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

Abstract

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.