Journal: PLOS Biology

Article Title: Biofilm dispersal patterns revealed using far-red fluorogenic probes

doi: 10.1371/journal.pbio.3002928

Figure Lengend Snippet: (A) Schematic for FAP-fluorogen interaction and initiation of fluorescence signal. (B) Left panel: Images of V . cholerae biofilm growth at low magnification (10× objective) for a strain constitutively expressing a representative fluorescent protein, mNeonGreen, fused to the FAP dL5, grown in the presence of 1 μM MGe and 100 μM norspermidine to stimulate biofilm formation. Right panel: quantification of whole-biofilm fluorescence intensity for the same images. Points represent averages and shaded regions represent standard deviations. N = 4 biological replicates. (C) Final time point bulk culture fluorescence for the same strain as in B grown for 16 h in the presence or absence of oxygen. Fluorescence for each channel is normalized to oxic conditions. N = 3 biological, 3 technical replicates. P = 2.5 × 10 −13 and 4.0 × 10 −6 for mNeonGreen and dL5-MGe, respectively, based on two-sided unpaired t tests relative to the oxic controls. (D) Left panels: Representative images of cells expressing cytoplasmic dL5, labeled with 1 μM MGe (top), 5 μM MHNe (middle), or fused to μNS and labeled with 1 μM MGe (bottom). Right panel: Field of view of cells expressing SS-dL5 labeled with 1 μM MG-2P. (E) Side-on (x-z) view of high-resolution confocal micrographs of V . cholerae biofilm cells expressing SS-dL5 labeled with 1 μM MG-2P over the course of biofilm dispersal. The entire biofilm was optically sectioned for 16 h with 10-min intervals. Images are displayed with a magenta-hot lookup table. a.u., arbitrary units. **** P < 0.0001. Underlying data for this figure can be found on Figshare ( https://figshare.com/s/e0978ade2bc95dccf357 ). FAP, fluorogen-activating protein; MGe, malachite green ester.

Article Snippet: For spinning disc confocal microscopy of biofilm dispersal, glass-bottom 96-well microtiter dishes (Mattek) were used.

Techniques: Fluorescence, Expressing, Labeling

Journal: PLOS Biology

Article Title: Biofilm dispersal patterns revealed using far-red fluorogenic probes

doi: 10.1371/journal.pbio.3002928

Figure Lengend Snippet: (A) Change in total biovolume (cellular volume inside a biofilm) of individual wild-type biofilms over time as measured from spinning-disc confocal sections. Data for each biofilm are normalized as fold-change relative to the biovolume at the initiation of biofilm dispersal. N = 5 biological replicates. For all panels pertaining to dispersal, time point zero is the time point of peak biofilm biomass. (B) Schematics of the proposed radial dispersal patterns. Timing of cell departures is indicated by shading, with light regions departing early and dark regions departing late. “Inside-out” corresponds to a scenario in which dispersal progresses from the core cells to the peripheral cells. “Outside-in” corresponds to a scenario in which dispersal progresses from the peripheral cells to the core cells. “Random” corresponds to a scenario in which cells depart the biofilm via a radially random process. (C) Representative kymograph of dispersal from a single wild-type experimental replicate, representing the change in local density at 10-min intervals and at the indicated distances from the biofilm core. Black line represents the biofilm boundary. (D) As in C for the “Random” model. The rate of overall cell departure was set to match the experimental data. (E) Centroid position of the density changes from the experimental data and the model of random cell departures over time. Lines and shading represent means and standard deviations of N = 5 biofilms, respectively. (F) Spatial distribution of the local density profiles for the data and random model at the completion of dispersal. Lines and shading represent means and standard deviations for N = 5 biofilms, respectively. a.u., arbitrary units. Underlying data for this figure can be found on Figshare ( https://figshare.com/s/e0978ade2bc95dccf357 ).

Article Snippet: For spinning disc confocal microscopy of biofilm dispersal, glass-bottom 96-well microtiter dishes (Mattek) were used.

Techniques:

Journal: PLOS Biology

Article Title: Biofilm dispersal patterns revealed using far-red fluorogenic probes

doi: 10.1371/journal.pbio.3002928

Figure Lengend Snippet: (A) Representative x-z spinning-disc confocal projections from of the first 2 h of dispersal of a wild-type V . cholerae biofilm highlighting compression of peripheral cells toward the core. The boundary of the biofilm is highlighted in white. Scale is as indicated. (B) Net displacement vectors during a ~1.5-h period preceding dispersal (“Growth”) and throughout dispersal (“Dispersal”). Color represents radial displacement (μm) with respect to the core of the biofilm. For ease of demonstration, a single projection on the x-z plane is shown. (C) Quantification of the radial components of the displacements during the growth and dispersal phases. N = 5 replicate biofilms. For each replicate, values are averaged over the entire biofilm. P = 1.06 × 10 −5 based on a two-sided unpaired t test relative to the “Growth” data. (D) Left panel: representative x-y slice of a spinning-disc confocal z-stack from a timelapse of wild-type V . cholerae dispersal, with net displacement vectors from the first 4 h of the timelapse overlayed. A length threshold was applied to isolate locally elevated displacements, taken to be a measure of dynamic regions in biofilms. Scale is as indicated. Right panel: quantification of dynamic region volume fraction during the 40 min preceding and throughout the dispersal phase for wild-type V . cholerae . Each line represents an independent replicate for N = 5 biofilms. a.u., arbitrary units. **** P < 0.0001. Underlying data for this figure can be found on Figshare ( https://figshare.com/s/e0978ade2bc95dccf357 ).

Article Snippet: For spinning disc confocal microscopy of biofilm dispersal, glass-bottom 96-well microtiter dishes (Mattek) were used.

Techniques:

Journal: PLOS Biology

Article Title: Biofilm dispersal patterns revealed using far-red fluorogenic probes

doi: 10.1371/journal.pbio.3002928

Figure Lengend Snippet: (A) Cartoon depiction of the proposed roles of CheY, LapG, and RbmB in V . cholerae biofilm dispersal. (B) Change in total biovolume (cellular volume inside a biofilm) of individual biofilms for the indicated strains over time based on spinning-disc confocal z-stacks. The data for each biofilm are normalized as fold-change relative to the biovolume at the first time point. N = 5 biological replicates. (C) Left panel: representative kymograph of dispersal data from a Δ cheY replicate, depicting the change in local density at 10-min intervals and at the indicated distances from the biofilm core. Black line represents the biofilm boundary. Middle panel: as in the left panel for a model of random cell departures. Right panel: centroids of the density changes from the experimental data and the model of random cell departures over time. Lines and shading represent means and standard deviations for N = 5 biofilms, respectively; (D) as in C for the Δ lapG mutant; (E) as in C for the Δ rbmB mutant. (F) Quantification of the radial components of the displacements during the dispersal phase for the indicated strains. Crossbars represent means and dots represent individual replicates for N = 5 biofilms, with the radial displacements averaged over each replicate biofilm. P = 0.054, 0.030, and 0.048 based on two-sided unpaired t tests of the Δ cheY , Δ lapG , and Δ rbmB data relative to the wild-type data, respectively. (G) Quantification of dynamic region volume fraction during the dispersal phase for the indicated strains. Data represent averages over the entire dispersal phase. Crossbars represent means and dots represent individual replicates for N = 5 biofilms. P = 0.291, 0.094, and 0.072 based on two-sided unpaired t tests of the Δ cheY , Δ lapG , and Δ rbmB data relative to the wild-type data, respectively. a.u., arbitrary units. * P < 0.05; n.s., P > 0.05. Underlying data for this figure can be found on Figshare ( https://figshare.com/s/e0978ade2bc95dccf357 ).

Article Snippet: For spinning disc confocal microscopy of biofilm dispersal, glass-bottom 96-well microtiter dishes (Mattek) were used.

Techniques: Mutagenesis

Journal: PLOS Biology

Article Title: Biofilm dispersal patterns revealed using far-red fluorogenic probes

doi: 10.1371/journal.pbio.3002928

Figure Lengend Snippet: (A) Cartoon depiction of the role of RbmA in forming cell–cell connections. (B) Change in total biovolume (cellular volume inside a biofilm) of individual biofilms for the indicated strains over time based on spinning-disc confocal z-stacks. The data for each biofilm are normalized as fold-change relative to the biovolume at the first time point. N = 5 biological replicates. (C) Left panel: representative kymograph of dispersal data from a ΔrbmA replicate, representing the change in local density at 10-min intervals and at the indicated distances from the biofilm core. Black line represents the biofilm boundary. Right panel: as in the left panel for a model of random cell departures. Bottom panel: centroids of the density changes from the experimental data and the model of random cell departures over time. Lines and shading represent means and standard deviations for N = 5 biofilms, respectively. (D) Quantification of the radial components of the displacements during the dispersal phase for the indicated strains. Crossbars represent means and dots represent individual replicates for N = 5 biofilms, with the radial displacements averaged over each biofilm. P = 0.151 based on a two-sided unpaired t test relative to the wild-type data. (E) Quantification of dynamic region volume fraction during the dispersal phase for the indicated strains. Crossbars represent means and dots represent individual replicates for N = 5 biofilms. P = 0.067 based on a two-sided unpaired t test relative to the wild-type data. a.u., arbitrary units. n.s., P > 0.05. Underlying data for this figure can be found on Figshare ( https://figshare.com/s/e0978ade2bc95dccf357 ).

Article Snippet: For spinning disc confocal microscopy of biofilm dispersal, glass-bottom 96-well microtiter dishes (Mattek) were used.

Techniques: