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Psl trails guide exploration and microcolony formation in Pseudomonas aeruginosa biofilms.

Zhao K, Tseng BS, Beckerman B, Jin F, Gibiansky ML, Harrison JJ, Luijten E, Parsek MR, Wong GC - Nature (2013)

Bottom Line: Bacterial biofilms are surface-associated, multicellular, morphologically complex microbial communities.This Pareto-type behaviour indicates that the bacterial community self-organizes in a manner analogous to a capitalist economic system, a 'rich-get-richer' mechanism of Psl accumulation that results in a small number of 'elite' cells becoming extremely enriched in communally produced Psl.Using engineered strains with inducible Psl production, we show that local Psl concentrations determine post-division cell fates and that high local Psl concentrations ultimately allow elite cells to serve as the founding population for initial microcolony development.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, University of California, Los Angeles, California 90095, USA.

ABSTRACT
Bacterial biofilms are surface-associated, multicellular, morphologically complex microbial communities. Biofilm-forming bacteria such as the opportunistic pathogen Pseudomonas aeruginosa are phenotypically distinct from their free-swimming, planktonic counterparts. Much work has focused on factors affecting surface adhesion, and it is known that P. aeruginosa secretes the Psl exopolysaccharide, which promotes surface attachment by acting as 'molecular glue'. However, how individual surface-attached bacteria self-organize into microcolonies, the first step in communal biofilm organization, is not well understood. Here we identify a new role for Psl in early biofilm development using a massively parallel cell-tracking algorithm to extract the motility history of every cell on a newly colonized surface. By combining this technique with fluorescent Psl staining and computer simulations, we show that P. aeruginosa deposits a trail of Psl as it moves on a surface, which influences the surface motility of subsequent cells that encounter these trails and thus generates positive feedback. Both experiments and simulations indicate that the web of secreted Psl controls the distribution of surface visit frequencies, which can be approximated by a power law. This Pareto-type behaviour indicates that the bacterial community self-organizes in a manner analogous to a capitalist economic system, a 'rich-get-richer' mechanism of Psl accumulation that results in a small number of 'elite' cells becoming extremely enriched in communally produced Psl. Using engineered strains with inducible Psl production, we show that local Psl concentrations determine post-division cell fates and that high local Psl concentrations ultimately allow elite cells to serve as the founding population for initial microcolony development.

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Local Psl levels determine post-division cell fatesa. Visit frequency map of Psl++ for the first 14 hours post inoculation. Microcolonies have already started to form. b. Bright-field image for Psl++ at t ~ 14 hours. c. Visit frequency distributions of Psl++ from (a) for experiments (left) and simulations (right) agree. Solid line is an exponential fit to the second part of the data (green). Inset shows a power-law fit to the first part of the data (red). d. Probability of post-division cells’ fates: “stay” (solid rod) or “leave” (dashed rod envelope) for WT (red), Psl++ (blue) and ΔpslD (green). Error bars are estimated from 1/√Ndiv, with Ndiv the total number of division events during the time period of interest (Ndiv ≥ 90). e. WT and Psl++ microcolonies have drastically different compositions, as depicted by color-coded cell division lineages at early stages of microcolony formation (top row). For Psl++ the microcolony is dominated by a single lineage, whereas the WT microcolony has 20 different lineages. Bottom row depicts more developed microcolonies at the same location 3.3 hours later. Scale bar is 10 μm.
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Figure 3: Local Psl levels determine post-division cell fatesa. Visit frequency map of Psl++ for the first 14 hours post inoculation. Microcolonies have already started to form. b. Bright-field image for Psl++ at t ~ 14 hours. c. Visit frequency distributions of Psl++ from (a) for experiments (left) and simulations (right) agree. Solid line is an exponential fit to the second part of the data (green). Inset shows a power-law fit to the first part of the data (red). d. Probability of post-division cells’ fates: “stay” (solid rod) or “leave” (dashed rod envelope) for WT (red), Psl++ (blue) and ΔpslD (green). Error bars are estimated from 1/√Ndiv, with Ndiv the total number of division events during the time period of interest (Ndiv ≥ 90). e. WT and Psl++ microcolonies have drastically different compositions, as depicted by color-coded cell division lineages at early stages of microcolony formation (top row). For Psl++ the microcolony is dominated by a single lineage, whereas the WT microcolony has 20 different lineages. Bottom row depicts more developed microcolonies at the same location 3.3 hours later. Scale bar is 10 μm.

Mentions: Power law relationships generally exist only over a limited range in nature and are often difficult to distinguish from other quantitatively similar relationships. Therefore, rather than concentrating on the approximate power law in the visit frequency distribution and the range over which it is observed, we focused instead on obtaining a deeper quantitative understanding of the processes that generate its functional form. Thus, we performed computer simulations of Psl-guided motility of Psl-secreting bacteria using experimentally measured parameters to stringently test the quantitative interplay between progressive Psl secretion, surface motility and the idea of surface exploration guided by positive feedback. Bacteria were modeled as non-overlapping line segments in a two-dimensional domain that, when unbiased by Psl, move according to a velocity distribution extracted from the experimental data for ΔPpsl/PBAD-psl at 0% arabinose. In each step, a fixed amount of Psl was secreted and the bacterial displacement was biased by the local Psl distribution. The bacterial concentration and dimensions as well as the sampling rate were all chosen in accordance with the experimental parameters (see Supplementary Methods). The simulations captured the complex distributions from experiments, in both the power-law regime and beyond (Figures 2e and 3c), including the dependence on Psl. The simulated visit frequency distributions exhibited a power-law behavior that agrees quantitatively with the tracked microscopy data. This is a striking confirmation of the role of Psl-biased motion as the underlying mechanism, since unbiased motion would give rise to nonlocalized, random-walk type behavior. As the Psl production rate was increased for both experiments (Figure 2d) and simulations (Figure 2e), the exponents increased over the same numerical range, from −3.1 to −1.8, confirming the shift to more hierarchical distributions in which the number of highly visited sites increased at the expense of visits to many of the rarely visited sites (Figures 2f–h). Likewise, both the lectin-stained Psl image observed in experiments (Figure 2i) and the simulated Psl map (Figure 2j) showed the hierarchical nature of the spatial distribution of Psl.


Psl trails guide exploration and microcolony formation in Pseudomonas aeruginosa biofilms.

Zhao K, Tseng BS, Beckerman B, Jin F, Gibiansky ML, Harrison JJ, Luijten E, Parsek MR, Wong GC - Nature (2013)

Local Psl levels determine post-division cell fatesa. Visit frequency map of Psl++ for the first 14 hours post inoculation. Microcolonies have already started to form. b. Bright-field image for Psl++ at t ~ 14 hours. c. Visit frequency distributions of Psl++ from (a) for experiments (left) and simulations (right) agree. Solid line is an exponential fit to the second part of the data (green). Inset shows a power-law fit to the first part of the data (red). d. Probability of post-division cells’ fates: “stay” (solid rod) or “leave” (dashed rod envelope) for WT (red), Psl++ (blue) and ΔpslD (green). Error bars are estimated from 1/√Ndiv, with Ndiv the total number of division events during the time period of interest (Ndiv ≥ 90). e. WT and Psl++ microcolonies have drastically different compositions, as depicted by color-coded cell division lineages at early stages of microcolony formation (top row). For Psl++ the microcolony is dominated by a single lineage, whereas the WT microcolony has 20 different lineages. Bottom row depicts more developed microcolonies at the same location 3.3 hours later. Scale bar is 10 μm.
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Figure 3: Local Psl levels determine post-division cell fatesa. Visit frequency map of Psl++ for the first 14 hours post inoculation. Microcolonies have already started to form. b. Bright-field image for Psl++ at t ~ 14 hours. c. Visit frequency distributions of Psl++ from (a) for experiments (left) and simulations (right) agree. Solid line is an exponential fit to the second part of the data (green). Inset shows a power-law fit to the first part of the data (red). d. Probability of post-division cells’ fates: “stay” (solid rod) or “leave” (dashed rod envelope) for WT (red), Psl++ (blue) and ΔpslD (green). Error bars are estimated from 1/√Ndiv, with Ndiv the total number of division events during the time period of interest (Ndiv ≥ 90). e. WT and Psl++ microcolonies have drastically different compositions, as depicted by color-coded cell division lineages at early stages of microcolony formation (top row). For Psl++ the microcolony is dominated by a single lineage, whereas the WT microcolony has 20 different lineages. Bottom row depicts more developed microcolonies at the same location 3.3 hours later. Scale bar is 10 μm.
Mentions: Power law relationships generally exist only over a limited range in nature and are often difficult to distinguish from other quantitatively similar relationships. Therefore, rather than concentrating on the approximate power law in the visit frequency distribution and the range over which it is observed, we focused instead on obtaining a deeper quantitative understanding of the processes that generate its functional form. Thus, we performed computer simulations of Psl-guided motility of Psl-secreting bacteria using experimentally measured parameters to stringently test the quantitative interplay between progressive Psl secretion, surface motility and the idea of surface exploration guided by positive feedback. Bacteria were modeled as non-overlapping line segments in a two-dimensional domain that, when unbiased by Psl, move according to a velocity distribution extracted from the experimental data for ΔPpsl/PBAD-psl at 0% arabinose. In each step, a fixed amount of Psl was secreted and the bacterial displacement was biased by the local Psl distribution. The bacterial concentration and dimensions as well as the sampling rate were all chosen in accordance with the experimental parameters (see Supplementary Methods). The simulations captured the complex distributions from experiments, in both the power-law regime and beyond (Figures 2e and 3c), including the dependence on Psl. The simulated visit frequency distributions exhibited a power-law behavior that agrees quantitatively with the tracked microscopy data. This is a striking confirmation of the role of Psl-biased motion as the underlying mechanism, since unbiased motion would give rise to nonlocalized, random-walk type behavior. As the Psl production rate was increased for both experiments (Figure 2d) and simulations (Figure 2e), the exponents increased over the same numerical range, from −3.1 to −1.8, confirming the shift to more hierarchical distributions in which the number of highly visited sites increased at the expense of visits to many of the rarely visited sites (Figures 2f–h). Likewise, both the lectin-stained Psl image observed in experiments (Figure 2i) and the simulated Psl map (Figure 2j) showed the hierarchical nature of the spatial distribution of Psl.

Bottom Line: Bacterial biofilms are surface-associated, multicellular, morphologically complex microbial communities.This Pareto-type behaviour indicates that the bacterial community self-organizes in a manner analogous to a capitalist economic system, a 'rich-get-richer' mechanism of Psl accumulation that results in a small number of 'elite' cells becoming extremely enriched in communally produced Psl.Using engineered strains with inducible Psl production, we show that local Psl concentrations determine post-division cell fates and that high local Psl concentrations ultimately allow elite cells to serve as the founding population for initial microcolony development.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, University of California, Los Angeles, California 90095, USA.

ABSTRACT
Bacterial biofilms are surface-associated, multicellular, morphologically complex microbial communities. Biofilm-forming bacteria such as the opportunistic pathogen Pseudomonas aeruginosa are phenotypically distinct from their free-swimming, planktonic counterparts. Much work has focused on factors affecting surface adhesion, and it is known that P. aeruginosa secretes the Psl exopolysaccharide, which promotes surface attachment by acting as 'molecular glue'. However, how individual surface-attached bacteria self-organize into microcolonies, the first step in communal biofilm organization, is not well understood. Here we identify a new role for Psl in early biofilm development using a massively parallel cell-tracking algorithm to extract the motility history of every cell on a newly colonized surface. By combining this technique with fluorescent Psl staining and computer simulations, we show that P. aeruginosa deposits a trail of Psl as it moves on a surface, which influences the surface motility of subsequent cells that encounter these trails and thus generates positive feedback. Both experiments and simulations indicate that the web of secreted Psl controls the distribution of surface visit frequencies, which can be approximated by a power law. This Pareto-type behaviour indicates that the bacterial community self-organizes in a manner analogous to a capitalist economic system, a 'rich-get-richer' mechanism of Psl accumulation that results in a small number of 'elite' cells becoming extremely enriched in communally produced Psl. Using engineered strains with inducible Psl production, we show that local Psl concentrations determine post-division cell fates and that high local Psl concentrations ultimately allow elite cells to serve as the founding population for initial microcolony development.

Show MeSH
Related in: MedlinePlus