Supplemental Figures

Supplemental Figure 1: P. aeruginosa culture kinetics is not differentially impacted by the presence of S. aureus or by co-culture substrate. Time course analyses were performed for the P. aeruginosa PA14 (Pa) population (quantified as log10(CFUs/well) at 16 hours) in planktonic (dashed lines) and biofilm (solid lines) phases. P. aeruginosa PA14 culture assays were performed on a CFBE monolayer (A) or plastic substrate (B) either in the presence of S. aureus 8325-4 (Sa) (black lines) or as a mono-culture (grey lines). The legend for panels A and B is shown below panel B.

Supplemental Figure 2: Increased planktonic S. aureus early in co-culture with P. aeruginosa coincides with a decreased S. aureus biofilm population, compared to S. aureus mono-culture. S. aureus 8325-4 (Sa) populations were analyzed after 6 hours in co-culture model with P. aeruginosa PA14 (Pa) or in mono-culture on plastic. A) Planktonic Sa population. B) Biofilm Sa population. Dotted lines connect paired samples (each measured in triplicate) from five independent assays.

Supplemental Figure 3: S. aureus-killing by P. aeruginosa is not dependent solely on P. aeruginosa growth or mucoidy but is variable between P. aeruginosa strains. Non-mucoid and mucoid laboratory strains or clinical isolates of P. aeruginosa (Pa) were grown in the presence or absence of S. aureus 8325-4 (Sa) in the co-culture model on plastic substrate. Sa (A) and Pa (B) viability was measured at 16 hours. A) Differential viability of Sa in co-culture with Pa was compared to Sa mono-culture using ANOVA and Dunnett’s multiple comparisons test (*,p<0.05; ns, not significant). B) Differential growth/survival of each Pa strain in mono-culture or co-culture with Sa was compared to Pa PA14 mono-culture. Statistical significance from Pa PA14 strain alone was determined using ANOVA and Dunnett’s multiple comparisons test (*,p<0.05; ns, not significant). No significant difference in Pa viability between mono-culture versus co-culture with Sa were determined for any strain pair (not shown above). Error bars represent SEM for six replicate samples from a single representative assay.

Supplemental Figure 4: Methicillin-sensitive and -resistant S. aureus strains are killed by P. aeruginosa. Laboratory strains and clinical isolates of S. aureus (Sa) were grown in the presence or absence of P. aeruginosa PA14 (Pa) in the co-culture model on plastic substrate. Sa viability in biofilms was quantified at 16 hours for a panel of methicillin-sensitive S. aureus (MSSA) strains (A) and methicillin-resistant S. aureus (MRSA) strains (B). Differential survival between co-culture and Sa mono-culture was compared for each Sa strain using multiple Student’s T-tests and the Holm-Sidak method for correcting for multiple comparisons in a representative assay with triplicate samples, (*,p<0.05). Error bars represent SEM.

Supplemental Figure 5: Global P. aeruginosa gene expression was not differentially impacted by the presence or absence of S. aureus. P. aeruginosa PA14 (Pa) gene expression analyzed by RNA-seq was compared in co-culture with S. aureus 8325-4 (Sa) versus Pa mono-culture. Total RNA was isolated from 6 hour biofilm fractions on plastic. Open circles correspond to individual genes expressed by Pa; dark grey, >10-fold decreased expression.

Supplemental Figure 6: P. aeruginosa preferentially consumes S. aureus-produced lactate over medium-provided glucose. P. aeruginosa PA14 (Pa) mono-culture was treated with high-lactate-containing culture supernatants from an S. aureus 8325-4 mono-culture treated with Pa supernatants (“Pa + (Sa + Pa) sup”). Glucose ( ) and lactate ( ) concentrations were quantified by HPLC from clarified supernatants collected throughout the assay at the indicated time points.

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Supplemental Figure 7: P. aeruginosa viability of WT P. aeruginosa PA14 or deletion mutant strains is not differentially impacted by the presence of S. aureus. P. aeruginosa PA14 (Pa) parent or mutant strain biofilm populations were quantified in the co-culture model on CFBE substrate at 16 hours in the presence or absence of S. aureus 8325-4 (Sa). Differential survival/growth of each Pa strain was compared between mono-culture versus co-culture with Sa using ANOVA and Sidak’s multiple comparisons post-test for average viability from triplicate samples in three experiments (ns, not significant). Error bars represent SEM.

Supplemental Figure 8: Late-stage S. aureus killing by P. aeruginosa requires P. aeruginosa-produced HQNO and siderophores in plastic-grown biofilms, but does not differentially impact P. aeruginosa survival. S. aureus 8325-4 (Sa) and P. aeruginosa PA14 (Pa) WT or specified deletion mutant biofilms were grown in mono-culture or co-culture on plastic substrate. Sa (A) and Pa (B) biofilm viability was measured at 16 hours. A) Differential survival of Sa in each co-culture was compared to Sa mono-culture using ANOVA and Dunnett’s multiple comparisons post-test (a, p<0.05). Differential survival of Sa mono-culture or Sa in co-culture with a Pa mutant is compared to Sa co-culture with WT Pa also using ANOVA and Dunnett’s multiple comparisons post-test (b, p<0.05) B) Differential survival/growth of each Pa strain was compared between mono-culture versus co-culture with Sa using ANOVA and Sidak’s multiple comparisons post-test (ns, not significant). Averages and errors (SEM) from three separate experiments, each performed in triplicate, are shown.

Supplemental Figure 9: Iron supplementation alters the efficiency of P. aeruginosa-mediated S. aureus killing. S. aureus 8325-4 (Sa) and P. aeruginosa PA14 (Pa) wildtype (WT) or specified deletion strain were grown in co-culture or as mono-culture on plastic substrate. MEM + L-gln was supplemented with 8 µM iron chloride (FeCl3). Sa (A) and Pa (B) biofilm viability was measured at 16 hours. A) Differential survival of Sa in each co-culture was compared to Sa mono-culture using ANOVA and Dunnett’s multiple comparisons post-test (*, p<0.05). B) Differential survival/growth of each Pa strain was compared between mono-culture versus co-culture with Sa using ANOVA and Sidak’s multiple comparisons post-test (ns, not significant). Averages and errors (SEM) from three separate experiments, each performed in triplicate, are shown.

Supplemental Figure 10: HQNO- and siderophore-dependent upregulation of S. aureus fermentation pathway gene expression by P. aeruginosa occurs independently of S. aureus strain or biofilm substrate. S. aureus (Sa) was grown as mono-culture or co-culture with P. aeruginosa PA14 (Pa) WT or deletion mutants. From 6 hour biofilms, Sa expression of marker genes from lactate (ldh, A and D), formate (pflB, B and E), and ethanol (adh, C and F) fermentation pathways was measured by qRT-PCR and normalized to a control gene, rpoB. A-C) Sa USA300 gene expression in biofilms grown on CFBE monolayers. D-F) Sa 8325-4 gene expression in plastic-grown biofilms. Gene expression from 6 (A-C) or 9 (D-F) replicate experiments was compared using Friedman rank test and Dunn’s multiple comparisons post-test. Differential Sa gene expression when in co-culture with Pa was compared to Sa mono-culture (a, p<0.05). Differential Sa gene expression from Sa mono-culture or Sa in co-culture with a Pa mutant was compared to Sa co-culture with WT Pa (b, p<0.05). Error bars represent SEM.