Abstract
Archaeal methanogenesis is a dynamic process regulated by various cellular and environmental signals. However, understanding this regulation is technically challenging due to the difficulty of measuring gene expression dynamics in individual archaeal cells. Here, we develop a multi-round hybridization chain reaction (HCR)-assisted single-molecule fluorescence in situ hybridization (FISH) method to quantify the transcriptional dynamics of 12 genes involved in methanogenesis in individual cells of Methanococcoides orientis. Under optimal growth condition, most of these genes appear to be expressed in a temporal order matching metabolic reaction order. Interestingly, an important environmental factor, Fe(III), stimulates cellular methane production without upregulating methanogenic gene expression, likely through a Fenton-reaction-triggered mechanism. Through single-cell clustering and kinetic analyses, we associate these gene expression patterns to a dynamic mixture of distinct cellular states, potentially regulated by a set of shared factors. Our work provides a quantitative framework for uncovering the mechanisms of metabolic regulation in archaea.