Quorum sensing and motility mediate interactions between Pseudomonas aeruginosa and Agrobacterium tumefaciens in biofilm cocultures

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   Fig. 1.

   Planktonic cocultures. Population dynamics of wild-type, mutant, and complemented mutant cocultures inoculated at a 1:1 ratio. Displayed on the y axis (at the left) is the percentage of A.t. present in the culture at four different points of the growth curve. Standard deviation of three replicates is indicated. Open circles indicate the OD₆₀₀ (at the right) of a representative growth curve at which a coculture sample was assayed. All coculture growth curves were nearly identical (data not shown). Quorum-sensing mutant strains were complemented by the exogenous addition of the indicated amount of purified acyl-HSLs.

   
   
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   Fig. 2.

   P.a. blankets A.t. in coculture biofilms. (A and B) Three-dimensional views of P.a. and A.t. wild-type pure-culture flow-cell biofilms. (C and D) Coculture biofilms at 24 and 164 h after inoculation. (Top) An x–y slice close to the attachment surface. (Bottom) Three-dimensional views. Red cells, P.a., green cells, A.t. (Scale bars, 20 μm; 1 unit in 3D view = 13.4 μm.) (E) A quantitative determination of biomass distributions along biofilm depth in a coculture biofilm at 96 h.

   
   
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   Fig. 3.

   Quorum sensing provides an advantage to P.a. in coculture biofilms. (A–C Top) An x–y slice close to the attachment surface. (A–C Bottom) Three-dimensional views. (A) A coculture biofilm of wild-type P.a. and A.t. (B) A coculture biofilm of P.a.-lasRrhlR and wild-type A.t. (C) A coculture biofilm of P.a.-lasRrhlR complemented with pDA1 and wild-type A.t. Red cells, P.a., green cells, A.t. (Scale bars, 20 μm; 1 unit in 3D view = 13.4 μm.) All micrographs were taken at 164 h. (D) comstat determination of the relative amount of A.t. biofilm biomass present in pure and coculture biofilms.

   
   
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   Fig. 4.

   P.a. motility is required for blanketing. (A and C) Three-dimensional views of a P.a.-pilA (A) and P.a.-flgK (C) pure-culture biofilms. (B and D) x–y slices close to the attachment surface and 3D views of a P.a.-pilA/A.t. (B) and P.a.-flgK/A.t. (D) Coculture biofilms. Red cells, P.a.; green cells, A.t. (Scale bars, 20 μm; 1 unit in 3D view, 13.4 μm.) All micrographs were taken at 164 h. (E) comstat determination of the relative amount of A.t. biofilm biomass present in cocultures. (F) A quantitative determination of biomass distributions along biofilm depth in P.a.-pilA/A.t. and P.a.-flgK/A.t. coculture biofilms at 96 h.

   
   
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   Fig. 5.

   Biofilm coculture phenotypes of an A.t.-fliR mutant strain. (A) A 3D-view of an A.t.-fliR pure-culture biofilm; (B) An x–y slice close to attachment surface and a 3D-view of a P.a./A.t.-fliR coculture biofilm; Red cells, P.a; green cells, A.t. (Scale bars, 20 μm; 1 unit in 3D view, 13.4 μm.) All micrographs were taken at 164 h. (C) comstat determination of the relative amount of A.t. biofilm biomass present in pure cultures and cocultures. (D) A quantitative determination of biomass distributions along biofilm depth in a P.a./A.t.-fliR. coculture biofilm at 96 h.

   
   
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   Fig. 6.

   Different biofilm time-lapse microscopy series. Yellow cells indicate bacteria that have not moved during the course of the time series. Green cells indicate bacteria that were present in the field of view at the beginning of the time course but not at the end. Red cells are bacteria present at the end of the time series that were not present at the start. (A and B) A 1-h time course of a newly inoculated pure-culture biofilm of wild-type A.t. (A) or A.t.-fliR (B). (Scale bar, 12 μm.) (C and D) A time course of wild-type A.t. in coculture with P.a. (P.a. cells are not visible). (C) A 3-h time course before blanketing has occurred. (D) A 3-h time course after complete blanketing has occurred. (Scale bar, 20.2 μm.)