Inhibition and dispersal of Agrobacterium tumefaciens biofilms by a small diffusible Pseudomonas aeruginosa exoproduct(s)

Abstract

Environmental biofilms often contain mixed populations of different species. In these dense communities, competition between biofilm residents for limited nutrients such as iron can be fierce, leading to the evolution of competitive factors that affect the ability of competitors to grow or form biofilms. We have discovered a compound(s) present in the conditioned culture fluids of Pseudomonas aeruginosa that disperses and inhibits the formation of biofilms produced by the facultative plant pathogen Agrobacterium tumefaciens. The inhibitory activity is strongly induced when P. aeruginosa is cultivated in iron-limited conditions, but it does not function through iron sequestration. In addition, the production of the biofilm inhibitory activity is not regulated by the global iron regulatory protein Fur, the iron-responsive extracytoplasmic function σ factor PvdS, or three of the recognized P. aeruginosa quorum-sensing systems. In addition, the compound(s) responsible for the inhibition and dispersal of A. tumefaciens biofilm formation is likely distinct from the recently identified P. aeruginosa dispersal factor, cis-2-decenoic acid (CDA), as dialysis of the culture fluids showed that the inhibitory compound was larger than CDA and culture fluids that dispersed and inhibited biofilm formation by A. tumefaciens had no effect on biofilm formation by P. aeruginosa.

Figure 1. The inhibitory activity of conditioned P. aeruginosa culture media on A. tumefaciens biofilm formation

Quantification of A) acetic acid solubilized CV biofilm stain, measured as A₆₀₀ and B) the planktonic growth, measured by OD₆₀₀, prior to staining, from 48 h PVC 96-well microtiter plate A. tumefaciens C58 biofilms. Increasing concentrations of culture fluids that were prepared from A. tumefaciens low iron cultures (open triangle), and P. aeruginosa cultures from, iron replete (closed circle) and iron limited (open circle) conditions were added to the biofilm assays. Data are normalized relative to unsupplemented cultures. Quantification of C) the acetic acid solubilized CV, measured by A₆₀₀ and D) the planktonic growth, measured by OD₆₀₀ prior to staining, from 48 h PVC 96-well microtiter plate A. tumefaciens biofilms with increasing concentrations of FeSO₄, with (open circles) or without (closed circles) 20% (vol/vol) P. aeruginosa culture fluids prepared from iron-limited cultures. Data are normalized relative to the cultures with 22 μM FeSO₄ but with no added fluids. In all cases, values are the means and standard deviations from five wells per condition.

Figure 2. Mutants in the global iron regulators Fur and PvdS retain iron dependent inhibition of A. tumefaciens biofilms

Quantification of A) acetic acid solubilized CV biofilm stain, measured as A₆₀₀ B) the planktonic growth, measured by OD₆₀₀ prior to staining, from 48 h PVC 96-well plate A. tumefaciens biofilms. Increasing concentrations of culture fluids prepared from cultures of wild type P. aeruginosa (circles), the global iron-responsive regulatory gene missense mutant furC6Tc (squares) and the iron responsive ECF σ-factor mutant ΔpvdS (triangles) grown with (closed symbols) and without (open symbols) 22 μM FeSO₄ were added to the biofilm assays. Values are normalized relative to cultures with no added fluids and are the means and standard deviations from five wells per condition.

Figure 3. The addition of P. aeruginosa culture fluids causes the dispersal of preformed A. tumefaciens biofilms

Static culture 24 h biofilms of A. tumefaciens. At time 0, ATGN (closed circles) or P. aeruginosa culture fluids (open circles) were added to the cultures resulting in treatment of the preformed biofilms with 50% (vol/vol) culture fluids or ATGN. At each time point A) the wells were stained with CV, incubated 10 min, the stain was solubilized with acetic acid, and measured at A₆₀₀ and B) the planktonic growth of the culture, OD₆₀₀ prior to staining. Values are normalized relative to the value at time 0, are the means and standard deviations from four wells per time-point.

Figure 4. P. aeruginosa biofilm formation is not influenced by its own culture fluids but is impacted by iron levels

P. aeruginosa static culture biofilms were grown in 12-well microtiter dishes with PCV coverslips sitting vertically in the wells. After incubating for 16 h, 40 h, and 64 h the coverslips were removed and the adherent biomass was stained with CV, and the OD₆₀₀ of the remaining planktonic culture was measured. The bacteria at the air-liquid interface were disrupted and photographed. The border between the pellicle suspended at the air-liquid interface (the lighter colored regions) and the planktonic bacteria (the darker colored regions) are marked with a white arrow. The images shown are representative of three coverslips. The OD₆₀₀ values presented are the mean and (standard deviation) from three wells.

Figure 5. Quorum sensing deficient mutants direct wild type levels of biofilm inhibition

A) acetic acid solubilized CV biofilm stain, measured as A₆₀₀ and B) the planktonic growth, measured by OD₆₀₀ prior to staining, from 48 h PVC 96-well plate A. tumefaciens biofilms. Increasing concentrations of culture fluids prepared from wild type (closed circles) and the five quorum sensing deficient mutants used in this study, ΔlasI (closed squares), Δ rhlI (closed triangles), pqsA::tet (open circles), Δ lasI Δ rhlI (open squares), and Δ lasR Δ rhlR (open triangles), prepared from iron limited cultures were added to the biofilm assays. Values are normalized relative to cultures with no added fluids and are the means and standard deviations of five wells per condition.

Figure 6. Dialysis of P. aeruginosa culture fluids reveals a low molecular size for the active compound(s

A) acetic acid solubilized CV biofilm stain, measured as A₆₀₀ and B) the planktonic growth, measured by OD₆₀₀ prior to staining, from 48 h PVC 96-well plate A. tumefaciens biofilms. Increasing concentrations of culture fluid that was either untreated (closed circles) or dialyzed across 2000 Da (open squares) or 7000 Da (closed squares) dialysis membranes into 4 liters of AT-N buffer were added to the biofilm assays. Values are normalized relative to cultures with no added fluids and are the means and standard deviations from five wells per condition.

Figure 7. CDA has a dialyzable biofilm inhibitory activity

A) acetic acid solubilized CV biofilm stain, measured as A₆₀₀ and B) the planktonic growth, measured by OD₆₀₀ prior to staining, from 48 h PVC 96-well plate A. tumefaciens biofilms. Increasing concentrations of CDA that was either untreated (closed circles) or dialyzed across 2000 Da (open squares) dialysis membrane into 4 liters of AT-N buffer were added to the biofilm assays. Values are normalized relative to cultures with no added CDA and are the means and standard deviations from three wells per condition.