Sunday, November 24, 2024
11/24/2024
Project Summary/Abstract Despite the tremendous amount of data that has been generated over the last 15 years regarding the human intestinal microbiota, we still know relatively little about energy generation processes of most of the abundant members of this ecosystem, including the Bacteroides. Our gap in this fundamental knowledge hinders our understanding of interactions between microbial members, how certain conditions in the gut environment affect microbial compositional changes, and how we may appropriately alter the composition of the ecosystem to improve human health. Bacteroides is one of the most abundant and stable bacterial genera of the human intestinal microbiota with strains colonizing their hosts for decades. During the first funding cycle of this project, we demonstrated that Bacteroides have a complex respiratory chain that provides substantial energy during both anaerobic and nanaerobic (0.1-0.15% O2) growth. Our studies revealed complexity at many steps in the respiration pathway and unexpected differences between Bacteroides species that will be pursued in this renewal application. These new aspects of respiration will be addressed in three specific aims. In Aim 1, we will study the important NUO complex that couples the transfer of electrons to menaquinone with creation of the proton gradient. The Bacteroides NUO complex is different than in most studied bacteria. In this aim, we will use genetics and biochemical analyses to conclusively identify the electron donor to NUO, determine the importance of NUO and Na+/H+ antiporters in maintaining the essential proton gradient, and determine the contribution of NUO and Na+/H+ antiporters to bacterial fitness in the mammalian gut. In Aim 2, we will study the acquisition and remodeling of the essential respiration component, menaquinone (MK). Most Bacteroides have all the genes necessary for the de novo synthesis of MK; however, certain Bacteroides species lack the primary men genes and must obtain and remodel dietary and/or microbial sources. We will explore unknown features of MK synthesis including how the isoprenoid chain is cleaved and remodeled, how Bacteroides species without the men operon obtain MK precursors, and the dietary and/or microbiota sources of these precursors. In Aim 3, we will study the NrfHA complex, the genes of which are among the most upregulated during nanaerobic growth. We predict NrfHA is an additional terminal electron donor that functions under nanaerobic conditions and donates electrons to both nitrite and nitric oxide (NO), detoxifying these molecules. We will study the regulation of the nrfHA operon revealing transcriptional factors that upregulate genes during nanaerobic growth, study its ability to donate electrons to both nitrite and NO, and determine if this complex allows Bacteroides to reduce host inflammation and better survive in the inflamed gut. The experiments of this proposal will reveal numerous aspects of basic physiology of the Bacteroides that can be translated for human health benefits.
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