7th joint conference "Joint Microbiology & Infection Conference 2024" of the DGHM and VAAM

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Tracing the gaps - Unraveling initial steps in hexose metabolism in Saccharolobus solfataricus using an improved stable isotope labeling approach

Archaea & Extremophiles

Archaea are often found in harsh habitats withstanding for instance high temperatures, salinity and acidic or alkaline environments making them valuable models to understand life under extreme conditions. Their metabolism is characterized by unique and unusual metabolic pathways and enzymes that differ from bacteria and eukaryotes. Among the Thermoproteota, Saccharolobus solfataricus represents an important model organism for archaeal metabolism. It thrives under thermoacidophilic conditions with an optimum temperature of 80 °C and a pH value between 2 and 4. Additionally, it is characterized by its metabolic versatility, which allows it to grow on a wide variety of carbon sources. For some hexoses like D-glucose and D-galactose the central carbohydrate metabolism has been extensively investigated, revealing a modified branched Entner-Doudoroff (ED) pathway for degradation instead of the Embden-Meyerhof-Parnas (EMP) pathway commonly used by other organisms. However, the degradation of other hexoses, such as D-mannose and D-fructose, is poorly understood so far. An initial 13C6-mannose spiking experiment indicated that the ED pathway is involved in D-mannose degradation. Due to the absence of homologues enzymes for known D-fructose degradation, a completely novel pathway is hypothesized. To unravel these metabolic blind spots, a GC-MS-based method has been optimized to intentionally slow down the metabolism of S. solfataricus. In this way, the metabolic flux of 13C6-labeled D-mannose and D-fructose can be traced in the first steps of degradation, paving the way for future elucidation and mapping of the degradation pathway.

Oral Presentation

Translational coupling via termination-reinitiation in archaea and bacteria

Translational coupling occurs at many gene pairs of archaea and bacteria. Translational coupling means that translation of the downstream gene on a polycistronic transcript strictly depends on translation of the upstream gene. One specific mechanism of coupling is called termination-reinitiation (TeRe). In this case, the translating ribosome (at least the small subunit) remains on the mRNA after terminating translation of the upstream gene, and it directly continues translation by reinitiating at the downstream gene. This mechanism typically occurs at gene pairs that have overlapping stop/start codons or very small intergenic distances.In previous studies we have shown that TeRe operates at several native gene pairs of Haloferax volcanii and Escherichia coli1 and that efficient coupling required very short intergenic distances2. Translational efficiencies were quantified using reporter genes fused to native gene pairs, enzyme assays and northern blotting were used to determine protein and transcript levels. In the present study, we investigated whether local mRNA structures at gene overlaps in E. coli are required to inhibit novel initiation at the downstream genes and guarantee coupling. To this end, we generated several constructs with truncated or mutated gene pairs, with the aim of destabilizing local mRNA structures close to the overlaps. The effects of these genetic perturbations on the efficiencies of translation and coupling will be reported.Additionally we created a random library with a 42 random nucleotide sequence directly upstream of the overlapping start/stop codon. In our two-reporter gene system, this library is used to select and screen for sequence motives that are important for efficient novel initiation at the downstream gene as well as translational coupling via TeRe.Taken together, translational coupling via TeRe operates at many gene pairs in archaea and bacteria, and various aspects of the molecular mechanism could be characterized using native gene pairs fused to reporter genes.1 Huber et al. (2019) Nature Comm. 10: 4006.2 Huber, Vogel et al. (2023) Front Microbiol. 14: 1291523.

Characterization of Methanosarcina mazei Solo-Cas4

The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) system and its associated (Cas) proteins serve as a defensive mechanism in prokaryotes against foreign genetic elements like viruses, plasmids, or transposons. Thereby, Cas proteins play a key role in CRISPR/Cas systems by recognizing invading nucleic acids and inserting fragments of the foreign nucleic acids (spacer) into the CRISPR array to reminisce overcome future infections. While most Cas proteins are encoded in the CRISPR locus, various cas genes exist that are located outside of CRISPR loci. The function of most of these stand-alone Cas proteins has yet not been investigated. Methanosarcina mazei, an anaerobic methanogenic archaeon, possesses two CRISPR loci together with an additional cas4 gene that has been identified outside of the CRISPR loci encoding a so-called solo-Cas4 protein. The function and structure of this solo-Cas4 protein and the potential connection to the CRISPR/Cas system or a function beyond have not been examined so far. The widespread occurrence of solo-Cas4 proteins even in organisms lacking CRISPR/Cas systems as well as encoded in viral genomes strengthen the hypothesis that solo-Cas4 have a potential function outside the prokaryotic defense.We heterologously expressed His-tagged SUMO fusion of solo-Cas4 and purified the tag-less protein. The purified solo-Cas4 protein showed a brown coloration and the characteristic absorption spectrum of a 4Fe4S-cluster with absorption peaks at approximately 330 nm and 420 nm. We further showed that M. mazei solo-Cas4 possesses a metal dependent exo- (from 3' to 5') and endo-nuclease activity against ssDNA substrates. The nuclease activity, as well as the 4Fe4S cluster, are characteristic features for Cas4 proteins. However, M. mazei solo-Cas4 showed only low sequence similarity to other Cas4 proteins. The detailed study of M. mazei solo-Cas4 may contribute to a broader understanding and elucidating a distinct role of solo-Cas4 proteins in archaea.

Oral Session
Archaeal Biology

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Oral SessionArchaeal Biology

Appointment

Chair

Topic

Programme

Oral Session
Archaeal Biology