Amino acid metabolism in Bacillus subtilis
by Janek Meißner
Date of Examination:2024-01-18
Date of issue:2024-03-05
Advisor:Prof. Dr. Jörg Stülke
Referee:Prof. Dr. Jörg Stülke
Referee:Prof. Dr. Rolf Daniel
Persistent Address:
http://dx.doi.org/10.53846/goediss-10387
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Abstract
English
Amino acid metabolism is a a central field of research aiming to understand the complex reactions that take place in a cell. Amino acids are the main building blocks for proteins, and they serve as carbon or nitrogen source and are therefore ubiquitous throughout all life forms. All amino acids apart from glycine possess an L- and D-enantiomer. While proteins are exclusively synthesized from L-amino acids, D-amino acids still play a big role in bacteria and are an upcoming field of research. Acquisition of amino acids occurs either by synthesis or transport. The gram positive soil bacterium Bacillus subtilis was used as a model organism for decades in research, as it is able to synthesize all amino acids by itself and also to take them up from the environment. However, the transporters for each amino acid are still not known. In fact, no transporters for glycine, phenylalanine, tyrosine and asparagine have been identified so far. Additionally, several amino acids are toxic to B. subtilis under specific conditions or even already to the wild type. This work used amino acid toxicity to uncover the mechanisms employed by the cells to deal with L-histidine, L-asparagine and D-asparagine. For this purpose, the c-di-AMP free (Δdac) strain was used in conjunction with amino acid stress in order to trigger the formation of suppressor mutants. When stressed with L-histidine, the cells responded with mutations in the azlB gene, encoding for the transcriptional repressor AzlB. The expression analysis revealed an overexpression of the bipartite amino acid exporter AzlCD, which was also verified as histidine exporter via liquid chromatography-mass spectrometry. When L-Asn stress was applied, the Δdac acquired mutations in the potassium transporter KtrD, showing a possible link between asparagine and potassium metabolism. Furthermore, deleting aimA also provided resistance to L-Asn, establishing it as L-Asn importer. In order to further elucidate asparagine metabolism in B. subtilis the reactions to L-asparagine were examined in an asparaginase-deficient strain (ΔansAB ΔansZ). The experiments again revealed suppressor mutations in azlB. This suggests a role of AzlCD in L-asparagine export and cements its role as a broad range amino acid exporter. Further adaptation experiments revealed mutations in aimA, as well as bcaP, which both led to increased resistance to the L-asparagine stress. These two were already known to be broad range transporters in B. subtilis. The data proves the role of AimA and BcaP in L-asparagine uptake. Suppressor screens with D-asparagine revealed mutations in mleN, coding for the malate/lactate antiporter MleN. Subsequent growth experiments could verify that MleN is the main transporter for D-asparagine, as the deletion strain fully complements growth during D-asparagine stress. A role of MleN in L-asparagine transport was not found. This work therefore suggests that stereo-enantiomeric amino acids are not necessarily taken up by the same uptake system. This work takes a step forward in the understudied field of amino acid transport and discusses amino acid toxicity, while providing strategies to identify novel transporters in the future.
Keywords: Amino acids; Bacillus subtilis; Import and export; toxic amino acids
