/pMMB206-BdcA) to verify the presence of the plasmid and to identify the host. The presence of the plasmid in recipient cells was confirmed via PCR of the bdcA gene with the bdcAc f-primer and the bdcAc r-primer, and the bacterial species of the recipient cells was confirmed via sequencing of the 16S rRNA genes (primers are the 1492R-primer 5'-GGTTACCTTGTTACGACTT-3' and the 27F-primer 5'-AGAGTTTGATCCTGGCTCAG-3' ).
Similarly, for BdcA dispersal in triple-species biofilms of E. coli, R. meliloti, and P. aeruginosa, pMMB206-BdcA was mobilized using E. coli DH5α/ pMMB206-BdcA/pRK2013 by adding 300 μL of turbidity 0.05 of these cells to existing double-species biofilms formed by R. meliloti and P. aeruginosa for 24 h. After another 11 h, 0.1 mM IPTG was added to produce BdcA, and cells were grown for an additional 24 h. To determine the percentage of each of the three strains in the biofilms, cells were taken from the air/liquid biofilm and from the liquid/bottom biofilm and diluted in LB for enumeration. The biofilm composition was determined based on the difference in rifampicin resistance of these three bacteria: 20 μg/mL inhibits E. coli, 100 μg/mL inhibits R. meliloti, and P. aeruginosa is resistant to 100 μg/mL.
Flow cell biofilms and image analysis
The flow cell experiments were performed as previously described . pP25-gfp  was used to produce the green fluorescent protein (GFP) for imaging each strain. The flow cells were inoculated with cultures at an initial turbidity at 600 nm of 0.05 at a flow rate of 10 mL/min for 2 h, then fresh LB medium with chloramphenicol and carbenicillin was added at 10 mL/min. IPTG (0.1 mM) was added to each flow cell system after 24 h, and images were taken 48 h and 96 h after IPTG addition for P. aeruginosa and 43 h and 91 h after IPTG addition for R. meliloti. Biofilm images from nine random positions were visualized with IMARIS confocal software (Bitplane, Zurich, Switzerland) and analyzed by COMSTAT confocal software as previously described . Two independent cultures were used for each strain; i.e., eight flow cell experiments were conducted.
Swarming motility, swimming motility, and EPS assays
The swarming motility of P. aeruginosa and P. fluorescens was assayed with BM-2 plates , while the swarming motility for R. meliloti was measured with 0.6% Eiken agar (Eiken Chemical Co., Tokyo, Japan) plates in LB medium with 0.5% glucose. Cells grown to a turbidity at 600 nm of ~1.0 were used for inoculation, and 0.1 mM IPTG was applied for each strain to induce the expression of bdcA. The swarming halo was measured after 15 h incubation for P. aeruginosa and R. meliloti and after 20 h incubation for P. fluorescens. Two independent cultures were used for each strain and at least three plates were used for each independent culture.
Swimming motility was performed as previously described . Overnight cultures were used to inoculate the plates (1% tryptone, 0.25% NaCl and 0.3% agar), and the swimming halo was measured after 11 h for P. aeruginosa, after 20 h for P. fluorescens, and after 15 h for R. meliloti. Two independent cultures were used for each strain, and at least three plates were used for each independent culture.
The amount of total EPS was determined as described previously . Briefly, 1 mL cell cultures grown in M9-0.4% mannitol medium were collected after 48 h and boiled in water for 10 min. The supernatants were then used for an anthrone-H2SO4 assay to determine EPS concentrations. This assay was performed with two independent cultures.
BdcA plasmid does not affect growth
In the absence of production of BdcA, all three pairs of bacteria grew at nearly the same rate (0.5860 ± 0.0003/h for P. aeruginosa/ pMMB206 vs. 0.571 ± 0.009/h for P. aeruginosa/ pMMB206-BdcA at 37°C; 0.512 ± 0.005/h for P. fluorescens/ pMMB206 vs. 0.506 ± 0.002/h for P. fluorescens/ pMMB206-BdcA at 30°C; and 0.511 ± 0.0008/h for R. meliloti/ pMMB206 vs. 0.51 ± 0.01/h for R. meliloti/ pMMB206-BdcA at 30°C) so the presence of bdcA on the plasmid does not affect growth. Hence, the bacteria are expected to form similar amounts of biofilm prior to production of BdcA for biofilm dispersal.
BdcA increases swarming and swimming motility and decreases EPS production
BdcA increases biofilm dispersal in microtitre plates
BdcA increases biofilm dispersal in flow cells
Flow cell statistical analysis of biofilm formation via COMSTAT
P. aeruginosa /pMMB206/pP25-gfp
P. aeruginosa /pMMB206-BdcA/pP25-gfp
3 ± 1
36 ± 7
6 ± 3
0.5 ± 0.3
4 ± 1
37 ± 9
8 ± 3
0.5 ± 0.2
3.1 ± 0.9
37 ± 8
7 ± 3
0.5 ± 0.2
0.2 ± 0.1
13 ± 4
0.5 ± 0.4
1.6 ± 0.2
R. meliloti /pMMB206/pP25-gfp
R. meliloti /pMMB206-BdcA/pP25-gfp
3 ± 1
34 ± 8
6 ± 2
0.6 ± 0.3
3 ± 1
40 ± 10
6 ± 2
0.7 ± 0.2
2.8 ± 0.7
30 ± 6
6 ± 2
0.5 ± 0.1
0.4 ± 0.3
17 ± 5
0.9 ± 0.7
1.3 ± 0.3
BdcA causes dispersal in mixed-species biofilms
To demonstrate that BdcA may be added to a mixed-species biofilm via conjugation, 96-well static biofilm cultures were used to form biofilms of R. meliloti and P. aeruginosa. After 24 h, BdcA was added to these biofilms by conjugating pMMB206-BdcA from E. coli DH5α/ pMMB206-BdcA/pRK2013; E. coli DH5α/ pMMB206/pRK2013 was used as the negative control which conjugates but does not provide BdcA. Upon producing BdcA for 26 h, total biofilm formation was reduced by 3.3 ± 0.6 for R. meliloti and 3.1 ± 0.6 for P. aeruginosa (Figure 3B). For these experiments, the efficiency of transferring pMMB206-BdcA to R. meliloti and P. aeruginosa was over 76% and over 80% respectively, probably due to vast excess of donor E. coli cells relative to the recipient cells of the very small biofilm when donor E. coli was added. After conjugation, we checked for the presence of the correct plasmid in recipient cells via PCR for the bdcA gene as well as identified each bacterial species via sequencing for 16S rRNA genes to confirm that the selective plating method was accurate. For both tests, all (10) cells from the selective antibiotic plates were found to have the bdcA plasmid (766 bp), and these cells were all determined to be either P. aeruginosa or R. meliloti, respectively.
In addition, we used the same strategy to treat an existing biofilm formed by both R. meliloti and P. aeruginosa and obtained a 3.1 ± 0.8-fold biofilm reduction based on BdcA production after conjugation from E. coli strain (Figure 3B). Hence, BdcA was able to disperse a multi-species biofilm that consisted of approximately 40% P. aeruginosa PA14, 23% R. meliloti, and 37% E. coli (the composition of control biofilm without BdcA was similar at 37% P. aeruginosa PA14, 26% R. meliloti, and 37% E. coli). Therefore, BdcA may be introduced into mixed biofilms via conjugation and used to reduce biofilm formation.
Given the near universal nature of c-di-GMP as a biofilm formation and dispersal signal, we investigated whether BdcA from E. coli would be effective in removing biofilms from various bacteria. To accomplish this goal, the broad host plasmid pMMB206 was used to express bdcA in different organisms including P. aeruginosa, P. fluorescens, and R. meliloti. As we had hoped, BdcA production increased swarming and swimming motility and reduced EPS production; these phenotypes indicate a reduced c-di-GMP concentration in these strains. The reduced c-di-GMP concentrations via BdcA proved extremely effective in dispersing biofilms including that of the opportunistic pathogen P. aeruginosa. This is the first report of reducing c-di-GMP via heterologous protein production to cause biofilm dispersal in these three strains. This is an important result in that it is a significant challenge to remove biofilms [30, 31] since cells in biofilms are cemented in place by the secreted polymer matrix consisting of polysaccharide, protein, DNA, and lipids ; this matrix of the biofilm colony makes most biofilms difficult or impossible to eradicate .
BdcA directly reduces bacterial c-di-GMP concentrations , and c-di-GMP is a signal that controls physiology of diverse bacteria . In R. meliloti, c-di-GMP controls growth, motility, EPS production, and even the interactions between bacteria and host plants . In P. aeruginosa, c-di-GMP controls various cell activities including biofilm formation, EPS production, and aggregation . Hence, BdcA may control biofilm dispersal in as many organisms as c-di-GMP functions as a second messenger. In addition, bdcA shows high sequence conservation with other species (including Pseudomonas and Rhizobium) and is well conserved in many bacteria . Hence, BdcA may be used for dispersal in many strains. As a preliminary proof of this principle, our results demonstrate the feasibility of introducing BdcA into existing non-E. coli biofilms (formed by P. aeruginosa and R. meliloti individually, as well as by mixed biofilms of these two bacteria) via conjugation of a broad-host-range plasmid) to reduce total biofilm formation. Therefore, in this study, we successfully disperse biofilms of disparate strains simply by producing BdcA. Hence, through this genetic approach, a single protein may be used to remove biofilms rather than by using traditional chemical or physical means.
List of abbreviations
green fluorescent protein.
This work was supported by the National Institutes of Health (R01 GM089999). T.W. is the T. Michael O'Connor Endowed Professor at Texas A & M University.
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