In the near future, we will set our sights on Mars, and even far more distant planets for explorations. As one of the essential prerequisites for colonizing on these planets, we first need to optimize the soil and increase the oxygen content to make its environment suitable for the growth of crops.

Clostridium tyrobutyricum, as a novel microbial preparation, is an obligate anaerobic bacterium, in order to make it survive long-term microaerobic events, we try to transform it into facultative anaerobic bacteria, improve its oxygen tolerance, so that it can perform functions in both aerobic and anaerobic environments.

It becomes more and more obvious that molecular oxygen itself is the harmful or lethal agent for these bacteria. More recent approaches suggested that obligate anaerobiosis is tightly linked to the central metabolism of these organ-isms.

Microorganisms occupy their particular habitats because their central metabolism is well- suited to degrading nutrients that are found there; a reciprocal hypothesis is that anaerobes cannot occupy fully aerobic habitats because O₂ is somehow incompatible with their core metabolic strategies.

Those strategies, of course, are diverse. Anaerobic respiration is a catch- all phrase that denotes electron transfer from a range of reductants (for example, molecular hydrogen, lactic acid and hydrogen sulfide) to a similarly broad range of acceptors (for example, carbon dioxide, sulfate and ferric iron).

The σB regulon and its role in the control of O₂ tolerance In most Firmicutes, a key actor of the general stress response is the sigma factor, σB, which provides protection to many types of stress and starvation conditions. These functions are important for maintenance of the metabolic

The Evolution of Defenses against ROS: Scavenging Systems.

The growing oxidative stress necessitated the evolution of antioxidant systems. The earliest forms of these systems may have been small molecules and metals such as manganese sulfur complexes and carotenoids some of these remain important parts of the antioxidant systems in prokaryotes and eukaryotes.

Stronger defenses arose with the evolution of enzymes that directly degrade O₂– and H₂O₂ In many bacteria, the [4Fe-4S] cofactors of dehydratases and the Fe(II) cofactors of a variety of enzymes are important targets of O₂– and H₂O₂ .

Microbes have evolved a variety of strategies to cope with this hazard, including the replacement of iron with manganese in mononuclear enzymes. Lactic acid bacteria which generate H₂O₂ as a routine by-product of their aerobic metabolism – routinely populate these enzymes with manganese.

After the GOE, manganese was more bioavailable than iron, and it is striking that it was recruited into manganese forms of SOD, catalase, and ribonucleotide reductase. The gradual displacement of iron by manganese may have been a broad feature of evolution after atmospheric oxygenation.

The third known threat of ROS stems from the reaction of H₂O₂ with the cytoplasmic pool of loose ferrous iron. Microbes lack polyunsaturated fatty acids and are thereby spared the lipid peroxidation that plagues eukaryote; however, their DNA is damaged, and it seems likely that this threat imposed pressure for the evolution of DNA-repair enzymes. During H₂O₂ stress an additional miniferritin, Dps, is induced and uses H₂O₂ itself as the co-reactant; the effect is to shrink the loose-iron pool and thereby avert Fenton chemistry.

Notably, these diverse defenses – scavenging enzymes, suppressed ROS formation, cluster repair, modified dehydratases, manganese import, DNA repair enzymes, ferritins and Dps – are all found in the same cells, demonstrating that nature evolved layers of tactics to suppress ROS stress.

Deletion of a peroxide repressor (perR)-homologous protein resulted in prolonged aerotolerance, limited growth under aerobic conditions and rapid consumption of oxygen from an aerobic environment.Several genes encoding the putative enzymes were upregulated and identified as members of the clostridial perR regulon, including the heat shock protein Hsp21, a reverse rubrerythrin which was massively produced and became the most abundant protein in the absence of perR.This multifunctional protein is proposed to play the crucial role in the oxidative stress defence.

Survival of C. acetobutylicum under oxidative stress. After anaerobic cultivation to an OD₆₀₀ of 0.6, exponentially growing cells of C. acetobutylicum perR+ (circles), C. acetobutylicum perR - (squares) or the complemented C. acetobutylicum DperR-perR+ (triangles) were incubated either (A) with different concentrations of H₂O₂ for 30 min, or (B) aerobically at 35°C and 200 r.p.m. on a rotary shaker. Survival of C. acetobutylicum perR+ in the presence of the iron chelator dipyridyl, and of C. acetobutylicum perR - at 100-fold lower cell density is indicated by open circles and open squares respectively. Samples were drawn at the indicated time points and the cfu were determined according to a non-stressed control (100%). Data represent the mean value of three independent experiments.

Hillmann F, Fischer RJ, Saint-Prix F, Girbal L, Bahl H. PerR acts as a switch for oxygen tolerance in the strict anaerobe Clostridium acetobutylicum. Mol Microbiol. 2008 May;68(4):848-60. doi: 10.1111/j.1365-2958.2008.06192.x. PMID: 18430081.

Khademian M, Imlay JA. How Microbes Evolved to Tolerate Oxygen. Trends Microbiol. 2021 May;29(5):428-440. doi: 10.1016/j.tim.2020.10.001. Epub 2020 Oct 24. PMID: 33109411; PMCID: PMC8043972.

Lu Z, Imlay JA. When anaerobes encounter oxygen: mechanisms of oxygen toxicity, tolerance and defence. Nat Rev Microbiol. 2021 Dec;19(12):774-785. doi: 10.1038/s41579-021-00583-y. Epub 2021 Jun 28. PMID: 34183820; PMCID: PMC9191689.