Life, Death, And Coexistence: Exploring And Manipulating The Respiratory Lifestyle Of Shewanella Oneidensis

Abstract

In their natural environment, microbes often exist in stressful, suboptimal, ever-changing conditions and have evolved innumerable and varied successful strategies for managing stresses and thriving in flux. Microbial ecosystems are defined not only by specialized members occupying defined narrow niches, but also members that move between niches and exist in a mode of constant opportunism. One such opportunistic group of organisms are those belonging to the genus Shewanella which are largely defined by their respiratory versatility. A particularly well-studied member of this genus, S. oneidensis, is the most versatile respiratory organism described to date, and is a model organism for extracellular electron transfer. This thesis explores the respiratory lifestyle of S. oniedensis primarily through the lens of cell physiology and competitive fitness under optimal growth conditions and those that yield catastrophic death. The second chapter of this thesis is a study in cofactor acquisition by a key respiratory enzyme in S. oneidensis MR-1. The periplasmic protein FccA is both a fumarate reductase and an electron carrier protein for extracellular electron transfer, that requires a flavin cofactor for its function. Through genetic manipulation, growth experiments, and biochemical experiments, we found that for S. oneidensis, self-secretion of flavins comes at minimal metabolic cost and is required for periplasmic flavoprotein cofactor acquisition. The third chapter is a probing of natural and engineered factors that enable survival under respiratory stress. Shewanella is considered an obligate respiring organism, and when placed under conditions in which respiration cannot normally function, it experiences massive loss in viability accompanied by cell lysis. Despite, 99-99.99% of cells undergoing death in this condition, many persist. This study leveraged synthetic proton motive force supplementation, which affords enhanced survival of S. oneidensis, to profile the innate strategies used to survive under respiratory stress. The key finding of this study is that the sodium motive force plays a key role in survival of S. oneidensis under respiratory stress, even when survival is enhanced by proton motive force supplementation. The fourth chapter of this thesis is a series of competition experiments reframing a central paradigm of the competitive exclusion principle: that two organisms occupying the same niche cannot coexist. This principle states that in this scenario any organism with a reproductive advantage will ultimately overtake a population. This study demonstrated that two engineered strains of S. oneidensis utilizing the same medium with the same food and nutrient sources, but growing a vastly different rates, can remain at stable frequencies when grown attached to an electrode as the sole sink of electrons in respiration. The primary reason for this stability is that the original parent population remains actively growing on a surface to which daughtered cells are constantly removed. While the scenario we have engineered to “break” the competitive exclusion principle could be considered a form of niche differentiation, it demonstrates an effective strategy to combat strain degeneration and contamination in industrial fermentation, by allowing a selected population of less competitively fit individuals to act indefinitely as a progenitor population. The work in this thesis brings special attention to the adaptation towards an obligate respiratory lifestyle in S. oneidenisis. The systems it has evolved to secrete flavins as both respiratory cofactors and intermediates and the marked death it experiences under respiratory stress together emphasize adaptations in Shewanella to thrive in redox stratified environments, supported by the wide variety of respiratory nodes it can utilize. Finally, this work highlights the utility of S. oneidensis as a test platform for ideas, with potential benefits for biotechnological and industrial applications.