Hydrogen Oxidation Influences Glycogen Accumulation in a Verrucomicrobial Methanotroph

Abstract

Metabolic flexibility in aerobic methane oxidizing bacteria (methanotrophs) enhances cell growth and survival in instances where resources are variable or limiting. Examples include the production of intracellular compounds (such as glycogen or polyhydroxyalkanoates) in response to unbalanced growth conditions and the use of some energy substrates, besides methane, when available. Indeed, recent studies show that verrucomicrobial methanotrophs can grow mixotrophically through oxidation of hydrogen and methane gases via respiratory membrane-bound group 1d [NiFe] hydrogenases and methane monooxygenases, respectively. Hydrogen metabolism is particularly important for adaptation to methane and oxygen limitation, suggesting this metabolic flexibility may confer growth and survival advantages. In this work, we provide evidence that, in adopting a mixotrophic growth strategy, the thermoacidophilic methanotroph, Methylacidiphilum sp. RTK17.1 changes its growth rate, biomass yields and the production of intracellular glycogen reservoirs. Under nitrogen-fixing conditions, removal of hydrogen from the feed-gas resulted in a 14% reduction in observed growth rates and a 144% increase in cellular glycogen content. Concomitant with increases in glycogen content, the total protein content of biomass decreased following the removal of hydrogen. Transcriptome analysis of Methylacidiphilum sp. RTK17.1 revealed a 3.5-fold upregulation of the Group 1d [NiFe] hydrogenase in response to oxygen limitation and a 4-fold upregulation of nitrogenase encoding genes (nifHDKENX) in response to nitrogen limitation. Genes associated with glycogen synthesis and degradation were expressed constitutively and did not display evidence of transcriptional regulation. Collectively these data further challenge the belief that hydrogen metabolism in methanotrophic bacteria is primarily associated with energy conservation during nitrogen fixation and suggests its utilization provides a competitive growth advantage within hypoxic habitats.

Keywords: extremophile; glycogen; hydrogenase; methanotroph; methylacidiphilum.

FIGURE 1

The production of intracellular glycogen in chemostat grown cultures of Methylacidiphilum sp. RTK17.1 is influenced by O₂ availability, and H₂ and nitrogen metabolism. (A) Total protein, ash and glycogen content of cells, (B) observed growth rates and biomass yields (CDW: cell dry weight), and (C) amino acid content profiles are shown relative to total biomass. For all chemostat growth conditions, Methylacidiphilum sp. RTK17.1 excess CH₄ was continuously supplied (3% v/v, 10 ml min^{– 1}). Displayed values represent the average of minimum triplicate samples, with error bars illustrating the standard deviation. Significant differences in cellular glycogen and protein content are shown next to squared brackets (^****p-value < 0.0001, ^*p-value < 0.05).

FIGURE 2

Differential gene expression profiles of chemostat-grown cultures of Methylacidiphilum sp. RTK17.1 grown under O₂-replete and O₂-limiting conditions in the presence of ammonium (NH₄⁺) or under N₂-fixing conditions. (A) Volcano plot showing differential gene expression changes following the transition from oxygen replete to O₂-limited growth. (B) Volcano plot showing differential gene expression changes following the transition from nitrogen-replete (NH₄⁺) to N₂-fixing growth conditions. Both volcano plots compare data generated from the same five transcriptomes with fold-change values (log₂FC) and false discovery rates (FDR) calculated using O₂-replete and nitrogen excess as reference conditions, respectively. Each gene is represented by a gray dot and genes of interest are highlighted as per the legend. (C) Heat map of transcript abundance for key genes encoding the structural subunits of enzymes participating in methane oxidation (pmoBAC; particulate methane monooxygenase), methanol oxidation (xoxFJ; methanol dehydrogenase, pqqABCDE; pyrroloquinoline biosynthesis), formate oxidation (fdsDAB; formate dehydrogenase), carbon-dioxide fixation (cbbsSL; Rubisco), hydrogen metabolism (hyaBA and hyhSL; encoding group 1d and 3b [NiFe]-hydrogenases respectively), nitrogen fixation (nifXNEKDH; nitrogenase), and glycogen biosynthesis (glgAABCPPP1). The fragment counts per kilobase million transcripts (FPKM) are shown for steady-state cultures. O₂-replete, O₂-limiting, nitrogen-replete (NH₄⁺), N₂-fixing and the supplementation of H₂ into the feed gas during chemostat operation is indicated.