2024 Theses Doctoral

Light affects metabolism in Pseudomonas aeruginosa biofilms

Eckartt, Kelly

Many species of bacteria naturally exist in multicellular structures called biofilms, which are formed when microbes excrete an adherent polymeric matrix. The biofilm lifestyle offers protection from environmental attacks. However, the high density of biomass within these structures also promotes the formation of resource gradients and therefore internal microenvironments with distinct conditions. Unlike the well-mixed liquid cultures routinely used for research, biofilms thus contain differentiated subpopulations that perform different metabolic processes. Such metabolic heterogeneity benefits multicellular systems by allowing for division of labor and cross-feeding of metabolites. Importantly, it also contributes to the robustness of the overall population because metabolic subpopulations commonly differ with respect to their abilities to survive environmental changes or drug treatments.

Pseudomonas aeruginosa is a chemotrophic opportunistic pathogen that avidly forms biofilms. It is a leading cause of infections in humans and can occupy a variety of sites, including burn and non-healing skin wounds. One factor that allows the bacterium to thrive in a wide range of environments is its metabolic versatility. P. aeruginosa is able to use oxygen and N-oxides as terminal electron acceptors and produces redox active small molecules called phenazines that support metabolic activity in oxygen-limited biofilm subzones. In many of the environments it inhabits P. aeruginosa is exposed to sunlight, which can act as an environmental cue and can damage light-sensitive enzymes. Light sensing proteins are found in diverse chemotrophic bacteria and have been studied structurally and biochemically for decades. In fact, the bacteriophytochrome BphP, purified from P. aeruginosa, was identified and biochemically characterized in the 1990’s. The physiological role of BphP, and light sensing in general, is still an active field of study. Recently light has been shown to play roles in inhibiting biofilm macrostructure formation, inhibiting aerobic respiration, and providing anticipatory protection from osmotic stress in various pseudomonads.

My thesis aims to investigate how light affects metabolism in P. aeruginosa biofilms. Chapter 1 provides the necessary background about bacterial multicellularity, light as an environmental factor, and the relevant aspects of P. aeruginosa metabolism. Chapter 2 explores the phenomenon of the inhibitory effect of light on aerobic respiration. Light/dark and temperature cycling elicits transcriptomically entrenched rings of high and low aerobic respiration which is not restricted to a singular color of light within the visible light spectrum. This chapter also highlights the role of the bacteriophytochrome BphP in red light-dependent respiratory switching. Chapter 3 further explores how the light effect is altered in response to changing the redox state of the biofilm. Light has distinct effects on the use of specific respiratory pathways, on oxygen consumption, and on metabolic activity based on the location in the biofilm and the availability of electron acceptors. Chapter 4 identifies the white light and red light-dependent proteome of P. aeruginosa biofilms and additionally determines the red light-dependent BphP regulon. This chapter also highlights how conversion of BphP between photostates is necessary for red light-dependent respiratory switching in P. aeruginosa biofilms.

Understanding how P. aeruginosa metabolism is modulated by light provides information as to how this bacterium thrives in diverse environments, and investigating the phenomenon in a biofilm model expands the relevance of this research. Because they define the relationships between light exposure and physiological responses in an important pathogen, the observations presented in this thesis constitute foundational work with the potential to inform treatment conditions for biofilm based infections.