Article Figures & Data
Figures
- Fig. 1.
Quantitative analysis showing the spatial distribution of the different organisms of the mixed-culture biofilm sampled at day 7. (A) Vertical profile through the biofilm showing percentage ofAcinetobacter sp. strain C6 (▵), P. putida RI (◊), and isolate D8 (□) relative to the total number of cells targeted with the general eubacterial 16S rRNA probe (EUB338 [1]). Each profile is an average of three images (each consisting of 31 optical sections) captured at three random locations in the biofilm. (B) Relative area covered by cells in each section, taken as an average over six images. Standard deviations are indicated by error bars.
- Fig. 2.
Time course analysis of the distribution of donor cells,P. putida RI, and transconjugants relative to the total number of cells collected from flow channel effluents. The donor (P. putida RI/TOLgfpmut3b) was introduced at day 2 in three different densities: 5 · 104 (▴), 5 · 105 (▪), and 5 · 106 (⧫) CFU/ml. Total counts (A) were enumerated on pure LB broth plates. TheP. putida RI cells (Nalr) (B), donor cells (Rifr) (C), and transconjugants (NalrKmr) (D) were enumerated on LB broth plates containing the appropriate antibiotics, and the numbers were taken relative to the total counts. Each data point is the average of two independent experiments.
- Fig. 3.
Time course analysis of the distribution of donor cells,P. putida RI, and transconjugants relative to the total number of cells collected from flow channel effluents. The donor (P. putida KT2442/TOLgfpmut3b) was introduced at day 2 in three different concentrations: 5 · 106(▴), 5 · 107 (▪), and 5 · 108(⧫) CFU/ml. Total counts (A) were enumerated on pure LB broth plates. The P. putida RI cells (Nalr) (B), donor cells (Rifr) (C), and transconjugants (NalrKmr) (D) were enumerated on LB broth plates containing the appropriate antibiotics, and the numbers were taken relative to the total counts. Each data point is the average of two independent experiments.
- Fig. 4.
Time course analysis of the distribution of donor cells,P. putida RI, and transconjugants relative to the total number of cells collected from flow channel effluents. Without changing the inoculation concentration of the two other isolates in the model community, P. putida RI was introduced in three different concentrations of 105 (▴), 107 (▪), and 108 (⧫) CFU/ml. Donor cells (P. putidaKT2442/TOLgfpmut3b) (5 · 108 CFU/ml) were introduced at day 2. Total counts (A) were enumerated on pure LB broth plates. The Nalr P. putida RI cells (B), Rifr donor cells (C), and Nalr Kmrtransconjugants (D) were enumerated on LB broth plates containing the appropriate antibiotics, and the numbers were taken relative to the total counts. Each point is the average of two individual experiments.
- Fig. 5.
On-line monitoring of transconjugant proliferation on microcolonies in the direction of flow at days 5 and 6 after donor introduction. The white patches are regions with strong green-fluorescent signal (microcolonies with transconjugants), and the gray regions are weak autofluorescent signals emitted from P. putida cells. This signal is easy to distinguish from the strong green-fluorescent signal emitted from cells expressing Gfp and could be used to visualize the location of noninfected microcolonies. On day 5 (A) a strongly green-fluorescent microcolony (solid arrow) was observed and other green-fluorescent microcolonies were located in a region straight downstream from this colony, but not upstream. On day 6 (B) more green-fluorescent colonies were observed. The scale bar also indicates the direction of flow. Open arrows indicate examples of microcolonies which had been infected with transconjugants from day 5 to day 6.
- Fig. 6.
(A) Spatial distribution of green-fluorescent transconjugants (green or yellow) relative to noninfected P. putida RI cells and Acinetobacter sp. strain C6 in a biofilm analyzed 8 days after introduction of donor cells. The organisms P. putida (red) and Acinetobacter sp. strain C6 (purple) were identified by hybridization. After hybridization, green-fluorescent transconjugants appear as either yellow or green, depending on the ratio between the green Gfp signal and the red hybridization signal. The x-y plot is presented as a SFP, where long shadows indicate a large and/or high microcolony. Shown to the right and below are vertical sections through the biofilm collected at the positions indicated by the white triangles. (B) Magnification of a P. putida colony with green-fluorescent cells covering the surface. Vertical sections through the colony are shown to the right and below. The microcolony is a SFP of a region 10 to 19 μm from the glass surface.
Tables
- Table 1.
Strains and plasmids
Strain or plasmid Relevant characteristics Source or reference E. coli K-12 CC118λpir Δ(ara-leu)araD ΔlacX74 galE galK phoA thi-1 rpsE rpoB argE(Am)recA λpir lysogen 21 HB101 E. coli K-12/B hybrid; Smr recA thi pro leu hsdRM+ 4 P. putida RI (SMO116) Natural isolate; γ subgroup of classProteobacteriaa 29 JB154 SMO116 Rifr This work JB156 SMO116 Nalr This work KT2440 hsdR/TOL K. Timmis KT2442 hsdR Rifr K. Timmis SM1443 KT2442 lacI q; nptremoved by site-specific recombination This work BBC443 SM1443/TOLgfpmut3b This work BBC458 JB156/TOLgfpmut3b This work BBC516 JB154/TOLgfpmut3b This work Acinetobacter sp. strain C6 (SMO112) Natural isolate; γ subclass of classProteobacteriab 29 Unknown genus D8 (SMO125) Natural isolate; β subclass of class Proteobacteriac 29 Plasmids RK600 Cmr ColE1oriV RP4 oriT; helper plasmid in triparental matings 22 pUC18Not Same as pUC18 (49) but with polylinker flanked with NotI sites 21 pUTKm AprKmr; transposon delivery vector for mini-Tn5Km 11 pCK242 mini-Tn5transposon delivery vector; npt gene flanked byres sites 23 pJBA27 SphI-HindIIIgfpmut3b fragment cloned downstream ofPA1/O4/O3 3 pJBA28 NotI fragmentPA1/O4/O3 ::gfpmut3b from pJBA27 cloned into pUT mini-Tn5 Km 3 pSM1431 lacI q fragment (38) cloned into pUC18Not (Sca-SalI) This work pSM1435 NotI fragment (lacI q) of pSM1431 inserted into pUTKm This work TOL Self-transmissible approximately 117-kb plasmid; fromP. putida mt-2 43 TOLgfpmut3b Integration ofPA1/O4/O3 ::gfpmut3b from pJBA28 into TOL This work
