5 AMINO ACID TRANSPORT
Although amino acids are the essential building blocks for proteins, there is still a lot to be uncovered, when it comes to the transport of amino acids. Many bacteria including B. subtilis can synthesize all 20 proteinogenic amino acids. However, the bacteria do not depend on amino acid synthesis, as they can also directly take up amino acids from their environment. In fact, none of the amino acid biosynthetic pathways is essential in B. subtilis, which suggests the presence of transporters for each amino acid. So far, no uptake systems have been identified for asparagine, phenylalanine, glycine, and tyrosine. Several problems make the identification of novel amino acid transporters a challenging task: (i) Multiple transporters exist for most amino acids, (ii) the substrate specificity of many transporters is low, allowing them to take up multiple different amino acids/ metabolites in a rather promiscuous way, and (iii) the affinity of multiple transporters for one amino acid may differ substantially, which results in some transporters only being active under specific conditions. Particularly the presence of multiple transporters for one amino acids (and additionally the existence of biosynthetic pathways) impedes the identification of amino acid transporters since mutants often have no phenotype.
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). Amino acid toxicity is even intensified in B. subtilisstrains lacking the second messenger cyclic di-AMP. This molecule controls potassium homeostasis, and too high potassium uptake is toxic for the cells (Gundlach et al., 2018). In strains lacking c-di-AMP, potassium is toxic even at low external concentrations in the presence of amino acids. This is caused by the activation of the potassium transporter KtrCD by the common product of amino acid catabolism, glutamate (Krüger et al., 2020). Toxicity and growth inhibition by amino acids can be used to isolate suppressor mutants that have often inactivated the major transport pathway. In this way, the major transporter for serine and glutamate, AimA, was identified (Klewing et al., 2020; Krüger et al., 2021b). Strikingly, the function of this important transporter has remained enigmatic until very recently!
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).
Although B. subtilis has been studied extensively, there are still several potential amino acid transporters with unknown function (Fig.2). Investigating the remaining uncharacterized transporters could yield valuable insights into overall amino acid metabolism. Moreover, a complete knowledge of amino acid uptake systems would help to understand the requirements that must be met to sustain life in a cell with a minimal genome (Reuß et al., 2016).
Another understudied facet of amino acid transport is the homeostasis of D-amino acids, the enantiomeric counterparts of the proteinogenic L-amino acids. D-amino acids were proposed to act as a bacterial antifungal defense mechanism, as they are integrated into fungicidal components like iturin, bacillomycin and mycosubtilin (Stein, 2005). They might also be metabolized by certain bacteria and act as carbon or nitrogen source, which makes them an interesting overall research topic. As far as uptake goes, it is known that certain transporters are able to transport both the D- and L-variant of an amino acid, as is the case for the alanine permease AlaP (Sidiq et al., 2021) The uptake of D-alanine is important, as it is an essential component in the bacterial cell wall. Still, B. subtilis is also able to import other D-amino acids such as D-methionine and D-asparagine (Hullo et al., 2004; our unpublished work), which serve no clear purpose within the cell and require further investigation.