Biology subjects

Stülke, J.

Publications and source records attributed to Stülke, J..

3 recordsLinked to original sources

bioRxiv2025

Photo-leucine incorporation in Bacillus subtilis: Identification of transporters and of a selectivity determinant in the Leu-tRNA synthetase

The elucidation of the intricate network of proteins is essential for our understanding of cellular function. In vivo crosslinking is a powerful tool for probing these interactions, with photoactivatable amino acids representing a promising next step. Here, we evaluated the application of photo-leucine in the gram-positive model organism Bacillus subtilis. This amino acid analog is incorporated into proteins, but high concentrations inhibit the growth of B. subtilis. We investigated photo-leucine homeostasis and identified the branched-chain amino acid importers BcaP and BraB as well as the bipartite exporter AzlCD as key players of its uptake and export, respectively. Additionally, we identified a previously uncharacterized exporter, AexB, a member of the "sleeping beauty" group of EamA amino acid exporters. Expression of the aexB gene is positively regulated by the transcription factor AerB, that is normally normally inactive. Selective pressure by photo-leucine triggered the acqusition of a point mutation in aerB, allowing AerB to activate aexB expression. Mutants highly resistant to photo-leucine carried a single amino acid exchange, Ala-494 to Thr, in the leucine-tRNA synthetase LeuS. Molecular docking analysis revealed that this mutation alters the leucine-binding pocket, in a manner that still allowed binding of leucine, but not photo-leucine. While high photo-leucine incorporation is beneficial for effective crosslinking, our results suggest that B. subtilis intrinsically limits its incorporation to maintain cellular tolerance. Thus, evolutionary processes provide adaptive mechanisms to adjust amino acid analogue incorporation to tolerable levels, making photo amino acids a candidate for life cell investigation of protein struture and interactions.

microbiology↗

bioRxiv2023

Control of three-carbon amino acid homeostasis by promiscuous importers and exporters in Bacillus subtilis: Role of the sleeping beauty family of amino acid exporters

The Gram-positive model bacterium Bacillus subtilis can acquire amino acids by import, de novo biosynthesis, or by degradation of proteins and peptides. The accumulation of several amino acids inhibits growth of B. subtilis, probably due to misincorporation into cellular macromolecules such as proteins or peptidoglycan or due to interference with other amino acid biosynthetic pathways. Here, we studied the adaptation of B. subtilis to toxic concentrations of the three-carbon amino acids L-alanine, {beta}-alanine, and 2,3-diaminopropionic acid as well as glycine. Resistance to the non-proteinogenic amino acid {beta}-alanine, which is a precursor for the vitamin B5 and thus for coenzyme A biosynthesis is achieved by mutations that either activate a cryptic amino acid exporter, AexA (previously YdeD), or inactivate the amino acid importers AimA, AimB (previously YbxG), and BcaP. The aexA gene is very poorly expressed under most conditions studied. However, mutations afecting the transcription factor AerA (previously YdeC), can result in strong constitutive aexA expression. AexA is the founding member of a conserved family of amino acid exporters in B. subtilis, which are all very poorly expressed. Therefore, we suggest to call this family "sleeping beauty family of amino acid exporters". 2,3-Diaminopropionic acid can also be exported by AexA, and this amino acid also seems to be a natural substrate of AerA/ AexA, as it can cause a slight but significant induction of aexA expression, and AexA also provides some natural resistance towards 2,3-diaminopropionic acid. Moreover, our work shows how low specificity amino acid transporters contribute to amino acid homeostasis in B. subtilis. IMPORTANCEEven though B. subtilis is of of the most-studied bacteria, amino acid homeostasis in this organism is not fully understood. We have identified import and export systems for the C2 and C3 amino acids. Our work demonstrates that the responsible amino acid permeases contribute in a rather promiscuitive way to amino acid uptake. In addition, we have discovered AexA, the first member of a family of very poorly expressed amino acid exporters, that we call "sleeping beauty amino acid exporters". The expression of these transporters is typically triggered by mutations in corresponding regulator genes that are acquired upon exposure to toxic amino acids. These exporters are ubiquitous in all domains of life. It is tempting to speculate that many of them are not expressed until the cells experience a selective pressure by toxic compounds and that the protect the cells from rare but potentially dangerous accounters with such compounds.

microbiology↗

bioRxiv2020

Essentiality of c-di-AMP in Bacillus subtilis: Bypassing mutations converge in potassium and glutamate homeostasis

In order to adjust to changing environmental conditions, bacteria use nucleotide second messengers to transduce external signals and translate them into a specific cellular response. Cyclic di-adenosine monophosphate (c-di-AMP) is the only known essential nucleotide second messenger. In addition to the well-established role of this second messenger in the control of potassium homeostasis, we observed that glutamate is as toxic as potassium for a c-di-AMP-free strain of the Gram-positive model bacterium Bacillus subtilis. In this work, we isolated suppressor mutants that allow growth of a c-di-AMP-free strain under these toxic conditions. Characterization of glutamate resistant suppressors revealed that they contain pairs of mutations, in most cases affecting glutamate and potassium homeostasis. Among these mutations, several independent mutations affected a novel glutamate transporter, AimA (Amino acid importer A, formerly YbeC). This protein is the major transporter for glutamate and serine in B. subtilis. Unexpectedly, some of the isolated suppressor mutants could suppress glutamate toxicity by a combination of mutations that affect phospholipid biosynthesis and a specific gain-of-function mutation of a mechanosensitive channel of small conductance (YfkC) suggesting the acquisition of a device for glutamate export. Cultivation of the c-di-AMP-free strain on complex medium was an even greater challenge because the amounts of potassium, glutamate, and other osmolytes are substantially higher than in minimal mediu. Suppressor mutants viable on complex medium could only be isolated under anaerobic conditions if one of the two c-di-AMP receptor proteins, DarA or DarB, was absent. Also on complex medium, potassium and osmolyte toxicity are the major bottlenecks for the growth of B. subtilis in the absence of c-di-AMP. Our results indicate that the essentiality of c-di-AMP in B. subtilis is caused by the global impact of the second messenger nucleotide on different aspects of cellular physiology. AUTHOR SUMMARYBacteria are exposed to constantly changing environmental conditions. In order to respond to these changes, they use nucleotide second messengers to transduce external signals and translate them into a specific cellular response. Among the repertoire of bacterial second messenger nucleotides, cyclic di-AMP (c-di-AMP) stands out as it is the only second messenger that is essential for the bacteria that produce it, including the Gram-positive model organism Bacillus subtilis. C-di-AMP plays a major role in the control of potassium homeostasis, and we found that glutamate is toxic to a B. subtilis strain lacking c-di-AMP to the same extent as potassium. These toxic conditions were the starting point for an extensive suppressor analysis, which led to the identification of a novel glutamate transporter (AimA). If the B. subtilis strain lacking c-di-AMP was cultivated on complex medium, the isolation of suppressor mutants was only possible under anaerobic conditions and if either of the two c-di-AMP-binding signal transduction proteins was absent. This suggests that these proteins are a major burden for the cell on complex medium in their c-di-AMP free state. Our result underline the complexity of c-di-AMP signaling and propose new directions for research.

microbiology↗