Fengping Wang’s research while affiliated with Shanghai Jiao Tong University and other places

Inward membrane budding, i.e., the bending of membrane towards the cytosol, is essential for forming and maintaining eukaryotic organelles. In eukaryotes, Arf GTPases initiate this inward budding. Our research shows that Asgard archaea genomes encode putative Arf proteins (AArfs). AArfs possess structural elements characteristic of their eukaryotic counterparts. When expressed in yeast and mammalian cells, some AArfs displayed GTP-dependent membrane targeting. In vitro, AArf associated with both eukaryotic and archaeal membranes. In yeast, AArfs interacted with and were regulated by key organelle biogenesis players. Expressing an AArf led to a massive proliferation of endomembrane organelles including the endoplasmic reticulum and Golgi. This AArf interacted with Sec23, a COPII vesicle coat component, in a GTP-dependent manner. These findings suggest certain AArfs are membrane-associating molecular switches with the functional potential to initiate organelle biogenesis, and the evolution of a functional coat could be the next critical step towards establishing eukaryotic cell architecture.

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.

Enrichment cultures of archaea and bacteria performing the anaerobic oxidation of methane (AOM) regularly contain persistent methanogens. Here, we isolated the marine methanogen Methanococcoides cohabitans sp. nov. strain LMO-2 T from a long-term AOM enrichment culture from the Northern Gulf of Mexico. Strain LMO-2 T is Gram-stain-negative, irregular 0.5–1 µm coccus without flagella. It utilizes a variety of methylated compounds including methanol, monomethylamine, dimethylamine and trimethylamine for growth and methanogenesis. However, it does not grow on formate, acetate, dimethyl sulphate, H 2 /CO 2 , betaine and choline. The optimal conditions for growth were observed within a temperature range of 30–35 °C, a pH range of 7.0–8.0 and a salinity range of 2–4% NaCl. Based on the similarity and phylogeny of the 16S rRNA gene and genomic sequence, strain LMO-2 T is classified within the genus Methanococcoides . Among the isolated type strains of the genus, strain LMO-2 T exhibited the highest 16S rRNA gene sequence identity with Methanococcoides vulcani SLH33 T (99.4%). The digital DNA–DNA hybridization and average nucleotide identity based on genome sequence showed that strain LMO-2 T shared the highest similarity with Methanococcoides orientis LMO-1 T , with values of 27.3% and 83.4%, respectively. In conclusion, we isolated a methylotrophic methanogen from an AOM culture, and the isolated strain LMO-2 T represented a novel species of the genus Methanococcoides , for which the name Methanococcoides cohabitans sp. nov. is proposed. The type strain is LMO-2 T (=CGMCC 1.18051 T =KCTC 25774 T ).

The metabolic transition from anaerobic to aerobic in prokaryotes reflects adaptations to oxidative stress. Methanogen, one of the earliest life forms on Earth, has evolved into three major groups within the Euryarchaeota, exhibiting different phylogenetic affiliations and metabolic characters. In comparison with other strictly anaerobic methanogenic groups, the Class II methanogens possess a better capability to adapt to limited oxygen pressure. Cyanobacteria is considered the first and only prokaryote evolving oxygenic photosynthesis and is responsible for the Great Oxidation Event on Earth. However, the connection between oxygenic Cyanobacteria and evolutionary adaptations to oxidative stress in prokaryotes remains elusive. Here, through the gene encoding structural maintenance of chromosomes (SMC) protein, which was horizontally transferred from ancient Class II methanogens to the last common ancestor of the crown-group Cyanobacteria, we demonstrate that the origin of extant Cyanobacteria was undoubtedly posterior to the occurrence of oxygen-tolerant Class II methanogens. In addition, we found that certain prokaryotic lineages had evolved the tolerance mechanisms against oxidative stress before the origin of extant Cyanobacteria. The contradiction that oxidative adaptations in Class II methanogens and other prokaryotes predating the crown-group oxygenic Cyanobacteria implies the existence of more ancient biological oxygenesis. We propose that these potential oxygenic organisms might represent the extinct phototrophs and first emerge during the Paleoarchean, contributing to the oxidative adaptations in the prokaryotic tree of life and facilitating the dispersal of reaction centers across the bacterial domain.

Understanding archaeal gene regulation is technically challenging due to the difficulty of measuring gene expression in individual archaeal cells. Here, we develop a multi-round smHCR-FISH method to quantify the transcription of multiple genes in single cells of a methanogenic archaeon.