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

Abstract

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. IMPORTANCE 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.

Metabolism of Soy Isoflavones by Intestinal Bacteria: Genome Analysis of an Adlercreutzia equolifaciens Strain That Does Not Produce Equol

“…The qPCR strategy targeting the tdr gene, however, did not result in the identification of any equol-producing isolate, even though 73 of them were initially considered as possibly positive. In agreement with their obligate anaerobic nature, most intestinal species possess large numbers of reductase-encoding genes [ 48 ]. As an example, 166 ORFs have been annotated as reductase-encoding genes in the genome of A. equolifaciens W18.34a [ 38 ].…”

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

“…The frd genes (BF638R_4499-4501) were deleted from the B. fragilis 638R (TM4000) genome by the following procedure. Regions flanking the deletion were PCR amplified with Phusion polymerase (NEB) and cloned directionally into the BamHI site of pLGB36 (49) using NEBuilder (NEB) and transformed into E. coli S17 λ pir. The plasmid was then conjugally transferred from E. coli to B. fragilis and cointegrates were selected on BHIS erythromycin plates.…”

Gene duplication drives genome expansion in Thaumarchaeota

“…The evolution of LA to the last common ancestor of Nitrosopumilales (LNP) involved gain of ubiquinone-dependent (EC:7.1.1.2) and ubiquinone-independent (EC:1.6.99.3) NADH dehydrogenases (Table S11 and S12), giving LNP all three families of respiratory NADH dehydrogenases, which regenerate NAD+ from NADH by catalysing the transfer of electrons from NADH to coenzyme Q10 in oxidative phosphorylation. The advantage of encoding all three families remains largely unknown 34-36 , but could provide a conservation of respiratory function under changing metabolic conditions.…”