Growth on Formic Acid Is Dependent on Intracellular pH Homeostasis for the Thermoacidophilic Methanotroph Methylacidiphilum sp. RTK17.1

Abstract

Members of the genus Methylacidiphilum, a clade of metabolically flexible thermoacidophilic methanotrophs from the phylum Verrucomicrobia, can utilize a variety of substrates including methane, methanol, and hydrogen for growth. However, despite sequentially oxidizing methane to carbon dioxide via methanol and formate intermediates, growth on formate as the only source of reducing equivalents (i.e., NADH) has not yet been demonstrated. In many acidophiles, the inability to grow on organic acids has presumed that diffusion of the protonated form (e.g., formic acid) into the cell is accompanied by deprotonation prompting cytosolic acidification, which leads to the denaturation of vital proteins and the collapse of the proton motive force. In this work, we used a combination of biochemical, physiological, chemostat, and transcriptomic approaches to demonstrate that Methylacidiphilum sp. RTK17.1 can utilize formate as a substrate when cells are able to maintain pH homeostasis. Our findings show that Methylacidiphilum sp. RTK17.1 grows optimally with a circumneutral intracellular pH (pH 6.52 ± 0.04) across an extracellular range of pH 1.5–3.0. In batch experiments, formic acid addition resulted in no observable cell growth and cell death due to acidification of the cytosol. Nevertheless, stable growth on formic acid as the only source of energy was demonstrated in continuous chemostat cultures (D = 0.0052 h−1, td = 133 h). During growth on formic acid, biomass yields remained nearly identical to methanol-grown chemostat cultures when normalized per mole electron equivalent. Transcriptome analysis revealed the key genes associated with stress response: methane, methanol, and formate metabolism were differentially expressed in response to growth on formic acid. Collectively, these results show formic acid represents a utilizable source of energy/carbon to the acidophilic methanotrophs within geothermal environments. Findings expand the known metabolic flexibility of verrucomicrobial methanotrophs to include organic acids and provide insight into potential survival strategies used by these species during methane starvation.

Verrucomicrobial methanotrophs grow on diverse C3 compounds and use a homolog of particulate methane monooxygenase to oxidize acetone

“…infernorum” strain V4 and a mesophilic “Methylacidimicrobium” isolate, strain B4 (Table S2 ). Strains IT5 and IT6 were able to grow on methane, methanol, and formate (at pH 4.5, which is above formate pKa, 3.8) [ 41 ] as energy sources, and their growth was highly dependent on CO 2 supplementation (Fig. S4A ), as previously observed in other “Methylacidiphilum” strains [ 9 , 13 , 41 , 72 , 73 ].…”

“…Strains IT5 and IT6 were able to grow on methane, methanol, and formate (at pH 4.5, which is above formate pKa, 3.8) [ 41 ] as energy sources, and their growth was highly dependent on CO 2 supplementation (Fig. S4A ), as previously observed in other “Methylacidiphilum” strains [ 9 , 13 , 41 , 72 , 73 ]. Propane or ethane (2 or 20%, v/v) alone did not support growth in either oxygen-replete or oxygen-limiting conditions, as expected for obligate methanotrophs [ 74 ].…”

“…RNA-sequencing results revealed that the expression levels of the pmoCAB1-2 operons in various “Methylacidiphilum” strains were tightly regulated in response to oxygen concentration, whereas the expression level of the most divergent paralog, pmoCAB3 , was very low or undetectable under methanotrophic growth conditions [ 38 – 40 ]. Recently, upregulation of pmoCAB3 in “Methylacidiphilum fumariolicum” SolV and “Methylacidiphilum sp.” RTK17.1 was observed in chemostat cultures grown on a mixture of methanol and propane [ 8 ] or on formic acid in the absence of methane [ 41 ], respectively. These results suggest that PMO3 plays some role in alkane degradation, although the precise nature of this role is not clear.…”

A Large-Scale Genome-Based Survey of Acidophilic Bacteria Suggests That Genome Streamlining Is an Adaption for Life at Low pH

“…The predominancy of autotrophic metabolism in acidophiles could result in an inclination of these organisms toward the biosynthesis of amino acids rather than uptake by active transporters. Additionally, uptake of amino acids could be harmful to acidophiles as organic acids carry protons into the cytoplasm of these organisms, thus short-circuiting acid resistance mechanisms ( Kishimoto et al, 1990 ; Lehtovirta-Morley et al, 2014 ; Carere et al, 2021 ). The current hypothesis is that organic acids are protonated in the extremely acid medium where acidophiles grow (pH < 3), becoming non-ionic and soluble in bacterial membranes and permitting diffusion into the cytoplasm where they uncouple from the proton.…”

Methanotrophs are vigorous H2S oxidizers using a sulfide:quinone oxidoreductase and a ba3-type terminal oxidase

“…Indeed, it is proposed that these hydrogenases can have sulfhydrogenase activity, which could be a mechanism to dispose of reducing equivalents (Ma et al, 1993; Søndergaard et al, 2016). Hence, the group 3b [NiFe] hydrogenase of M. fumariolicum SolV might be responsible for the conversion of S 0 to H 2 S. Culturing in a continuous culture again proved to be a very powerful tool to investigate the metabolism of methanotrophs (Mohammadi et al, 2017; Carere et al, 2021). Through adaption, M. fumariolicum SolV was able to respire H 2 S at a rate five times higher than non-adapted cells, presumably due to the upregulation of SQR and the ba 3 -type terminal oxidase.…”