Pyocins are drivers of biofilm formation in co-cultures.

<p>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-null mutant (PA14 <i>prtR</i>^<i>S153A</i>) 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-null 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 <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002191#pbio.1002191.s001" target="_blank">S1 Data</a>.