Carbon monoxide (CO) is a ubiquitous atmospheric trace gas produced by natural and anthropogenic sources. Some aerobic bacteria can oxidize atmospheric CO and, collectively, they account for the net loss of ~250 teragrams of CO from the atmosphere each year. However, the physiological role, genetic basis, and ecological distribution of this process remain incompletely resolved. In this work, we addressed these knowledge gaps through culture-based and culture-independent work. We confirmed through shotgun proteomic and transcriptional analysis that the genetically tractable aerobic soil actinobacterium Mycobacterium smegmatis upregulates expression of a form I molydenum–copper carbon monoxide dehydrogenase by 50-fold when exhausted for organic carbon substrates. Whole-cell biochemical assays in wild-type and mutant backgrounds confirmed that this organism aerobically respires CO, including at sub-atmospheric concentrations, using the enzyme. Contrary to current paradigms on CO oxidation, the enzyme did not support chemolithoautotrophic growth and was dispensable for CO detoxification. However, it significantly enhanced long-term survival, suggesting that atmospheric CO serves a supplemental energy source during organic carbon starvation. Phylogenetic analysis indicated that atmospheric CO oxidation is widespread and an ancestral trait of CO dehydrogenases. Homologous enzymes are encoded by 685 sequenced species of bacteria and archaea, including from seven dominant soil phyla, and we confirmed genes encoding this enzyme are abundant and expressed in terrestrial and marine environments. On this basis, we propose a new survival-centric model for the evolution of aerobic CO oxidation and conclude that, like atmospheric H2, atmospheric CO is a major energy source supporting persistence of aerobic heterotrophic bacteria in deprived or changeable environments.

Diverse aerobic bacteria persist by consuming atmospheric hydrogen (H2) using group 1h [NiFe]-hydrogenases. However, other hydrogenase classes are also distributed in aerobes, including the group 2a [NiFe]-hydrogenase. Based on studies focused on Cyanobacteria, the reported physiological role of the group 2a [NiFe]-hydrogenase is to recycle H2 produced by nitrogenase. However, given this hydrogenase is also present in various heterotrophs and lithoautotrophs lacking nitrogenases, it may play a wider role in bacterial metabolism. Here we investigated the role of this enzyme in three species from different phylogenetic lineages and ecological niches: Acidithiobacillus ferrooxidans (phylum Proteobacteria), Chloroflexus aggregans (phylum Chloroflexota), and Gemmatimonas aurantiaca (phylum Gemmatimonadota). qRT-PCR analysis revealed that the group 2a [NiFe]-hydrogenase of all three species is significantly upregulated during exponential growth compared to stationary phase, in contrast to the profile of the persistence-linked group 1h [NiFe]-hydrogenase. Whole-cell biochemical assays confirmed that all three strains aerobically respire H2 to sub-atmospheric levels, and oxidation rates were much higher during growth. Moreover, the oxidation of H2 supported mixotrophic growth of the carbon-fixing strains C. aggregans and A. ferrooxidans. Finally, we used phylogenomic analyses to show that this hydrogenase is widely distributed and is encoded by 13 bacterial phyla. These findings challenge the current persistence-centric model of the physiological role of atmospheric H2 oxidation and extend this process to two more phyla, Proteobacteria and Gemmatimonadota. In turn, these findings have broader relevance for understanding how bacteria conserve energy in different environments and control the biogeochemical cycling of atmospheric trace gases.

18 19 Diverse aerobic bacteria persist by consuming atmospheric hydrogen (H2) using group 20 1h [NiFe]-hydrogenases. However, other hydrogenase classes are also distributed in 21 aerobes, including the group 2a [NiFe]-hydrogenase. Based on studies focused on 22 Cyanobacteria, the reported physiological role of the group 2a [NiFe]-hydrogenase is to 23 recycle H2 produced by nitrogenase. However, given this hydrogenase is also present in 24 various heterotrophs and lithoautotrophs lacking nitrogenases, it may play a wider role in 25 bacterial metabolism. Here we investigated the role of this enzyme in three species from 26 different phylogenetic lineages and ecological niches: Acidithiobacillus ferrooxidans 27 (phylum Proteobacteria), Chloroflexus aggregans (phylum Chloroflexota), and 28 Gemmatimonas aurantiaca (phylum Gemmatimonadota). qRT-PCR analysis revealed 29 that the group 2a [NiFe]-hydrogenase of all three species is significantly upregulated 30 during exponential growth compared to stationary phase, in contrast to the profile of the 31 persistence-linked group 1h [NiFe]-hydrogenase. Whole-cell biochemical assays 32confirmed that all three strains aerobically respire H2 to sub-atmospheric levels, and 33 oxidation rates were much higher during growth. Moreover, the oxidation of H2 supported 34 mixotrophic growth of the carbon-fixing strains C. aggregans and A. ferrooxidans. Finally, 35 we used phylogenomic analyses to show that this hydrogenase is widely distributed and 36 is encoded by 13 bacterial phyla. These findings challenge the current persistence-centric 37 model of the physiological role of atmospheric H2 oxidation and extends this process to 38 two more phyla, Proteobacteria and Gemmatimonadota. In turn, these findings have 39 broader relevance for understanding how bacteria conserve energy in different 40 environments and control the biogeochemical cycling of atmospheric trace gases. 41 42 43 Aerobic bacteria mediate the biogeochemically and ecologically important process of 44 atmospheric hydrogen (H2) oxidation [1]. Terrestrial bacteria constitute the largest sink 45 of this gas and mediate the net consumption of approximately 70 million tonnes of 46 atmospheric H2 per year [2, 3]. The energy derived from this process appears to be 47 critical for sustaining the productivity and biodiversity of ecosystems with low organic 48 carbon inputs [4-9]. Atmospheric H2 oxidation is thought to be primarily mediated by 49 group 1h [NiFe]-hydrogenases, a specialised oxygen-tolerant, high-affinity class of 50 hydrogenases [4, 10-13]. To date, aerobic heterotrophic bacteria from four distinct 51 bacterial phyla, the Actinobacteriota [10, 12, 14, 15], Acidobacteriota [16, 17], 52 Chloroflexota [18], and Verrucomicrobiota [19], have been experimentally shown to 53 consume atmospheric H2 using this enzyme. This process has been primarily linked 54 to energy conservation during persistence. Reflecting this, the expression and activity 55 of the group 1h hydrogenase is induced by carbon starvation across a wide...

19Carbon monoxide (CO) is a ubiquitous atmospheric trace gas produced by natural and 20 anthropogenic sources. Some aerobic bacteria can oxidize atmospheric CO and, 21 collectively, they account for the net loss of ~250 teragrams of CO from the 22 atmosphere each year. However, the physiological role, genetic basis, and ecological 23 distribution of this process remain incompletely resolved. In this work, we addressed 24 these knowledge gaps through culture-based and culture-independent work. We 25 confirmed through shotgun proteomic and transcriptional analysis that the genetically 26 tractable aerobic soil actinobacterium Mycobacterium smegmatis upregulates 27 expression of a carbon monoxide dehydrogenase by 50-fold when exhausted for 28 organic carbon substrates. Whole-cell biochemical assays in wild-type and mutant 29 backgrounds confirmed that this organism aerobically respires CO, including at sub-30 atmospheric concentrations, using the enzyme. Contrary to current paradigms on CO 31 oxidation, the enzyme did not support chemolithoautotrophic growth and was 32 dispensable for CO detoxification. However, it significantly enhanced long-term 33 survival, suggesting that atmospheric CO serves a supplemental energy source during 34 organic carbon starvation. Phylogenetic analysis indicated that atmospheric CO 35 oxidation is widespread and an ancestral trait of CO dehydrogenases. Homologous 36 enzymes are encoded by 685 sequenced species of bacteria and archaea, including 37 from seven dominant soil phyla, and we confirmed genes encoding this enzyme are 38 abundant and expressed in terrestrial and marine environments. On this basis, we 39 propose a new survival-centric model for the evolution of CO oxidation and conclude 40 that, like atmospheric H2, atmospheric CO is a major energy source supporting 41 persistence of aerobic heterotrophic bacteria in deprived or changeable environments.42 43 45 natural processes and anthropogenic pollution. The average global mixing ratio of this 46 gas is approximately 90 ppbv in the troposphere (lower atmosphere), though this 47 concentration greatly varies across time and space, with levels particularly high in 48 urban areas [1-4]. Currently, human activity is responsible for approximately 60% of 49 emissions, with the remainder attributable to natural processes [1]. Counteracting 50these emissions, CO is rapidly removed from the atmosphere (lifetime of two months) 51 by two major processes: geochemical oxidation by atmospheric hydroxyl radicals 52 (85%) and biological oxidation by soil microorganisms (10%) [1, 5]. Soil 53 microorganisms account for the net consumption of approximately 250 teragrams of 54 atmospheric CO [1, 5, 6]; on a molar basis, this amount is seven times higher than the 55 amount of methane consumed by soil bacteria [7]. Aerobic CO-oxidizing 56 microorganisms are also abundant in the oceans; while oceans are a minor source of 57 atmospheric CO overall [8,9], this reflects that substantial amounts of the gas are 58 produced photochemically within...