Interspecies surfactants serve as public goods enabling surface motility in Pseudomonas aeruginosa

In most natural environments, bacteria live in polymicrobial communities where secreted molecules from neighboring species alter bacterial behaviors including motility, but such interactions are understudied. Pseudomonas aeruginosa is a motile opportunistic pathogen that exists in diverse multispecies environments such as the soil and is frequently found in human wound and respiratory tract co-infections with other bacteria including Staphylococcus aureus. Here we show that P. aeruginosa can co-opt secreted surfactants from other species for flagellar-based surface motility. We found that exogenous surfactants from S. aureus, other bacteria, and interkingdom species enabled P. aeruginosa to switch from swarming to an alternative surface spreading motility on semi-solid surfaces and allowed for the emergence of surface motility on hard agar where P. aeruginosa was otherwise unable to move. This motility was distinct from the response of other motile bacteria in the presence of exogenous surfactants. Mutant analysis indicated that this P. aeruginosa motility was similar to a previously described mucin-based motility, ‘surfing’, albeit with divergent regulation. Thus, our study demonstrates that secreted surfactants from the host as well as neighboring bacterial and interkingdom species act as public goods facilitating P. aeruginosa flagella-mediated surfing-like surface motility, thereby allowing it to access different environmental niches.


SUPPLEMENTAL FIGURES AND FIGURE LEGENDS
plates containing 25% media salts as a control or S. aureus supernatant, and motility was imaged after 24 hours incubation.Three independent replicates are shown.Supplemental Figure 12.Surface spreading is not a universal bacterial response to exogenous surfactants.The indicated species -P.aeruginosa PA14, Burkholderia cenocepacia K56-2, K. pneumoniae, V. cholerae, or E. coliwere inoculated on semi-solid LB agar plates containing 25% media salts as a control, S. aureus supernatant, or 25% media salts with the addition of 0.1% Triton X-100 or 25 µg•mL -1 saponin and motility was imaged after 24 hours incubation.Three independent replicates are shown.for movement on mucin.The indicated P. aeruginosa strains were inoculated on semi-solid agar plates containing 25% of S. aureus supernatant or 25% media salts with the addition of 0.4% mucin, and images were taken after 24 hours incubation.Three independent replicates shown.

Figure 1 .Supplemental Figure 9 .
S. aureus secreted products alter P. aeruginosa twitching and swimming motility.P. aeruginosa was inoculated on (A) twitch plates and (B) swim plates containing 25% media salts as a control or S. aureus supernatant.(A) Twitch plates were incubated for 48 hours, visualized by crystal violet staining, and imaged.(B) Swim plates were imaged after 24 hours incubation.Arrows indicate the swim boundaries.(A,B) Three independent replicates are shown.Pili function and rhamnolipids are not required for surface spreading on hard agar.(A,B) The indicated P. aeruginosa strains were inoculated on hard agar plates containing 25% media salts as a control or S. aureus supernatant, and motility was imaged after 24 hours incubation.Three independent replicates are shown.Supplemental Figure 10.The P. aeruginosa quorum sensing systems are not required for surface spreading motility.The indicated P. aeruginosa strains were inoculated on (A) semisolid or (B) hard agar plates containing 25% media salts as a control or S. aureus supernatant, and motility was imaged after 24 hours incubation.(A,B) Three independent replicates are shown.Supplemental Figure 11.Several P. aeruginosa regulators are not required for surface spreading motility.The indicated P. aeruginosa strains were inoculated on semi-solid agar

Figure 13 .
P. aeruginosa, B. subtilis ZK3814, E. coli, and B. cenocepacia demonstrate swimming motility.(A,B) The indicated species -P.aeruginosa PA14, B. subtilis ZK3814 and PY79, B. cenocepacia K56-2, K. pneumoniae, V. cholerae, or E. coliwere inoculated on LB swim plates containing 25% media salts as a control.Plates were imaged after (A) 24 hours or (B) 48 hours incubation.(A,B) Arrows indicate the swim boundaries.Three independent replicates are shown.Supplemental Figure 14.Exogenous surfactants restore swarming in surfactant-deficient B. subtilis.The indicated species -P.aeruginosa PA14 or B. subtilis ZK3814 and PY79were inoculated on semi-solid LB agar plates containing 25% media salts as a control, S. aureus supernatant, or 25% media salts with the addition of 50 µg•mL -1 rhamnolipids or 25 µg•mL -1 saponin and motility was imaged after 24 hours incubation.Three independent replicates are shown.Supplemental Figure 15.P. aeruginosa differentially regulates genes when undergoing surface spreading vs. planktonic growth in the presence of S. aureus secreted products.(A) WT P. aeruginosa cells were scraped from the leading edge on semi-solid and hard agar plates containing 25% media salts as a control or S. aureus supernatant after 17 hours of incubation.Both independent replicates used for the RNA-seq are shown prior to scraping.(B) Volcano plots of -log10(p-value) vs. log2(fold-change) transcript levels after exposure to S. aureus supernatant compared to media salts control after 17 hours on (left) semi-solid or (right) hard agar.Genes shown as upregulated or downregulated have p < 0.05 and log2(fold-change) ≥ 1 or ≤ −1 respectively.(C) Scatter plots of log2(fold-change) transcript levels in P. aeruginosa after 17 hours on (left) semi-solid or (right) hard agar containing S. aureus supernatant compared to 2 hours exposure to S. aureus supernatant in planktonic culture (1).Genes shown as upregulated or downregulated have p < 0.05 and log2(fold-change) ≥ 1 or ≤ −1 respectively.(D,E) Gene Ontology (GO) enrichment (2-4) of P. aeruginosa genes downregulated after 17 hours incubation on (D, top) semi-solid and (D, bottom) hard agar plates or commonly downregulated on (E) both agar plates containing S. aureus supernatant.Nonredundant categories shown.Supplemental Figure 16.Quorum sensing and flagella, but not pili, are required for motility on mucin.(A,B) The indicated P. aeruginosa strains were inoculated on semi-solid agar plates containing 25% S. aureus supernatant or 25% media salts with the addition of 0.4% mucin, and motility was imaged after 24 hours incubation.Three independent replicates are shown.Supplemental Figure 17.Known P. aeruginosa motility regulatory systems are not required

Table 2 . Gene expression in P. aeruginosa from semi-solid or hard agar at 17 hours after exposure to S. aureus supernatant or media salts control determined from RNA-seq analysis. (Excel)
Gene expression was analyzed by RNA-seq from RNA purified from two biological replicates of each treatment at 17 hours (semi-solid or hard agar with addition of S. aureus supernatant or media salts control).

Table 3 . Differentially expressed genes between P. aeruginosa from semi-solid or hard agar plates after exposure to S. aureus supernatant or media salts control determined from RNA-seq analysis
. (Excel) Gene expression was analyzed by RNA-seq from purified RNA from two biological replicates of each treatment after 17 hours (semi-solid or hard agar with addition of S. aureus supernatant or media salts control).Fold change indicates the mean expression of the supernatant-exposed P. aeruginosa over the control.Supp.

Table 4 . Differentially expressed genes in common between P. aeruginosa from semi-solid and hard agar plates after exposure to S. aureus supernatant or media salts control determined from RNA-seq analysis
. (Excel) Gene expression was analyzed by RNAseq from purified RNA from two biological replicates of each treatment after 17 hours (semisolid or hard agar with addition of S. aureus supernatant or media salts control).Genes commonly upregulated or downregulated from the semi-solid and hard agar conditions are listed.Fold change indicates the mean expression of the supernatant-exposed P. aeruginosa over the control.Supp.

Table 5 . Differentially expressed genes in common between P. aeruginosa from semi-solid or hard agar and planktonic growth, or semi-solid and hard agar exclusively, after
each treatment after 17 hours (semi-solid or hard agar and planktonic growth with addition of S. aureus supernatant or media salts control).Genes commonly upregulated or downregulated from the semi-solid or hard agar and planktonic growth conditions, or semi-solid and hard agar exclusively, are listed.Fold change indicates the mean expression of the supernatant-exposed P. aeruginosa over the control.
exposure to S. aureus supernatant or media salts control determined from RNA-seq analysis.(Excel) Gene expression was analyzed by RNA-seq from purified RNA from two biological replicates of

Table 6 . Gene ontology (GO) enrichment of upregulated and downregulated genes after
P. aeruginosa exposure to S. aureus exoproducts.(Excel)GO enrichment of P. aeruginosa genes differentially expressed on semi-solid and hard agar plates individually as well as in both, in common with planktonic cells, as well as exclusively in both motility conditions (but not in planktonic cells).Supp.

Table 7 . Primers used in this study*. 181
Up = upstream arm of gene; down = downstream arm of gene.
# Primers utilized for Sanger sequencing.^ Site is underlined in primer sequence.