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Biofilm Formation As a Response to Ecological Competition.

Oliveira NM, Oliveria NM, Martinez-Garcia E, Xavier J, Durham WM, Kolter R, Kim W, Foster KR - PLoS Biol. (2015)

Bottom Line: We co-cultured a diverse collection of natural isolates of the opportunistic pathogen Pseudomonas aeruginosa and studied the effect on biofilm formation.This is inconsistent with the idea that sub-lethal concentrations of antibiotics are cooperative signals that coordinate microbial communities, as is often concluded.Instead, our work is consistent with competition sensing where low-levels of antibiotics are used to detect and respond to the competing genotypes that produce them.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, University of Oxford, Oxford, United Kingdom; Oxford Centre for Integrative Systems Biology, University of Oxford, Oxford, United Kingdom.

ABSTRACT
Bacteria form dense surface-associated communities known as biofilms that are central to their persistence and how they affect us. Biofilm formation is commonly viewed as a cooperative enterprise, where strains and species work together for a common goal. Here we explore an alternative model: biofilm formation is a response to ecological competition. We co-cultured a diverse collection of natural isolates of the opportunistic pathogen Pseudomonas aeruginosa and studied the effect on biofilm formation. We show that strain mixing reliably increases biofilm formation compared to unmixed conditions. Importantly, strain mixing leads to strong competition: one strain dominates and largely excludes the other from the biofilm. Furthermore, we show that pyocins, narrow-spectrum antibiotics made by other P. aeruginosa strains, can stimulate biofilm formation by increasing the attachment of cells. Side-by-side comparisons using microfluidic assays suggest that the increase in biofilm occurs due to a general response to cellular damage: a comparable biofilm response occurs for pyocins that disrupt membranes as for commercial antibiotics that damage DNA, inhibit protein synthesis or transcription. Our data show that bacteria increase biofilm formation in response to ecological competition that is detected by antibiotic stress. This is inconsistent with the idea that sub-lethal concentrations of antibiotics are cooperative signals that coordinate microbial communities, as is often concluded. Instead, our work is consistent with competition sensing where low-levels of antibiotics are used to detect and respond to the competing genotypes that produce them.

No MeSH data available.


Related in: MedlinePlus

Pyocins are drivers of biofilm formation in co-cultures.A) Size-based separation of the cell-free culture supernatant from strain 69 isolates fractions that induce biofilm formation at low concentrations and growth inhibition at high concentrations. Adding the unfractionated supernatant (“full sup”) induces biofilm in strain 4, which is comparable to direct mixtures of strains 4 and 69 (here labeled as “4 + 69”). Mass spectrometry analysis of fraction C9 (arrow) identified three proteins that are homologous to R and F pyocins in strain PA14. Compared to manufacturer size standards for the column, fraction C9 is expected to be >600 kDa. B) Cell-free culture supernatant at 2.5% in tryptone broth from PA14 (“+”) causes increased biofilm formation in strain 4 relative to tryptone broth with the same amount of water added (“-”), but this effect is not seen for strains 1, 7, or 69. C) The supernatant from PA14 is toxic to only strain 4, causing a strong population growth lag. This suggests that some degree of cell damage is needed for the biofilm response. Supernatant from a PA14 pyocin- mutant (PA14 prtRS153A) does not cause any growth lag in strain 4 (red curve), confirming that the toxicity to strain 4 is driven by pyocins. D) R pyocins, and F pyocins to a lesser degree, drive the biofilm response in strain 4. At 2.5%, the supernatant from both PA14 (“s14”) and F-pyocin- mutant (“s14ΔFpyo”) elicit a strong biofilm response in strain 4, which is largely removed by mutating either a master regulator of pyocins (“s14Δpyo”) or R pyocins (“s14ΔRpyo”) in PA14 (left panel). At a higher concentration (25%), supernatant that contains R pyocins is strongly toxic to strain 4, and R pyocin free supernatant now induces biofilm formation, which is consistent with the effects of other pyocins. Data from biofilm assays are means and 95% confidence intervals. Growth curves are means of eight replicates. Find numerical values in S1 Data.
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pbio.1002191.g004: Pyocins are drivers of biofilm formation in co-cultures.A) Size-based separation of the cell-free culture supernatant from strain 69 isolates fractions that induce biofilm formation at low concentrations and growth inhibition at high concentrations. Adding the unfractionated supernatant (“full sup”) induces biofilm in strain 4, which is comparable to direct mixtures of strains 4 and 69 (here labeled as “4 + 69”). Mass spectrometry analysis of fraction C9 (arrow) identified three proteins that are homologous to R and F pyocins in strain PA14. Compared to manufacturer size standards for the column, fraction C9 is expected to be >600 kDa. B) Cell-free culture supernatant at 2.5% in tryptone broth from PA14 (“+”) causes increased biofilm formation in strain 4 relative to tryptone broth with the same amount of water added (“-”), but this effect is not seen for strains 1, 7, or 69. C) The supernatant from PA14 is toxic to only strain 4, causing a strong population growth lag. This suggests that some degree of cell damage is needed for the biofilm response. Supernatant from a PA14 pyocin- mutant (PA14 prtRS153A) does not cause any growth lag in strain 4 (red curve), confirming that the toxicity to strain 4 is driven by pyocins. D) R pyocins, and F pyocins to a lesser degree, drive the biofilm response in strain 4. At 2.5%, the supernatant from both PA14 (“s14”) and F-pyocin- mutant (“s14ΔFpyo”) elicit a strong biofilm response in strain 4, which is largely removed by mutating either a master regulator of pyocins (“s14Δpyo”) or R pyocins (“s14ΔRpyo”) in PA14 (left panel). At a higher concentration (25%), supernatant that contains R pyocins is strongly toxic to strain 4, and R pyocin free supernatant now induces biofilm formation, which is consistent with the effects of other pyocins. Data from biofilm assays are means and 95% confidence intervals. Growth curves are means of eight replicates. Find numerical values in S1 Data.

Mentions: In order to identify the active component within the supernatant, we first performed anion exchange chromatography on the supernatant of strain 69 as it generates a robust response in strain 4 (Fig 3B). The active fraction that induced biofilm formation in strain 4 was further separated based on size by gel filtration. A single fraction (C9) appeared to contain the bulk of the active component as it strongly inhibited growth in strain 4, while the two flanking fractions C8 and C10 (i.e., lower concentrations of the effector) induced the biofilm response (Fig 4A). We subjected fraction C9 to protein mass spectrometry (see Methods). Of the proteins identified, three are notable for their known role in microbial interactions: homologs (PA14_8070, PA14_8090, PA14_8210) of the R and F pyocins in P. aeruginosa strain PA14 (S1 Table) [51]. Given that PA14 is a well-characterized strain, we next sought to explore how the pyocins of PA14 induce the biofilm response in our natural isolates.


Biofilm Formation As a Response to Ecological Competition.

Oliveira NM, Oliveria NM, Martinez-Garcia E, Xavier J, Durham WM, Kolter R, Kim W, Foster KR - PLoS Biol. (2015)

Pyocins are drivers of biofilm formation in co-cultures.A) Size-based separation of the cell-free culture supernatant from strain 69 isolates fractions that induce biofilm formation at low concentrations and growth inhibition at high concentrations. Adding the unfractionated supernatant (“full sup”) induces biofilm in strain 4, which is comparable to direct mixtures of strains 4 and 69 (here labeled as “4 + 69”). Mass spectrometry analysis of fraction C9 (arrow) identified three proteins that are homologous to R and F pyocins in strain PA14. Compared to manufacturer size standards for the column, fraction C9 is expected to be >600 kDa. B) Cell-free culture supernatant at 2.5% in tryptone broth from PA14 (“+”) causes increased biofilm formation in strain 4 relative to tryptone broth with the same amount of water added (“-”), but this effect is not seen for strains 1, 7, or 69. C) The supernatant from PA14 is toxic to only strain 4, causing a strong population growth lag. This suggests that some degree of cell damage is needed for the biofilm response. Supernatant from a PA14 pyocin- mutant (PA14 prtRS153A) does not cause any growth lag in strain 4 (red curve), confirming that the toxicity to strain 4 is driven by pyocins. D) R pyocins, and F pyocins to a lesser degree, drive the biofilm response in strain 4. At 2.5%, the supernatant from both PA14 (“s14”) and F-pyocin- mutant (“s14ΔFpyo”) elicit a strong biofilm response in strain 4, which is largely removed by mutating either a master regulator of pyocins (“s14Δpyo”) or R pyocins (“s14ΔRpyo”) in PA14 (left panel). At a higher concentration (25%), supernatant that contains R pyocins is strongly toxic to strain 4, and R pyocin free supernatant now induces biofilm formation, which is consistent with the effects of other pyocins. Data from biofilm assays are means and 95% confidence intervals. Growth curves are means of eight replicates. Find numerical values in S1 Data.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4497666&req=5

pbio.1002191.g004: Pyocins are drivers of biofilm formation in co-cultures.A) Size-based separation of the cell-free culture supernatant from strain 69 isolates fractions that induce biofilm formation at low concentrations and growth inhibition at high concentrations. Adding the unfractionated supernatant (“full sup”) induces biofilm in strain 4, which is comparable to direct mixtures of strains 4 and 69 (here labeled as “4 + 69”). Mass spectrometry analysis of fraction C9 (arrow) identified three proteins that are homologous to R and F pyocins in strain PA14. Compared to manufacturer size standards for the column, fraction C9 is expected to be >600 kDa. B) Cell-free culture supernatant at 2.5% in tryptone broth from PA14 (“+”) causes increased biofilm formation in strain 4 relative to tryptone broth with the same amount of water added (“-”), but this effect is not seen for strains 1, 7, or 69. C) The supernatant from PA14 is toxic to only strain 4, causing a strong population growth lag. This suggests that some degree of cell damage is needed for the biofilm response. Supernatant from a PA14 pyocin- mutant (PA14 prtRS153A) does not cause any growth lag in strain 4 (red curve), confirming that the toxicity to strain 4 is driven by pyocins. D) R pyocins, and F pyocins to a lesser degree, drive the biofilm response in strain 4. At 2.5%, the supernatant from both PA14 (“s14”) and F-pyocin- mutant (“s14ΔFpyo”) elicit a strong biofilm response in strain 4, which is largely removed by mutating either a master regulator of pyocins (“s14Δpyo”) or R pyocins (“s14ΔRpyo”) in PA14 (left panel). At a higher concentration (25%), supernatant that contains R pyocins is strongly toxic to strain 4, and R pyocin free supernatant now induces biofilm formation, which is consistent with the effects of other pyocins. Data from biofilm assays are means and 95% confidence intervals. Growth curves are means of eight replicates. Find numerical values in S1 Data.
Mentions: In order to identify the active component within the supernatant, we first performed anion exchange chromatography on the supernatant of strain 69 as it generates a robust response in strain 4 (Fig 3B). The active fraction that induced biofilm formation in strain 4 was further separated based on size by gel filtration. A single fraction (C9) appeared to contain the bulk of the active component as it strongly inhibited growth in strain 4, while the two flanking fractions C8 and C10 (i.e., lower concentrations of the effector) induced the biofilm response (Fig 4A). We subjected fraction C9 to protein mass spectrometry (see Methods). Of the proteins identified, three are notable for their known role in microbial interactions: homologs (PA14_8070, PA14_8090, PA14_8210) of the R and F pyocins in P. aeruginosa strain PA14 (S1 Table) [51]. Given that PA14 is a well-characterized strain, we next sought to explore how the pyocins of PA14 induce the biofilm response in our natural isolates.

Bottom Line: We co-cultured a diverse collection of natural isolates of the opportunistic pathogen Pseudomonas aeruginosa and studied the effect on biofilm formation.This is inconsistent with the idea that sub-lethal concentrations of antibiotics are cooperative signals that coordinate microbial communities, as is often concluded.Instead, our work is consistent with competition sensing where low-levels of antibiotics are used to detect and respond to the competing genotypes that produce them.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, University of Oxford, Oxford, United Kingdom; Oxford Centre for Integrative Systems Biology, University of Oxford, Oxford, United Kingdom.

ABSTRACT
Bacteria form dense surface-associated communities known as biofilms that are central to their persistence and how they affect us. Biofilm formation is commonly viewed as a cooperative enterprise, where strains and species work together for a common goal. Here we explore an alternative model: biofilm formation is a response to ecological competition. We co-cultured a diverse collection of natural isolates of the opportunistic pathogen Pseudomonas aeruginosa and studied the effect on biofilm formation. We show that strain mixing reliably increases biofilm formation compared to unmixed conditions. Importantly, strain mixing leads to strong competition: one strain dominates and largely excludes the other from the biofilm. Furthermore, we show that pyocins, narrow-spectrum antibiotics made by other P. aeruginosa strains, can stimulate biofilm formation by increasing the attachment of cells. Side-by-side comparisons using microfluidic assays suggest that the increase in biofilm occurs due to a general response to cellular damage: a comparable biofilm response occurs for pyocins that disrupt membranes as for commercial antibiotics that damage DNA, inhibit protein synthesis or transcription. Our data show that bacteria increase biofilm formation in response to ecological competition that is detected by antibiotic stress. This is inconsistent with the idea that sub-lethal concentrations of antibiotics are cooperative signals that coordinate microbial communities, as is often concluded. Instead, our work is consistent with competition sensing where low-levels of antibiotics are used to detect and respond to the competing genotypes that produce them.

No MeSH data available.


Related in: MedlinePlus