Control of asparagine homeostasis in Bacillus subtilis : identification of promiscuous amino acid importers and exporters

Abstract

The Gram-positive model bacterium B. subtilis is able to import all proteinogenic amino acids from the environment as well as to synthesize them. However, the players involved in the acquisition of asparagine have not yet been identified for this bacterium. In this work, we used d -asparagine as a toxic analog of l -asparagine to identify asparagine transporters. This revealed that d - but not l -asparagine is taken up by the malate/lactate antiporter MleN. Specific strains that are sensitive to the presence of l -asparagine due to the lack of the second messenger cyclic di-AMP or due to the intracellular accumulation of this amino acid were used to isolate and characterize suppressor mutants that were resistant to the presence of otherwise growth-inhibiting concentrations of l -asparagine. These screens identified the broad-spectrum amino acid importers AimA and BcaP as responsible for the acquisition of l -asparagine. The amino acid exporter AzlCD allows detoxification of l -asparagine in addition to 4-azaleucine and histidine. This work supports the idea that amino acids are often transported by promiscuous importers and exporters. However, our work also shows that even stereo-enantiomeric amino acids do not necessarily use the same transport systems. IMPORTANCE Transport of amino acid is a poorly studied function in many bacteria, including the model organism Bacillus subtilis . The identification of transporters is hampered by the redundancy of transport systems for most amino acids as well as by the poor specificity of the transporters. Here, we apply several strategies to use the growth-inhibitive effect of many amino acids under defined conditions to isolate suppressor mutants that exhibit either reduced uptake or enhanced export of asparagine, resulting in the identification of uptake and export systems for l -asparagine. The approaches used here may be useful for the identification of transporters for other amino acids both in B. subtilis and in other bacteria.

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

“…6 , AexA expression resulted in increased tolerance to all three amino acids indicating that AexA is also active in the export of these amino acids. The contribution of an aerA * mutation to serine resistance has been described in a recent study ( 23 ), and the results reported here demonstrate that this is caused by the increased expression of AexA in the mutant. We can thus conclude that AexA is a non-specific amino acid exporter for C3-amino acids.…”

“…2 ). This observation suggests that AimA also imports β-alanine in addition to serine, glutamate, and aspartate ( 12 , 15 , 23 ).…”

“…Amino acid transporters, both importers and exporters, tend to be promiscuous and can transport multiple amino acids ( 24 ). This has been extensively studied for the amino acid importer AimA and the exporter AzlCD which are involved in either the uptake of serine, glutamate, and asparagine or in the export of 4-azaleucine, histidine, and asparagine ( 12 , 15 , 18 , 19 , 23 ). We, therefore, considered the possibility that AexA might also export amino acids other than β-alanine.…”

“…The remaining two 2,3-diaminopropionic acid-tolerant mutants both had mutations affecting the transcription repressor AzlB that controls the expression of the well-studied amino acid exporter AzlCD ( 18 , 19 , 23 ) (see Fig. 2 ).…”

“…The use of toxic amino acid analogs or the identification of conditions under which a given amino acid becomes growth-inhibiting is a powerful tool for the identification of amino acid transporters. This approach has been used to identify transporters for serine, threonine, proline, glutamate, and asparagine, as well as for the toxic inhibitors of amino acid biosyntheses, glyphosate and glufosinate, in B. subtilis ( 12 , 15 , 20 – 23 ). This strategy, however, has limited application as some amino acids are imported by multiple transporters ( 19 , 24 ), and thus, the simultaneous inactivation of all of them would be required to prevent the uptake.…”