Paul Rodrigo F. Cordero

Chemolithoautotrophic nitrite-oxidizing bacteria (NOB) of the genus Nitrospira contribute to nitrification in diverse natural environments and engineered systems. Nitrospira are thought to be well-adapted to substrate limitation owing to their high affinity for nitrite and capacity to use alternative energy sources. Here, we demonstrate that the canonical nitrite oxidizer Nitrospira moscoviensis oxidizes hydrogen (H ₂ ) below atmospheric levels using a high-affinity group 2a nickel-iron hydrogenase [ K _m(app) = 32 nM]. Atmospheric H ₂ oxidation occurred under both nitrite-replete and nitrite-deplete conditions, suggesting low-potential electrons derived from H ₂ oxidation promote nitrite-dependent growth and enable survival during nitrite limitation. Proteomic analyses confirmed the hydrogenase was abundant under both conditions and indicated extensive metabolic changes occur to reduce energy expenditure and growth under nitrite-deplete conditions. Respirometry analysis indicates the hydrogenase and nitrite oxidoreductase are bona fide components of the aerobic respiratory chain of N. moscoviensis , though they transfer electrons to distinct electron carriers in accord with the contrasting redox potentials of their substrates. Collectively, this study suggests atmospheric H ₂ oxidation enhances the growth and survival of NOB in amid variability of nitrite supply. These findings also extend the phenomenon of atmospheric H ₂ oxidation to a seventh phylum (Nitrospirota) and reveal unexpected new links between the global hydrogen and nitrogen cycles.

Carbon monoxide (CO) gas is infamous for its acute toxicity. This toxicity predominantly stems from its tendency to form carbonyl complexes with transition metals, thus inhibiting the heme-prosthetic groups of proteins, including respiratory terminal oxidases. While CO has been proposed as an antibacterial agent, the evidence supporting its toxicity toward bacteria is equivocal, and its cellular targets remain poorly defined. In this work, we investigate the physiological response of mycobacteria to CO. We show that Mycobacterium smegmatis is highly resistant to the toxic effects of CO, exhibiting only minor inhibition of growth when cultured in its presence. We profiled the proteome of M. smegmatis during growth in CO, identifying strong induction of cytochrome bd oxidase and members of the dos regulon, but relatively few other changes. We show that the activity of cytochrome bd oxidase is resistant to CO, whereas cytochrome bcc-aa₃ oxidase is strongly inhibited by this gas. Consistent with these findings, growth analysis shows that M. smegmatis lacking cytochrome bd oxidase displays a significant growth defect in the presence of CO, while induction of the dos regulon appears to be unimportant for adaptation to CO. Altogether, our findings indicate that M. smegmatis has considerable resistance to CO and benefits from respiratory flexibility to withstand its inhibitory effects. IMPORTANCE Carbon monoxide has an infamous reputation as a toxic gas, and it has been suggested that it has potential as an antibacterial agent. Despite this, how bacteria resist its toxic effects is not well understood. In this study, we investigated how CO influences growth, proteome, and aerobic respiration of wild-type and mutant strains of Mycobacterium smegmatis. We show that this bacterium produces the CO-resistant cytochrome bd oxidase to tolerate poisoning of its CO-sensitive complex IV homolog. Further, we show that aside from this remodeling of its respiratory chain, M. smegmatis makes few other functional changes to its proteome, suggesting it has a high level of inherent resistance to CO.

Carbon monoxide (CO) is a gas infamous for its acute toxicity. The toxicity of CO predominantly stems from its tendency to form carbonyl complexes with transition metals, thus inhibiting the heme-prosthetic groups of proteins, including the terminal oxidases of the respiratory chain. While CO has been proposed as an antibacterial agent, the evidence supporting its toxicity towards bacteria is equivocal, and its cellular targets remain poorly defined. In this work, we investigate the physiological response of mycobacteria to CO. We show that Mycobacterium smegmatis is highly resistant to the toxic effects of CO, exhibiting normal growth parameters when cultured in its presence. We profiled the proteome of M. smegmatis during growth in CO, identifying strong induction of cytochrome bd oxidase and members of the dos regulon, but relatively few other changes. We show that the activity of cytochrome bd oxidase is resistant to CO, whereas cytochrome bcc-aa ₃ oxidase is strongly inhibited by this gas. Consistent with these findings, growth analysis shows that M. smegmatis lacking cytochrome bd oxidase displays a significant growth defect in the presence of CO, while induction of the dos regulon appears to be unimportant for adaption to CO. Altogether, our findings suggest that M. smegmatis has considerable resistance to CO and benefits from respiratory flexibility to withstand its inhibitory effects. Importance: Carbon monoxide has an infamous reputation as a toxic gas and it has been suggested that it has potential as an antibacterial agent. Despite this, the means by which bacteria resist its toxic effects are not well understood. In this study we determine the physiological response of Mycobacterium smegmatis to growth in CO. We show for the first time that the cytochrome bd oxidase is inherently resistant to CO and is deployed by M. smegmatis to tolerate the presence of this gas. Further, we show that aside from this remodelling of its respiratory chain, M. smegmatis makes few other functional changes to its proteome, suggesting it has a high level of inherent resistance to CO.

F ₄₂₀ is a low-potential redox cofactor used by diverse bacteria and archaea. In mycobacteria, this cofactor has multiple roles, including adaptation to redox stress, cell wall biosynthesis, and activation of the clinical antitubercular prodrugs pretomanid and delamanid. A recent biochemical study proposed a revised biosynthesis pathway for F ₄₂₀ in mycobacteria; it was suggested that phosphoenolpyruvate served as a metabolic precursor for this pathway, rather than 2-phospholactate as long proposed, but these findings were subsequently challenged. In this work, we combined metabolomic, genetic, and structural analyses to resolve these discrepancies and determine the basis of F ₄₂₀ biosynthesis in mycobacterial cells. We show that, in whole cells of Mycobacterium smegmatis , phosphoenolpyruvate rather than 2-phospholactate stimulates F ₄₂₀ biosynthesis. Analysis of F ₄₂₀ biosynthesis intermediates present in M. smegmatis cells harboring genetic deletions at each step of the biosynthetic pathway confirmed that phosphoenolpyruvate is then used to produce the novel precursor compound dehydro-F ₄₂₀ -0. To determine the structural basis of dehydro-F ₄₂₀ -0 production, we solved high-resolution crystal structures of the enzyme responsible (FbiA) in apo, substrate, and product bound forms. These data show the essential role of a single divalent cation in coordinating the catalytic pre-complex of this enzyme and demonstrate that dehydro-F ₄₂₀ -0 synthesis occurs through a direct substrate transfer mechanism. Together, these findings resolve the biosynthetic pathway of F ₄₂₀ in mycobacteria and have significant implications for understanding the emergence of antitubercular prodrug resistance.

Aerobic soil bacteria persist by scavenging molecular hydrogen (H₂) from the atmosphere. This key process is the primary sink in the biogeochemical hydrogen cycle and supports the productivity of oligotrophic ecosystems. In Mycobacterium smegmatis, atmospheric H₂ oxidation is catalyzed by two phylogenetically distinct [NiFe]-hydrogenases, Huc (group 2a) and Hhy (group 1h). However, it is currently unresolved how these enzymes transfer electrons derived from H₂ oxidation into the aerobic respiratory chain. In this work, we used genetic approaches to confirm that two putative iron-sulfur cluster proteins encoded on the hydrogenase structural operons, HucE and HhyE, are required for H₂ consumption in M. smegmatis. Sequence analysis show that these proteins, while homologous, fall into distinct phylogenetic clades and have distinct metal-binding motifs. H₂ oxidation was reduced when the genes encoding these proteins were deleted individually and was eliminated when they were deleted in combination. In turn, the growth yield and long-term survival of these deletion strains was modestly but significantly reduced compared to the parent strain. In both biochemical and phenotypic assays, the mutant strains lacking the putative iron-sulfur proteins phenocopied those of hydrogenase structural subunit mutants. We hypothesize that these proteins mediate electron transfer between the catalytic subunits of the hydrogenases and the menaquinone pool of the M. smegmatis respiratory chain; however, other roles (e.g., in maturation) are also plausible and further work is required to resolve their role. The conserved nature of these proteins within most Hhy- or Huc-encoding organisms suggests that these proteins are important determinants of atmospheric H₂ oxidation.

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.

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 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 CO oxidation and conclude that, like atmospheric H ₂ , 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.

Bacteria within aerated environments often exist within a variety of dormant forms. In these states, bacteria endure adverse environmental conditions such as organic carbon starvation by decreasing metabolic expenditure and using alternative energy sources. In this study, we investigated the energy sources that facilitate the persistence of the environmentally widespread but understudied bacterial phylum Chloroflexi. A transcriptome study revealed that Thermomicrobium roseum (class Chloroflexia) extensively remodels its respiratory chain upon entry into stationary phase due to organic carbon limitation. Whereas primary dehydrogenases associated with heterotrophic respiration were downregulated, putative operons encoding enzymes involved in molecular hydrogen (H ₂ ), carbon monoxide (CO), and sulfur compound oxidation were significantly upregulated. Gas chromatography and microsensor experiments were used to show that T. roseum aerobically respires H ₂ and CO at a range of environmentally relevant concentrations to sub-atmospheric levels. Phylogenetic analysis suggests that the enzymes mediating atmospheric H ₂ and CO oxidation, namely group 1h [NiFe]-hydrogenases and type I carbon monoxide dehydrogenases, are widely distributed in Chloroflexi genomes and have been acquired on at least two occasions through separate horizontal gene transfer events. Consistently, we confirmed that the sporulating isolate Thermogemmatispora sp. T81 (class Ktedonobacteria) also oxidises atmospheric H ₂ and CO during persistence. This study provides the first axenic culture evidence that atmospheric CO supports bacterial persistence and reports the third phylum to be experimentally shown to mediate the biogeochemically and ecologically important process of atmospheric H ₂ oxidation. This adds to the growing body of evidence that atmospheric trace gases serve as dependable energy sources for the survival of dormant microorganisms.