PROJECT SUMMARY/ABSTRACT Bacteroidales is the most abundant order of bacteria in the human gut, yet we still know little about energy generating processes of these bacteria and how these systems support their fitness in the gut and affect host processes. We have demonstrated that Bacteroides have a complex respiratory chain that provides substantial energy during both anaerobic and nanaerobic growth. Our preliminary results reveal new and unexpected complexities of the respiratory pathway, new terminal electron acceptors that we predict contribute to bacterial and host fitness, and differences between Bacteroides species that likely impact microbiota composition. These important features and complexities of respiration in the gut Bacteroides will be addressed in three specific aims. In Aim 1, we will study the NUO complex, likely the most important generator of the proton gradient, which provides the energy for transport functions including TonB- dependent import processes such as acquisition of dietary polysaccharides. Unlike most NUO enzymes, Bacteroides has the NUO-11 variant that does not accept electrons from NADH. In this aim, we will use genetics, biochemistry, and mouse models to conclusively identify the electron donor to NUO-11, determine the importance of NUO and Na+/H+ antiporters in maintaining the essential proton gradient, and determine their contributions to bacterial fitness. In Aim 2, we will study the acquisition and remodeling of the essential respiratory 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 MK from dietary or microbial sources. This remodeling requires cleavage of the hydrophobic side chain by an unknown enzyme that also is likely necessary for the synthesis of MK-4 (Vitamin K2) by humans. We will explore unknown features of MK synthesis including identification and characterization of the enzyme that cleaves the isoprenoid chain in the remodeling process, how 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, expression of which is among the most upregulated during nanaerobic growth. We predict NrfHA is an additional terminal carrier that donates electrons to both nitrite and nitric oxide (NO). We predict NrfHA protects Bacteroides against NO produced in the normal and inflamed gut. We will study the regulation of the nrfHA operon during nanaerobic growth, study the ability of NrfHA to donate electrons to both nitrite and NO, and determine if this complex allows Bacteroides to better survive in the inflamed gut with concomitant protective effects for the host. The data obtained from the experiments of this proposal will reveal several new aspects of the physiology of the Bacteroides that can be translated for human health benefits.

PROJECT NARRATIVE Bacteroides species are prevalent and abundant in the human intestinal microbiota providing the host with numerous properties essential for health. In this proposal, we will examine new pathways and factors that these bacteria use to generate energy and their contribution to human health and disease prevention. These studies will ultimately support the design of better probiotic formulations and inform about factors important in the restoration of a healthy gut community to various patient populations.