Function and homeostasis of non-proteinogenic amino acids in Bacillus subtilis
by Robert Warneke
Date of Examination:2024-06-07
Date of issue:2024-07-23
Advisor:Prof. Dr. Jörg Stülke
Referee:Prof. Dr. Ralf Ficner
Referee:Prof. Dr. Gerhard Braus
Persistent Address:
http://dx.doi.org/10.53846/goediss-10626
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Abstract
English
The gram-positive soil bacterium Bacillus subtilis lives in a constantly changing environment, requiring rapid adaptation to survive and thrive. This forces the bacteria to adapt to nutrient availability or stressful situations. For many of these situations, it is necessary to adjust the intracellular metabolite pool through targeted synthesis or import and export processes. In this adaptation, the non-proteinogenic amino acids play an important role. In this thesis, the homeostasis and functions of non-proteinogenic amino acids in B. subtilis are investigated. Beyond their role as precursors in biosynthetic pathways, non-proteinogenic amino acids can have regulatory or protective properties for the cell. We demonstrate such a regulatory role for ornithine that is bound by a transcription factor and activates the expression of the arginine degradative enzymes. Additionally, non-proteinogenic amino acids can also act as protective agents for the cell. We show that the loss of the c-di-AMP receptor DarA can be compensated by the accumulation of citrulline. On the other hand, non-proteinogenic amino acids can also be toxic for the cell, as in the case for the coenzyme A precursor -alanine. This highlights the importance of their tightly regulated homeostasis. However, the transport of non-proteinogenic has only been poorly investigated. We identified the uptake of the coenzyme A precursor -alanine to be facilitated by the broad range amino acid transporter AimA and the alanine permease AlaP. Bacteria can also defend themselves against toxic amino acids by exporting them. We identified a novel group of amino acid exporters, dubbed "sleeping beauty exporters," due to their low expression under various conditions. We show that AexA and AexB can export numerous amino acids, thereby preventing toxic accumulation within the cell. We identify the transcriptional activators AerA and AerB, which tightly control expression either through ligand binding or through mutation independent of an inducer. In the context of exploring the homeostasis of β-alanine, we have elucidated the complete pathways for the acquisition of pantothenate and coenzyme A. As an alternative to synthesis, the cells can take up pantothenate from the environment by an ECF transporter with the S component YhfU, which we identified as a high-affinity transporter and consequently rename it PanU (Pantothenate Uptake). We isolate suppressors that have overcome pantothenate biosynthesis deficiency via the utilization of the newly identified metabolite cysteinopantetheine. This metabolite is utilized via a previously unknown salvage pathway and provides another example of the metabolic flexibility of B. subtilis. Taken together, the findings of this thesis shed light on the functions, as well as the uptake and export mechanisms of both proteinogenic and non-proteinogenic amino acids in B. subtilis. This showcases the remarkable adaptability and resilience of this versatile bacterium in response to changing environmental conditions.
Keywords: Bacillus subtilis; amino acids; coenzyme A; non proteinogenic; transport; homeostasis; beta alanine; PII
