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Supplementary information for Harrison & Buckling: Materials & Methods
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Bacterial Strains
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Eight P. aeruginosa clones were used in this study: the wild type PA01, the methionine
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auxotroph PA06049 (Rella, 1985), the siderophore-deficient transposon mutant pvdF
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(University of Washington: Jacobs et al., 2003) and five siderophore-deficient mutants which
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arose de novo from strain PA06049 during a previous experiment in our laboratory (Harrison
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et al., 2005). These last are denoted Cheat 1-5. They were isolated from populations that
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had evolved for 100 or 200 generations in iron-limited medium and produce pyoverdine at
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≤26% of the wild type level (Harrison, 2007). Clones were identified as siderophore cheats
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using three criteria: their presence in mixed populations decreased iron-limited growth
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(Harrison & Buckling, 2005); monoculture growth in shaken broth was increased by iron
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supplementation; and they could invade iron-limited cooperator populations from rare
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(Harrison, 2007).
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siderophores at c. 50% of the wild type level and showed the negative frequency-dependent
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ability to invade wild type populations that is typical of cheats (Fredrik Inglis, personal
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communication).
The pvdF mutant carried a tetracycline-resistance marker, produced
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Biofilm growth
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Biofilms were grown using a microplate/peg lid system as described in (Moskowitz et al.,
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2004). For single-strain biofilms, c.107 fresh overnight culture cells of each strain were
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inoculated into twenty wells of a 96-well microplate containing 150µl Casamino acids broth
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(5g Casamino acids, 1.18g K2HPO4.3H2O, 0.25g MgSO4.7H2O/L) supplemented with 20mM
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sodium bicarbonate (necessary for iron chelator activity: (Meyer et al., 1996)). Ten wells
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were made iron limited by the addition of 70µg ml -1 human apotransferrin (Sigma Aldrich,
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Gillingham, UK) and the remaining ten were supplemented with 5µM Fe(III)Cl 3. For mixed-
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strain biofilms, c.107 fresh overnight culture cells of a 1:1 mix of the strains to be tested were
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inoculated. Nunc Immuno TSP microplate lids were placed on all microplates and the plates
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incubated at 37ºC for 36 hours.
The lids have plastic pegs which sit in the wells of the
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microplate and on which biofilms form. This system allows large numbers of biofilms to be
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grown and assayed simply and rapidly.
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6.4.3 Assessment of biofilm growth
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Peg lids were washed with M9 minimal salts, transferred to a microplate containing 150µl
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crystal violet solution (Pro-Lab Diagnostics, Neston, UK) per well, incubated for 15 minutes at
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room temperature, rinsed twice with ddH2O and dried in a laminar flow hood for 30 minutes.
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The crystal violet was then solubilised by placing peg lids in a microplate containing 150µl 95%
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ethanol per well and incubating for 15 minutes at room temperature. The absorbance at
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590nm of each well was measured and this value was taken as a measure of absolute biofilm
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density.
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For single-strain populations, the cell density of the planktonic subpopulation was
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assayed by reading the absorbance at 650nm of each broth culture and comparing the
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value obtained with a strain-specific calibration curve. A measure of the relative allocation
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of cells to the biofilm in each well was calculated by dividing the A 590 of each crystal violet-
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treated peg by the number of colony-forming units present in the corresponding well.
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The single-species biofilm data revealed a positive correlation between biofilm mass
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(crystal violet stain) and relative biofilm allocation (ANOVA: mass = clone + biofilm allocation
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+ clone*biofilm allocation; iron-limited F1,48=161.91, p<0.001; iron-supplemented F1,48=3.91,
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p<0.001). Therefore, we analysed only total biofilm mass.
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6.4.3 Morphological study
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Seven populations of PA01 and ten populations of each of the mixes were grown as above.
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Aliquots of diluted planktonic and biofilm subpopulations were plated on casamino acids
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agar plates. The primary siderophore of Pseudomonas spp., pyoverdine, fluoresces yellow-
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green under visible light and so allows easy visual scoring of pyoverdine-producing and
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pyoverdine-negative colonies after overnight growth at 37ºC. Because the pvdF insertion
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mutant produced siderophores at c. 50% of the wild-type level, visual scoring was not feasible
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for PA01+pvdF biofilms; instead, aliquots of planktonic and biofilm subpopulations were
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replica plated on casamino acids agar plates with and without 60µg ml -1 tetracycline.
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6.4.4 Statistical analyses
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Data were analysed using Minitab 14. Data for planktonic CFU under both iron regimes were
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arcsine square root transformed. Data for biofilm mass and iron-limited/iron-supplemented
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ratios were transformed using the natural logarithm. These transformations provided the best
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fit to the assumptions of parametric statistical tests in each case. Cheat frequency data
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were not transformed and were analysed non-parametrically.
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under iron limitation, the expected biofilm mass for each population was calculated as
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p(x mutan t )  (1 p)(x PA01) , where p =proportion of cheats in the biofilm, x mutan t =mean biofilm
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 69
mass obtained in pure cultures of that mutant clone and
mass.
For mixed biofilms grown
x PA01 =mean PA01 pure biofilm


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Where multiple T-tests were performed to compare
the evolved clones with PA06049,

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the value of alpha was adjusted using the false discovery rate method described by
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Benjamini & Hochberg (1995).
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Legends for supplementary figures
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Figure S1
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axis shows A590 of crystal-violet stained biofilms. White bars represent single-strain biofilms,
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grey bars PA01+cheat mixtures; bars show mean ± standard error (N=10 in each case). pvdF
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had reduced biofilm mass compared with its ancestor, PA01 (T10=7.5, p<0.001). When the five
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evolved clones were compared with their ancestor, PA06049, there was no effect of
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siderophore production on biofilm mass (T≥2.26, p≤0.021).
Single- and mixed-strain biofilm mass in iron-supplemented growth medium. Y-
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Figure S2
Growth of the planktonic subpopulations in wild-type and cheat pure cultures
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under iron limitation (white bars) and iron supplementation (grey bars); bars show mean ±
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standard error. Compared with PA01, pvdF showed reduced planktonic growth under iron
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limitation (T17=2.56, p=0.010) but not under iron supplementation (T 9=0.63, p=0.727).
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Under
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iron limitation, Clone 2 showed reduced planktonic growth relative to PA06049 (T11=3.48,
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p=0.003), Clone 3 showed increased growth (T9=3.80, p=0.002) and the remainder showed
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similar growth to PA06049 (T≤0.46, p>0.4). Under iron supplementation, Clones 1, 2, 3 and 5
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showed increased planktonic growth relative to PA06049 (T≥2.15, p≤0.027) while Clone 4
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showed decreased growth (T17=2.08, p=0.027).
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References
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