Fukuyama, Y. | Search bioRxiv

bioRxiv2023

Development of a rapid and highly accurate method for 13C tracer-based metabolomics and its application on a hydrogenotrophic methanogen

Microfluidic capillary electrophoresis-mass spectrometry (CE-MS) is a rapid and highly accurate method to determine isotopomer patterns in isotopically labeled compounds. Here, we developed a novel method for tracer-based metabolomics using CE-MS for underivatized proteinogenic amino acids. The method consisting of a ZipChip CE system and a high-resolution Orbitrap Fusion Tribrid mass spectrometer allows us to obtain highly accurate data from 1 L of 100 nmol/L mol amino acids comparable to a mere 1 x 104-5 prokaryotic cells. To validate the capability of the CE-MS method, we analyzed 16 protein-derived amino acids from a methanogenic archaeon Methanothermobacter thermautotrophicus as a model organism, and the mass spectra showed sharp peaks with low mass errors and background noise. Tracer-based metabolome analysis was then performed to identify the central carbon metabolism in M. thermautotrophicus using 13C-labeled substrates. The mass isotopomer distributions of serine, aspartate, and glutamate revealed the co-occurrence of the Wood-Ljungdahl pathway and an incomplete reductive TCA cycle for carbon fixation. In addition, biosynthesis pathways of 15 amino acids were constructed based on the mass isotopomer distributions of the detected protein-derived amino acid, genomic information, and public database. Among them, the presence of the alternative enzymes of alanine dehydrogenase, ornithine cyclodeaminase, and homoserine kinase was suggested in the biosynthesis pathways of alanine, proline, and threonine, respectively. To our knowledge, the novel 13C tracer-based metabolomics using CE-MS is the most efficient method to identify central carbon metabolism and amino acid biosynthesis pathways and is applicable to in any kind of isolated microbe.

microbiology↗

bioRxiv2021

Isolation and characterization of novel temperate virus Aeropyrum globular virus 1 infecting hyperthermophilic archaeon Aeropyrum

We isolate a novel archaeal temperate virus named Aeropyrum globular virus 1 (AGV1) from the host Aeropyrum culture. Reproduction of AGV1 was induced by adding 20 mM tris-acetate buffer to exponentially growing host cells. Negatively stained virions showed spherical morphology (60 {+/-}2 nm in diameter) similar to Globuloviridae viruses. The double-stranded circular DNA genome of AGV1 contains 18,222 bp encoding 34 open-reading frames. No ORFs showed significant similarity with Globuloviridae viruses. AGV1 shares three genes, including an integrase gene, with reported spindle-shaped temperate viruses. However we couldnt detect its integration site in the host genome. Moreover AGV1 seemed not to replicate autonomously because there are no origin recognition boxes in the genome. qPCR results showed that the genome copy number of AGV1 was lower than that of the host genome (10-3 copies per host genome). Upon the addition of tris-acetate buffer, a steep increase in the AGV1 genome copy number (9.5-26 copies per host genome at 2 days post-treatment) was observed although clustered regularly interspaced short palindromic repeat (CRISPR) elements of the host genome showed significant matches with AGV1 protospacers. Our findings suggest that AGV1 is a novel globular virus exhibiting an unstable carrier state in the growing host and in that way AGV1 can escape from the host defense system and propagate under stressful host conditions. ImportanceStudying archaeal viruses yields novel insights into the roles of virospheres and viruses in the evolutionary process of their hosts. Here, we isolated a novel spherical virus named Aeropyrum globular virus 1. AGV1 has integrase gene but its genome is not integrated into the host genome. AGV1 could not replicate autonomously due to the lack of origin recognition boxes and thus its copy number was too low (10-3 copies per host genome) without any inducing stimulus. However, upon the addition of tris-acetate buffer, the AGV1 genome copy number steeply increased instead of a perfect sequence match between the spacer of the host CRISPR/Cas system and the protospacer. Our findings suggest that AGV1 can escape from the host defense system and propagate under stressful conditions for the host by establishing an unstable carrier state. These results reveals a novel aspect of host-virus interactions in extreme environments.

microbiology↗