Genetic and Biochemical Analysis of Anaerobic Respiration in Bacteroides fragilis and Its Importance In Vivo

Bacteroides species are abundant in the human intestine and provide numerous beneficial properties to their hosts. The ability of Bacteroides species to convert host and dietary glycans and polysaccharides to energy is paramount to their success in the human gut. We know a great deal about the molecules that these bacteria extract from the human gut but much less about how they convert those molecules into energy. Here, we show that B. fragilis has a complex respiratory pathway with two different enzymes that transfer electrons from NADH to quinone and a third enzyme complex that may use an electron donor other than NADH. Although fermentation has generally been believed to be the main mechanism of energy generation in Bacteroides, we found that a mutant lacking one of the NADH:quinone oxidoreductases was unable to compete with the wild type in the mammalian gut, revealing the importance of respiration to these abundant gut symbionts.In bacteria, the respiratory pathways that drive molecular transport and ATP synthesis include a variety of enzyme complexes that utilize different electron donors and acceptors. This property allows them to vary the efficiency of energy conservation and to generate different types of electrochemical gradients (H+ or Na+). We know little about the respiratory pathways in Bacteroides species, which are abundant in the human gut, and whether they have a simple or a branched pathway. Here, we combined genetics, enzyme activity measurements, and mammalian gut colonization assays to better understand the first committed step in respiration, the transfer of electrons from NADH to quinone. We found that a model gut Bacteroides species, Bacteroides fragilis, has all three types of putative NADH dehydrogenases that typically transfer electrons from the highly reducing molecule NADH to quinone. Analyses of NADH oxidation and quinone reduction in wild-type and deletion mutants showed that two of these enzymes, Na+-pumping NADH:quinone oxidoreductase (NQR) and NADH dehydrogenase II (NDH2), have NADH dehydrogenase activity, whereas H+-pumping NADH:ubiquinone oxidoreductase (NUO) does not. Under anaerobic conditions, NQR contributes more than 65% of the NADH:quinone oxidoreductase activity. When grown in rich medium, none of the single deletion mutants had a significant growth defect; however, the double Δnqr Δndh2 mutant, which lacked almost all NADH:quinone oxidoreductase activity, had a significantly increased doubling time. Despite unaltered in vitro growth, the single nqr deletion mutant was unable to competitively colonize the gnotobiotic mouse gut, confirming the importance of NQR to respiration in B. fragilis and the overall importance of respiration to this abundant gut symbiont

Nanaerobic growth enables direct visualization of dynamic cellular processes in human gut symbionts

Mechanistic studies of anaerobic gut bacteria have been hindered by the lack of a fluorescent protein system to track and visualize proteins and dynamic cellular processes in actively growing bacteria. Although underappreciated, many gut "anaerobes" are able to respire using oxygen as the terminal electron acceptor. The oxygen continually released from gut epithelial cells creates an oxygen gradient from the mucus layer to the anaerobic lumen [L. Albenberg et al., Gastroenterology 147, 1055-1063.e8 (2014)], with oxygen available to bacteria growing at the mucus layer. Here, we show that; Bacteroides; species are metabolically and energetically robust and do not mount stress responses in the presence of 0.10 to 0.14% oxygen, defined as nanaerobic conditions [A. D. Baughn, M. H. Malamy, Nature 427, 441-444 (2004)]. Taking advantage of this metabolic capability, we show that nanaerobic growth provides sufficient oxygen for the maturation of oxygen-requiring fluorescent proteins in; Bacteroides; species. Type strains of four different; Bacteroides; species show bright GFP fluorescence when grown nanaerobically versus anaerobically. We compared four different red fluorescent proteins and found that mKate2 yields the highest red fluorescence intensity in our assay. We show that GFP-tagged proteins can be localized in nanaerobically growing bacteria. In addition, we used time-lapse fluorescence microscopy to image dynamic type VI secretion system processes in metabolically active; Bacteroides fragilis; The ability to visualize fluorescently labeled; Bacteroides; and fluorescently linked proteins in actively growing nanaerobic gut symbionts ushers in an age of imaging analyses not previously possible in these bacteria