The gut anaerobe Faecalibacterium prausnitzii uses an extracellular electron shuttle to grow at oxic-anoxic interphases

Abstract

Faecalibacterium prausnitzii is one of the most abundant bacteria in the human gut ecosystem and it is an important supplier of butyrate to the colonic epithelium. Low numbers of faecalibacteria have been associated with inflammatory bowel disease. Despite being extremely oxygen sensitive, F. prausnitzii is found adherent to the gut mucosa where oxygen diffuses from epithelial cells. This paradox is now explained on the basis of gas tube experiments, flavin-dependent reduction of 5,5'-dithiobis-2-nitrobenzoate and microbial fuel cell experiments. The results show that F. prausnitzii employs an extracellular electron shuttle of flavins and thiols to transfer electrons to oxygen. Both compounds are present in the healthy human gut. Our observations may have important implications for the treatment of patients with Crohn's disease, for example, with flavin- or antioxidant rich diets, and they provide a novel key insight in host-microbe interactions at the gut barrier.

Figures

Figure 1

Presumed pathways for the production of butyrate and other short-chain fatty acids from the glucose utilized by F. prausnitzii strains. The diagram also depicts the net acetate consumption and cycling of general intracellular electron carriers (blue). Adapted from diagrams described by Herrmann et al. (2008) and by Seedorf et al. (2008). An alternative pathway for the use of fumarate as terminal electron acceptor and carbon source is indicated in the grey box with red dotted arrows. The colour reproduction of this figure is available at the ISME Journal online.

Figure 2

Growth of anaerobic gut bacteria after 16 h in agar medium with and without an oxygenated gas phase. (a) Gas tube experiments are based on glass tubes with two gas phases at the opposite ends of an agar medium plug. The gas phase at the top is composed of ∼5 ml oxygen-containing air and the gas phase at the bottom is an anaerobic gas- mixture of ∼81% N2, ∼11% H2 and ∼8% CO2. (b) F. prausnitzii A2–165 showing enhanced growth in a rim at the oxygenated end and moderate growth in small colonies throughout the rest of the agar plug. (c) R. inulinivorans A2–194 showing growth in colonies throughout the agar plug except at the oxygenated zone. (d) Un-inoculated medium showing a color change of resazurin due to oxygen diffusion. (e) Schematic interpretation of the gas tube oxygen gradient on the human gut lumen and mucosal layer (Van den Abbeele et al., 2011). F. prausnitzii can be found in the transition zone or adherent to gut-mucosa (Swidsinski et al., 2008).

Figure 3

The faecalibacterial extracellular flavin-thiol electron shuttle. (a) Bacterial cell, flavin–thiol electron shuttle experiment. In this experiment the bacterial cells (schematically represented) reduce riboflavin with the help of intracellular electron carriers (see Figure 1). The electrons are subsequently transferred from riboflavin to the disulfide bond in DTNB, resulting in the yellow-colored compound NTB−. No yellow color is formed when the cells are incubated with DTNB in the absence of riboflavins. (b) Model for extracellular electron transfer to oxygen via a flavin–thiol redox shuttle. In the absence of terminal electron acceptor, reducing equivalents are used to produce SCFAs such as lactate, formate or butyrate. If fumarate is available during anaerobic growth, a membrane-associated fumarate reductase transfers electrons to fumarate, resulting in the production of succinate.

Figure 4

(a) Biomass and organic acids produced or acetate consumed (mmoles) per mmole of glucose by two different strains of F. prausnitzii. The strains were cultivated in YCFAG medium with a nitrogen or oxygen gas phase. Alternatively, fumarate was added to the medium of the strains cultivated with a nitrogen head. Note that both oxygen and fumarate can serve as terminal electron acceptors. (b) Oxygen or fumarate consumption per mmol of glucose, and succinate production by two isolates of F. prausnitzii. The data presented here is the mean of three independent replicates.

Figure 5

F. prausnitzii requires riboflavin for electron transfer to the anode of a microbial fuel cell. The diagram shows current production profiles for the human gut bacteria F. prausnitzii, B. fragilis and R. inulinivorans. (I) Bacterial cells were added to the anode chamber containing potassium phosphate buffer (50 mM) and glucose (0.1 M). A voltage of −100 mV versus Ag/AgCl reference electrode was applied and the current was measured at 30 s intervals. (II) After 10 min of incubation at 37 °C, 200 μM of riboflavin was injected as an extracellular redox mediator into the anode chamber. Under these conditions only F. prausnitzii generates a measurable current wave.