Environmental and Genetic Drivers of Swarming and Surface Competitiveness in Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen. It is known for its multidrug resistance and ability to cause severe infections, including pneumonia, urinary tract infections, and diabetic foot ulcers. P. aeruginosa is a highly adaptive bacterium with a large gene pool encoding various virulence factors, including quorum-sensing (QS) machinery. QS enables collective behavior, giving it a competitive advantage in times of nutrient scarcity. In this study, I have investigated the ecological and behavioral strategies employed by P. aeruginosa to thrive in polymicrobial and nutrient-limited environments. To study the polymicrobial interactions, I examined the interactions of P. aeruginosa with co-occurring pathogens, including Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Serratia marcescens, and Staphylococcus aureus. I identified a unique mechanism employed by P. aeruginosa specifically against K. pneumoniae to displace it from solid surfaces. Interestingly, P. aeruginosa employs neither proteases nor toxic secondary metabolites against K. pneumoniae. I found the production of rhamnolipid biosurfactant under the control of the RhlR quorum-sensing system to be the primary factor required by P. aeruginosa to displace K. pneumoniae effectively. Under conditions of iron limitation, both bacteria produce iron-scavenging molecules. However, P. aeruginosa also produces rhamnolipid biosurfactant, which allows it to push K. pneumoniae cells away from the substratum. Our study describes a unique quorum and iron-responsive mechanism in P. aeruginosa to support its growth during resource competition on a solid surface. P. aeruginosa exhibits QS-regulated swarming motility in response to external cues, such as ethanol, phosphate limitation, and iron limitation, forming branch-like patterns on semi-solid surfaces. In this study, I tested whether ecologically important microbially produced alcohols could induce swarming in P. aeruginosa. I found that butanol induces swarming as effectively as ethanol in mPGM media, but unlike ethanol, butanol-induced swarming was independent of ErdR regulation. However, mutants in genes downstream of ErdR, such as sensor kinase mutants ercS and eraS, and ExaA, a quinoproteinethanol dehydrogenase, were required in both ethanol and butanol conditions. The data suggest that while both alcohols use overlapping signaling networks, butanol employs an alternative, ErdR-independent regulatory pathway, potentially involving FixR. I also investigated how P. aeruginosa responds to changes in nutrient availability, with a focus on the role of iron availability in regulating swarming motility. Using iron-limiting M9 medium, I conducted RNA-seq to analyze gene expression in swarming bacteria with and without iron supplementation. Iron limitation promoted swarming by activating quorum sensing (rhlR, rhlA, rhlB), siderophore synthesis (pvd genes), and stress-response pathways, whereas iron supplementation suppressed these processes and downregulated motility-related genes. Collectively, these findings reveal how P. aeruginosa integrates chemical and nutrient cues to fine-tune its motility, optimizing surface colonization and survival in polymicrobial and nutrient-variable environments. To understand the relevance of swarming under clinical conditions, I also tested the swarming phenotype of P. aeruginosa reference panel strains under iron-limited (M9) and ethanol-rich (PGM) conditions. While most clinical strains swarmed on both media conditions, some strains didn't swarm in any condition. By performing swimming motility assays, I demonstrated that the swarming-deficient phenotype in the clinical strains was associated with defects in flagellar motility. Sequential cystic fibrosis isolates revealed temporal shifts in motility, reflecting within-host adaptation. Iron supplementation suppressed swarming across all strains, confirming iron limitation as a key inducer. Overall, swarming emerges as a niche-dependent trait influenced by nutrient availability and environmental signals, highlighting how P. aeruginosa fine-tunes motility to persist in complex and changing habitats. In summary, I highlighted the sophisticated survival strategies of P. aeruginosa, including physical displacement of competitors, environmental sensing, and social motility, providing insights that could inform the development of anti-virulence therapies targeting its ecological fitness and social behavior.