Studies of the spatial organization of metabolism in Shewanella oneidensis and Pseudomonas aeruginosa biofilms

Abstract

Bacteria grow in the environment as surface-attached microbial communities. These communities are pervasive and resilient in the face of changing and challenging environmental conditions. Because of their community organization and three-dimensional structure, conditions within a biofilm are heterogenous, exposing the bacterial cells to individual microenvironments depending on their location in the biofilm and the biomass of the structure. Communities are therefore thought to be metabolically stratified. To understand how communities are organized with regard to growth activity and metabolic state and what role endogenous compounds might play in this organization, this thesis explores the spatiometabolic organization and dynamics of Shewanella oneidensis biofilms and the roles that acyl-homeserine lactones and phenazines might have in Pseudomonas aeruginosa communities. Using unstable fluorescent reporters to measure growth activity and protein synthesis and conducting quantitative image analysis, domains of activity were determined for developing S. oneidensis biofilms. Biofilm structures reproducibly stratify with respect to growth activity and metabolism as a function of structure size. Within domains of growth-inactive cells, genes upregulated under anaerobic conditions are expressed demonstrating that cells in the nutrient-limited regions of the biofilm are not dead, but are capable of generating enough energy to persist. To determine if these growth-inactive cells are able to respond dynamically to changes in environmental conditions and what types of nutrients affect growth activity profiles, S. oneidensis biofilms were exposed to increased concentrations of an electron acceptor and an electron donor. Cells in the growth-inactive regions were able to respond to nutrient changes, but were more affected by a change in electron acceptor than electron donor. To investigate the role of small molecules in biofilm community organization, the degradation of acyl-homoserine lactone (AHL), was studied. This molecule is an important part of the quorum sensing signaling network in P. aeruginosa, where the bacteria both produce and sense this molecule. When bacteria sense a specific concentration of the AHL, they are induced to form a biofilm or initiate a community wide response. To determine what role AHL degradation has on the community response, a mutant that constitutively degrades the compound was characterized and expression profiles for degradation were compared between this strain and wild type communities. Genes for AHL degradation were expressed in the middle of biofilm colonies suggesting that degradation may be an important part of the community response network. It was also shown that AHLs can be used as a substrate for growth, so nutrient-limited cells might also be able to use AHLs to generate energy. Finally, to investigate whether endogenously produced redox-active small molecules could potentially play a role in energy maintenance in communities, the SoxR sensing system was studied. This system is typically thought to regulate the response to superoxide radicals. In P. aeruginosa and other organisms outside the class of enterics, however, recent evidence suggested that they may instead play a role in the sensing of redox-active small molecules produced under conditions of low nutrients and high cell density. To determine the ubiquity of this response mechanism, bioinformatic analyses were conducted to discover SoxR binding sites across all genomes containing SoxR. Predictions for binding sites and the mechanism of regulation, redox-active molecule induction, were confirmed in the Gram-positive bacterium Streptomyces coelicolor. This work brings us closer to understanding how cells persist and retain the capacity to dynamically regulate their metabolism in biofilm communities. Using reporter assays and quantitative analyses, studies can be done to determine metabolic organization within communities and further investigate the role that endogenous small molecules can play in community organization.