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.

Most aerobic bacteria exist in dormant states within natural environments. In these states, they endure adverse environmental conditions such as nutrient starvation by decreasing metabolic expenditure and using alternative energy sources. In this study, we investigated the energy sources that support persistence of two aerobic thermophilic strains of the environmentally widespread but understudied phylum Chloroflexi. A transcriptome study revealed that Thermomicrobium roseum (class Chloroflexia) extensively remodels its respiratory chain upon entry into stationary phase due to nutrient limitation. Whereas primary dehydrogenases associated with heterotrophic respiration were downregulated, putative operons encoding enzymes involved in molecular hydrogen (H2), carbon monoxide (CO), and sulfur compound oxidation were significantly upregulated. Gas chromatography and microsensor experiments showed that T. roseum aerobically respires H2 and CO at a range of environmentally relevant concentrations to sub-atmospheric levels. Phylogenetic analysis suggests that the hydrogenases and carbon monoxide dehydrogenases mediating these processes are widely distributed in Chloroflexi genomes and have probably been horizontally acquired on more than one occasion. Consistently, we confirmed that the sporulating isolate Thermogemmatispora sp. T81 (class Ktedonobacteria) also oxidises atmospheric H2 and CO during persistence, though further studies are required to determine if these findings extend to mesophilic strains. This study provides axenic culture evidence that atmospheric CO supports bacterial persistence and reports the third phylum, following Actinobacteria and Acidobacteria, to be experimentally shown to mediate the biogeochemically and ecologically important process of atmospheric H2 oxidation. This adds to the growing body of evidence that atmospheric trace gases are dependable energy sources for bacterial persistence.

23Bacteria within aerated environments often exist within a variety of dormant forms. In 24 these states, bacteria endure adverse environmental conditions such as organic 25 carbon starvation by decreasing metabolic expenditure and using alternative energy 26 sources. In this study, we investigated the energy sources that facilitate the 27 persistence of the environmentally widespread but understudied bacterial phylum 28 Chloroflexi. A transcriptome study revealed that Thermomicrobium roseum (class 29 Chloroflexia) extensively remodels its respiratory chain upon entry into stationary 30 phase due to organic carbon limitation. Whereas primary dehydrogenases associated 31 with heterotrophic respiration were downregulated, putative operons encoding 32 enzymes involved in molecular hydrogen (H2), carbon monoxide (CO), and sulfur 33 compound oxidation were significantly upregulated. Gas chromatography and 34 microsensor experiments were used to show that T. roseum aerobically respires H2 35 and CO at a range of environmentally relevant concentrations to sub-atmospheric 36 levels. Phylogenetic analysis suggests that the enzymes mediating atmospheric H2 37 and CO oxidation, namely group 1h [NiFe]-hydrogenases and type I carbon monoxide 38 dehydrogenases, are widely distributed in Chloroflexi genomes and have been 39 acquired on at least two occasions through separate horizontal gene transfer events. 40Consistently, we confirmed that the sporulating isolate Thermogemmatispora sp. T81 41(class Ktedonobacteria) also oxidises atmospheric H2 and CO during persistence. This 42 study provides the first axenic culture evidence that atmospheric CO supports bacterial 43 persistence and reports the third phylum to be experimentally shown to mediate the 44 biogeochemically and ecologically important process of atmospheric H2 oxidation. This 45 adds to the growing body of evidence that atmospheric trace gases serve as 46 dependable energy sources for the survival of dormant microorganisms. 47 48 49 Bacteria from the phylum Chloroflexi are widespread and abundant in free-living 50 microbial communities [1-4]. One reason for their success is their metabolic diversity; 51 cultured strains from the phylum include heterotrophs, lithotrophs, and phototrophs 52 adapted to both oxic and anoxic environments [5]. Cultured representatives of the 53 phylum are classified into four classes by the genome taxonomy database [6], the 54 primarily aerobic Chloroflexia and Ktedonobacteria and the anaerobic Anaerolineae 55 and Dehalococcoidia [5]. Numerous studies have provided insight into the metabolic 56 strategies anaerobic classes within Chloroflexi use to adapt to oligotrophic niches [7, 57 8]. Surprisingly little, however, is known about how aerobic heterotrophic bacteria 58 within this phylum colonise oxic environments. Global surveys have reported that 59 Chloroflexi comprise 4.3% of soil bacteria [2] and 3.2% of marine bacteria [3]. However, 60 the most dominant lineages within these ecosystems (notably Ellin6524 and SAR202) 61 have proven...

To persist when nutrient sources are limited, aerobic soil bacteria metabolize atmospheric hydrogen (H 2 ). This process is the primary sink in the global H 2 cycle and supports the productivity of microbes in oligotrophic environments. H 2 -metabolizing bacteria possess [NiFe] hydrogenases that oxidize H 2 to subatmospheric concentrations. The soil saprophyte Mycobacterium smegmatis has two such [NiFe] hydrogenases, designated Huc and Hhy, that belong to different phylogenetic subgroups. Both Huc and Hhy are oxygen-tolerant, oxidize H 2 to subatmospheric concentrations, and enhance bacterial survival during hypoxia and carbon limitation. Why does M. smegmatis require two hydrogenases with a seemingly similar function? In this work, we resolved this question by showing that Huc and Hhy are differentially expressed, localized, and integrated into the respiratory chain. Huc is active in late exponential and early stationary phases, supporting energy conservation during mixotrophic growth and transition into dormancy.In contrast, Hhy is most active during long-term persistence, providing energy for maintenance processes following carbon exhaustion. We also show that Huc and Hhy are obligately linked to the aerobic respiratory chain via the menaquinone pool and are differentially affected by respiratory uncouplers. Consistently, these two enzymes interacted differentially with the respiratory terminal oxidases. Huc exclusively donated electrons to, and possibly physically associated with, the proton-pumping cytochrome bcc-aa 3 supercomplex. In contrast, the more promiscuous Hhy also provided electrons to the cytochrome bd oxidase complex. These results indicate that, despite their similar characteristics, Huc and Hhy perform distinct functions during mycobacterial growth and survival.Earth's soils consume vast amounts of H 2 from the atmosphere (1, 2). Over the past decade, research by a number of This work was supported by Australian Research Council DECRA Fellowship DE170100310 (to C. G.), National Health and Medical Research Council New Investigator Grant APP5191146 (to C. G.), a Monash University Science-Medicine seed grant (to C. G. and M. J. C.), and a Monash University doctoral scholarship (to P. R. F. C.). The authors declare that they have no conflicts of interest with the contents of this article. This article contains Figs. S1 and S2 and Tables S1-S5. The raw mass spectrometry data have been deposited in Figshare with accession numbers F1CH20190327_WT_Top.raw and F1CH20190327_WT_down.raw.

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