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.

Thermal food processing leads to the formation of advanced glycation end products (AGE) such as Nε-carboxymethyllysine (CML). Accordingly, these non-canonical amino acids are an important part of the human diet. However, CML is only partially decomposed by our gut microbiota and up to 30% are excreted via faeces and, hence, enter the environment. In frame of this study, we isolated a soil bacterium that can grow on CML as well as its higher homologue Nε-carboxyethyllysine (CEL) as sole source of carbon. Bioinformatic analyses upon whole-genome sequencing revealed a subspecies of Pseudomonas asiatica, which we named ‘bavariensis’. We performed a metabolite screening of P. asiatica subsp. bavariensis str. JM1 grown either on CML or CEL and identified N-carboxymethylaminopentanoic acid and N-carboxyethylaminopentanoic acid respectively. We further detected α-aminoadipate as intermediate in the metabolism of CML. These reaction products suggest two routes of degradation: While CEL seems to be predominantly processed from the α-C-atom, decomposition of CML can also be initiated with cleavage of the carboxymethyl group and under the release of acetate. Thus, our study provides novel insights into the metabolism of two important AGEs and how these are processed by environmental bacteria.

The alarming rise of antibacterial drug resistance and the dwindling supply of novel antibiotics highlight the need for innovative approaches in combating bacterial infections. Traditionally, antibacterial drug discovery campaigns have largely been conducted in rich media. Such growth conditions are not representative of the host environment and render many metabolic pathways, otherwise needed for survival and infection, dispensable. Such pathways have been overlooked in conventional drug discovery campaigns despite their validity as potential antibacterial targets. The work presented in this thesis focuses on the development and validation of a screening strategy for the identification and mechanism of action determination of novel inhibitors of metabolic pathways in bacteria under nutrient-limited conditions. This screen led to the identification of MAC168425, MAC173979 and MAC13772 as inhibitors that target glycine metabolism, p-aminobenzoic acid biosynthesis and biotin biosynthesis, respectively. Moreover, it established this approach as a general platform that can be applied for different organisms with synthetic or natural product libraries. Additional mechanistic studies of the biotin biosynthesis inhibitor, MAC13772, resulted in solving the crystal structure of BioA in complex with MAC13772. Analysis of the co-structure confirmed our proposed mode of inhibition and provided information for strategies for rational drug design. Investigation of the antibacterial activity of MAC13772 revealed its potency against a number of pathogens. Furthermore, we show how MAC13772 acts synergistically with rifampicin in clearing growing mycobacterial cultures. The potential of this inhibitor as a lead for preclinical pharmacokinetic studies and for antibacterial drug development is discussed. We also discuss our current efforts to develop a metabolomic platform for the characterization of novel antibacterials that can be used in concert with our current approach to chart the metabolic response of bacteria to chemical perturbants and to generate testable hypotheses regarding the mode of action of novel inhibitors of bacterial metabolism. / Thesis / Doctor of Philosophy (PhD)