Specific Inhibitors of Microbial Sulfate Reduction: Dynamics of Inhibition and Mechanisms of Resistance across Sulfate-Reducing Microorganisms

Sulfate-reducing microorganisms (SRM) gain energy by coupling the oxidation of organic matter or hydrogen to dissimilatory sulfate-reduction. This use of sulfate as a terminal electron acceptor for growth results in the generation of the toxic and corrosive compound, hydrogen sulfide. SRM are ubiquitous, and their metabolism forms a critical component of the global carbon and sulfur cycles. The activity of these microbes is also of economic importance: SRM are implicated in the bio-corrosion of steel and concrete. The petroleum industry is particularly susceptible to these costs; “sour” oil, i.e. oil contaminated with SRM-produced sulfide, represents a corrosion threat to transportation infrastructure as well as a health threat for workers. The identification and characterization of specific and potent inhibitors of SRM is critical to preventing these outcomes. This thesis characterizes the specific inhibition of SRM in pure culture and in sulfate-reducing communities, both in terms of how inhibitors behave in complex systems as well as how resistance to inhibition arises./nSpecific inhibitors are compounds that disrupt the metabolism of one group of organisms, with little or no effect on the rest of the community. The first chapter of this dissertation reviews the current understanding of the specific inhibition of SRM: how we define and measure specific inhibition and how we gain insight into cellular targets of inhibitors. I review mechanisms of known specific inhibitors, which target sulfate transport systems and key enzymes of the sulfate-reduction pathway. This chapter also highlights several knowledge gaps: a quantitative understanding of the “specificity” of inhibition across diverse SRM in ecological contexts, and characterizations of the activity of key sulfate transport systems. Finally, emphasis on additional targets of inhibitors will improve our basic understanding of this metabolism and our ability to control it across environmental systems./nThe second chapter of this dissertation aims to quantitatively dissect SRM inhibition by the specific inhibitor, perchlorate. The inhibitory dynamics of perchlorate (i.e. rate and community effects of inhibition and rate/community structure post cessation of inhibition) are characterized in continuous-flow sulfate-reducing systems. Empirical data supports a model where perchlorate acts largely to decrease SRM growth rates, rendering planktonic SRM increasingly susceptible to wash-out. Several other key factors putatively affecting the inhibitory dynamics of perchlorate are further investigated: surface attachment, indirect effects mediated by dissimilatory perchlorate-reducing bacteria, and effects related to the donor used for growth. Inhibition in this mixed community is compared to inhibition in a pure-culture of the dominant SRM isolated from the community, Desulfovibrio sp. BMSR. In contrast to community-level experiments, pure culture D. sp. BMSR adapts to high concentrations of perchlorate./nThe third chapter of this dissertation represents work further exploring the ability of pure cultures of SRM to adapt to perchlorate stress. By passaging model SRM, Desulfovibrio alaskensis G20 in increasing concentrations of perchlorate we obtain a population capable of robust growth in 100 mM of the inhibitor. Whole genome-sequencing and comparison to the ancestral strain reveals a single base-pair substitution in the sulfate adenylyltransferase (sat) and biochemical analysis of the mutant Sat variant confirms its decreased susceptibility to perchlorate inhibition. Further adaptation experiments demonstrate the prevalence of this mutation across ten independently adapting populations of D. alaskensis G20. We also confirm that this mutation increases perchlorate resistance in another model SRM, Desulfovibrio vulgaris Hildenborough. Thus, sat represents a primary target of perchlorate stress and pure cultures of SRM can adapt to this stress via mutations in this critical enzyme./nPrevious chapters fail to detect adaptation to perchlorate in community-embedded SRM. Thus, the fourth chapter of this dissertation explores the adaptation sulfate-reducing communities to perchlorate. Laboratory evolution experiments generate communities capable of robust sulfidogenesis in 100 mM perchlorate. Using 16s rRNA amplicon sequencing, isolation, whole genome sequencing, and molecular and analytic techniques, we describe the ecological and evolutionary mechanisms of adaptation in these communities. In contrast to pure-culture adaptations, which target sat, we identify anion transport systems as primary targets of adaptation in community-embedded SRM faced with perchlorate inhibition./nIn an attempt to elucidate additional targets of specific inhibitors, chapter five of this dissertation again makes use of adaptive laboratory evolution in D. alaskensis G20. Specifically, we examine adaptations required for robust growth of D. alaskensis G20 under nitrate, monofluorophosphate and tungstate stress. This work highlights the importance of sulfate and other anion transport as a primary target of all sulfate analogs examined. Further targets of each inhibitor are addressed: monofluorophosphate stress necessitates overexpression of a cluster of genes likely involved in the turnover of fluorinated intermediates, and tungstate stress generates mutations in the sat, likely altering the specificity of this enzyme./nThe final sixth chapter of this dissertation summarizes conclusions arising from the above described studies and highlights future research needed for a thorough understanding of this ubiquitous microbial metabolism, and a wider application of SRM-specific inhibitors across areas of human and environmental health.

Advisor: Coates, John D