How to deal with toxic amino acids: the bipartite AzlCD complex exports histidine in Bacillus subtilis

Abstract

In the Gram-positive model bacterium Bacillus subtilis, the presence of the amino acid glutamate triggers potassium uptake due to the glutamate-mediated activation of the potassium channel KtrCD. As a result, the intracellular accumulation of glutamate is toxic in strains lacking the second messenger cyclic di-AMP since these cells are unable to limit potassium uptake. We observed that the presence of histidine, which is degraded to glutamate, is also toxic for a B. subtilis strain that lacks all three c-di-AMP synthesizing enzymes. However, suppressor mutants emerged, and whole genome sequencing revealed mutations in the azlB gene encoding the repressor of the azl operon. This operon encodes an exporter and an importer for branched-chain amino acids. The suppressor mutations result in overexpression of the azl operon. Deletion of the azlCD genes encoding the branched chain amino acid exporter restored the toxicity of histidine indicating that this exporter is required for histidine export and resistance to otherwise toxic levels of the amino acid. The higher abundance of the amino acid exporter AzlCD increased the extracellular concentration of histidine, thus confirming the new function of AzlCD as a histidine exporter. The azl operon encodes transporters for the uptake and export of amino acids. Unexpectedly, AzlB-mediated repression of the operon remains active even in the presence of amino acids suggesting that expression of the azl operon requires mutational activation by inactivation of AzlB.

Understudied Proteins and Understudied Functions in the Model Bacterium Bacillus subtilis -- a Major Challenge in Current Research

“…It is important to note that amino acids can also pose a threat to bacteria: histidine inhibits growth of B. subtilis in minimal medium (Meißner et al, 2022), and serine and threonine are even toxic to the bacteria under these conditions (Klewing et al, 2020). The toxicity of some amino acids is caused by their high reactivity or by the ability to interfere with other pathways as shown for inhibition of threonine synthesis by serine (de Lorenzo et al, 2015;Mundhada et al, 2017).…”

“…However, it seems that some amino acids do not have one major uptake system, but instead multiple transporters, which contribute to their acquisition. This idea is supported by the fact that suppressors obtained under histidine pressure acquire mutations in the transcriptional repressor AzlB, which allows overexpression of AzlCD, a histidine exporter (Meißner et al, 2022).…”