Biology subjects

Kiessling, C.

Publications and source records attributed to Kiessling, C..

3 recordsLinked to original sources

bioRxiv2025

Small subunits MttS and MttQ of the MttP transporter regulate trimethylamine transport in Methanosarcina mazei

Small proteins (<100 aa) have moved in the focus of science after being overlooked for decades due to bioinformatical and biochemical challenges. While mass spectrometry-coupled ribosome profiling of the mesophilic methanoarchaeon Methanosarcina mazei has recently unveiled a wealth of novel small ORFs, the functional roles of most of their products remain unknown. Here, we report the characterization of MttQ (98 aa) and MttS (49 aa), products of small ORFs situated in an operon alongside genes encoding a drug-metabolite-efflux (DME) family transporter (mttP) and other enzymes involved in trimethylamine (TMA) degradation. MttS and MttQ interact with MttP to form a stable oligomeric complex spanning the cytoplasmic membrane. TMA transport activity of the MttQ/MttS/MttP-complex is demonstrated via in vivo Escherichia coli cells heterologously expressing this system. Based on the reduced growth of a M. mazei mutant lacking mttS, on TMA as sole carbon source, we conclude that MttS governs the specificity of TMA transport. We posit that interactions between a DME transporter, e.g., MttP, and small proteins fueled evolution of the MttPQS complex and the advent of selective TMA uptake in methylotrophic methanoarchaea. These findings suggest an evolutionary mechanism on how small accessory proteins can alter conserved core functions in order to explore new ecological niches. Further, given that TMA levels in the human bloodstream influence cardiovascular disease risk but can be degraded by host-associated methanoarchaea containing a homolog of the MttPQS-complex, our findings present insight into an archaeal pathway with relevance to human health.

microbiology↗

bioRxiv2025

Small ORF16 modulates the Mtr complex activity in Methanosarcina mazei Goe1 depending on the molecular hydrogen availability

Until recently, small open reading frame (sORF)-encoded proteins of fewer than 100 amino acids, have attracted increasing attention over the past decade after being overlooked due to limitations in conventional detection methodologies. While numerous previously unannotated sORFs have recently been identified in the mesophilic archaeal model system Methanosarcina mazei, the physiological roles of most of their encoded small proteins remain unknown. We report here the functional characterization of sORF16 encoded small protein MtrR (49 amino acids) and show that it localizes oligomerically at the cytoplasmic membrane. There, it interacts with and influences the activity of tetrahydrosarcinapterin S-methyltransferase (Mtr), a key membrane-bound complex involved in energy metabolism. In vitro interaction and in vivo copurification assays revealed interactions between MtrR and the Mtr-complex, and microscale thermophoresis showed specific interactions with the MtrA subunit. Mutant strains lacking sORF16 exhibited significantly impaired growth in the presence of molecular hydrogen (H2), irrespective of the carbon source. We posit that by modulating the activity of the Mtr-complex, MtrR enables the archaeon adapt to changing environmental H2 conditions.

microbiology↗

bioRxiv2020

Small protein 26 interacts and enhances glutamine synthetase activity in Methanosarcina mazei

Small ORFs (sORF) encoded small proteins have been overlooked for a long time due to challenges in prediction and distinguishing between coding and non-coding predicted sORFs and in their biochemical detection and characterization. We report on the first biochemical and functional characterization of a small protein (sP26) in the archaeal model organism Methanosarcina mazei, comprising 23 amino acids. The corresponding encoding leaderless mRNA (spRNA26) is highly conserved within numerous Methanosarcina strains on the amino acid as well as on nucleotide level strongly arguing for a cellular function of the small protein. spRNA26 is significantly enhanced under nitrogen limitation, but also under oxygen and salt stress conditions. His-tagged sP26 was heterologously expressed and purified by fractionated ammonium sulfate precipitation, affinity chromatography and size exclusion centrifugation. Using independent biochemical approaches (pull-down by affinity chromatography followed by MS analysis, revers pull-down, microscale thermophoresis and size exclusion chromatography) we observed that sP26 interacts and forms complexes with M. mazei glutamine synthetase (GlnA1) with high affinity (app. KD = 45 +/- 14 {micro}M). Upon interaction with sP26, GlnA1 activity was significantly stimulated independently and in addition to the known activation by the metabolite 2-oxoglutarate. Besides strong interaction of sP26 with the PII-like protein GlnK1 was demonstrated (KD= 1.4 {micro}M +/- 0.9 {micro}M). On the basis of these findings, we hypothesize that in addition to 2-oxoglutarate, sP26 activates GlnA1 activity under nitrogen limitation most likely by stabilizing the dodecameric structure of GlnA1.

biochemistry↗