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Exploring the hidden world of Methanosarcina mazei: Unravelling the secrets of its small proteome and its nitrogen-dependent regulation

ABSTRACT: The mesophilic methanogenic archaeal model organism Methanosarcina mazei strain Gö1, is crucial for climate and environmental research due to its ability to produce methane from H2 plus CO2, acetate, and methylamines. Here, we established the first Ribo-seq protocol for M. mazei strain Gö1 under two growth conditions (nitrogen sufficiency (+N) versus nitrogen limitation (-N)). Translation of 93 previously annotated and 311 novel small open reading frames (sORFs), coding for proteins ≤ 70 amino acids in length was predicted with high confidence based on Ribo-seq data. Epitope tagging followed by immunoblotting analysis confirmed the translation of 12 out of 15 selected novel sORFs, the remaining three were validated by LC-MS-MS analysis. In total, LC-MS-MS analysis validated translation for 60 annotated sORFs and 26 novel sORFs. A comprehensive differential expression analysis revealed that 29 of 311 novel sORFs were differentially regulated in response to nitrogen availability at the transcriptional and 49 at the translational level. Several reported sRNAs are now emerging as dual-functional sRNAs, including sRNA154, the central regulatory sRNA in regulation of nitrogen metabolism. Numerous of the novel sORFs identified in this study are conserved in Methanosarcina species, some of which showed a phenotype when overproduced, pinpointing important physiological functions. Overall, the comprehensive analysis opens a new avenue to start elucidating the function(s) of small proteins in M. mazei.

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive Plus

ORGANISM(S): Methanosarcina Mazei Go1

SUBMITTER: Andreas Tholey

LAB HEAD: Andreas Tholey

PROVIDER: PXD045039 | Pride | 2024-08-02

REPOSITORIES: Pride

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