When anaerobes encounter oxygen: mechanisms of oxygen toxicity, tolerance and defence

Abstract

The defining trait of obligate anaerobes is that oxygen blocks their growth, yet the underlying mechanisms are unclear. A popular hypothesis was that these microorganisms failed to evolve defences to protect themselves from reactive oxygen species (ROS) such as superoxide and hydrogen peroxide, and that this failure is what prevents their expansion to oxic habitats. However, studies reveal that anaerobes actually wield most of the same defences that aerobes possess, and many of them have the capacity to tolerate substantial levels of oxygen. Therefore, to understand the structures and real-world dynamics of microbial communities, investigators have examined how anaerobes such as Bacteroides, Desulfovibrio, Pyrococcus and Clostridium spp. struggle and cope with oxygen. The hypoxic environments in which these organisms dwell - including the mammalian gut, sulfur vents and deep sediments - experience episodic oxygenation. In this Review, we explore the molecular mechanisms by which oxygen impairs anaerobes and the degree to which bacteria protect their metabolic pathways from it. The emergent view of anaerobiosis is that optimal strategies of anaerobic metabolism depend upon radical chemistry and low-potential metal centres. Such catalytic sites are intrinsically vulnerable to direct poisoning by molecular oxygen and ROS. Observations suggest that anaerobes have evolved tactics that either minimize the extent to which oxygen disrupts their metabolism or restore function shortly after the stress has dissipated.

Fig. 1. Lifestyles of anaerobes.

Obligate anaerobes are principal occupants of diverse environments that have limited rates of convective oxygenation. Natural systems such as the intestinal tract, rumen, buried sediments and hot springs have slow oxygen (O₂) entry and rapid consumption by peripheral microbes, thereby establishing hypoxic or anoxic conditions.

Fig. 2. O₂-dependent respiration in anaerobes.

Two types of oxygen (O₂)-directed respiration are found in anaerobes. The membrane-bound cytochrome bd oxidase (Cyd) receives electrons from menaquinone (MQ) and upstream dehydrogenases; and the soluble rubredoxin:oxygen oxidoreductase (ROO) obtains electrons from reduced rubredoxin. NDH, NADH dehydrogenase; NROR, NADH:rubredoxin oxidoreductase; Rd, rubredoxin.

Fig. 3. O₂ inactivates pyruvate formate-lyase.

a | Pyruvate formate-lyase (PFL) catalyses pyruvate breakdown without producing NADH; this strategy is energetically economical because it avoids consuming acetyl-CoA to reoxidize NADH. Acetyl-CoA is then available for ATP synthesis (not shown). b | PFL is a prototype of the family of glycyl-radical enzymes. The glycyl radical is produced as a post-translational modification by PFL activating enzyme (activase), which activates PFL through a S-denosylmethionine (SAM)-dependent reaction, in which the monoelectronic reduction of SAM generates a 5-deoxyadenosyl radical (5′-dA) and methionine as by-products. Oxygen (O₂) inactivates PFL by adducting the glycyl radical, forming a hydroperoxyl radical species that finally cleaves the polypeptide.

Fig. 4. Structures of O₂-sensitive metalloenzymes.

Molecular oxygen (O₂) directly inactivates diverse metalloenzymes featuring low-potential metal centres at or near the protein surface. The structures of representative O₂-sensitive metalloenzymes are shown (left), alongside their pivotal positions in anaerobic metabolism (right): aconitase B from Escherichia coli (Protein Data Bank (PDB) ID 1L5J); pyruvate:ferredoxin oxidoreductase (PFOR) from Moorella thermoacetica (PDB ID 6CIN); iron-only hydrogenase (Hyd) from Clostridium pasteurianum (PDB ID 1FEH); and methyl-coenzyme M reductase (MCR) from Methanothermobacter marburgensis Marburg (PDB ID 1MRO). Iron–sulfur clusters are represented by yellow and red balls, nickel-containing F430 of MCR is shown in red with nickel in green. CoM-SH, coenzyme M; CoB-SH, coenzyme B; Fd^ox, oxidized ferredoxin; Fd^red, reduced ferredoxin.

Fig. 5. Metabolism in Bacteroides spp. is blocked upon aeration and resumes in anoxia.

Oxygen (O₂) poisoning results from inactivation of several enzymes. a | In aerated cells, endogenous O₂⁻ inactivates fumarase in the redox-balancing succinate branch of central metabolism. At the same time, molecular O₂ directly inactivates both pyruvate formate-lyase (PFL) and pyruvate:ferredoxin oxidoreductase (PFOR), creating a bottleneck in the pathway leading to acetate. Either block is sufficient to prohibit growth. Enzymes indicated in square brackets. b | Cell viability is not lost during the period of O₂ exposure. When anoxia is restored, growth soon resumes (drop in OD₆₀₀ reflects dilution into anoxic medium). Fd^ox, oxidized ferredoxin; Fd^red, reduced ferredoxin; FRD, fumarate reductase; Fum, fumarase; Hyd, hydrogenase; OAA, oxaloacetic acid; PEP, phosphoenolpyruvate. Part b reprinted from REF., CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/).