Resistance to serine inBacillus subtilis: Identification of the serine transporter YbeC and of a metabolic network that links serine and threonine metabolism

Abstract

22 promiscuity 23 24 2 Originality-Significance Statement 25Serine is an important precursor for many biosynthetic reactions, and lack of this amino acid can induce 26 biofilm formation in Bacillus subtilis. However, serine is toxic for the growth of B. subtilis. To 27 understand the reason(s) for this toxicity and to identify the so far unknown serine transporter(s) of 28 this bacterium, we performed exhaustive mutant screens to isolate serine-resistant mutants. This 29 screen identified YbeC, the major serine transporter of B. subtilis. Moreover, we observed an intimate 30 link between serine and threonine metabolism that is responsible for serine toxicity by inhibiting 31 threonine biosynthesis. 33 Summary 34The Gram-positive bacterium Bacillus subtilis uses serine not only as building block for proteins but 35 also as an important precursor in many anabolic reactions. Moreover, a lack of serine results in the 36 initiation of biofilm formation. However, in excess serine inhibits the growth of B. subtilis. To unravel 37 the underlying mechanisms, we isolated suppressor mutants that can tolerate toxic serine 38 concentrations by three targeted and non-targeted genome-wide screens. All screens as well as 39 genetic complementation in Escherichia coli identified the so far uncharacterized permease YbeC as 40 the major serine transporter of B. subtilis. In addition to YbeC, the threonine transporters BcaP and 41 YbxG make minor contributions to serine uptake. A strain lacking these three transporters was able to 42 tolerate 100 mM serine whereas the wild type strain was already inhibited by 1 mM of the amino acid. 43The screen for serine-resistant mutants also identified mutations that result in increased serine 44 degradation and in increased expression of threonine biosynthetic enzymes suggesting that serine 45 toxicity results from interference with threonine biosynthesis. 46 47 4 acid transporter families do actually transport other substrates, such as the recently described 74 potassium transporter KimA (Gundlach et al., 2017). A complete overview on the known and potential 75 amino acid transporters of B. subtilis can be found in Table S1 (see also http://subtiwiki.uni-76 goettingen.de/v3/category/view/SW%201.2, Zhu and Stülke, 2018). Importantly, no transporters have 77 so far been identified or proposed for alanine, glycine, serine, asparagine, and the aromatic amino 78 acids phenylalanine and tyrosine. The identification of new amino acid transporters is hampered by 79 two peculiarities: For one amino acid, there are often multiple transporters, as has been shown for 80 arginine, proline, or the branched-chain amino acids (Calogero et al., 1994; Gardan et al., 1995; 81 Sekowska et al., 2001; Zaprasis et al., 2014; Belitsky, 2015). On the other hand, many permeases have 82 a relatively weak substrate specificity, i. e. they are able to transport multiple amino acids, as shown 83 for BcaP or GltT (Belitsky 2015; Zaprasis et al., 2015). 84We are interested in the identification of the functions that are r...

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

“…It is likely that these larger colonies were formed bysuppressor mutants that were resistant to histidine in the medium. We hypothesized that mutations could affect uptake systems for histidine as already observed for glutamate, serine, or threonine (9, 10, 14). Indeed, we were able to indentify mutations in two isolates by whole genome sequencing.…”

“…Third, suppressor mutations may facilitate detoxification of the toxic amino acid by degradation or modification to a non-toxic metabolite as observed for glutamate and serine (Belitsky & Sonenshein, 1998;Commichau et al, 2008;Klewing et al, 2020;Krüger et al, 2021). Finally, the target of the toxic metabolite may be modified in a way that it becomes resistant escapes inactivation by other mechanisms such as imceased expression as has been reported for serine and the anti-metabolite glyphosate (Klewing et al, 2020;Wicke et al, 2019).…”

“…Amino acid toxicity is not only relevant for the design of minimal genomes but it is also an important tool for the identification of components involved in amino acid metabolism. While some amino acids such as serine or threonine are toxic already for wild type strains (Belitsky, 2015;De Lorenzo et al, 2015; Klewing et al, 2020), others are well tolerated. In this case, the corresponding D-amino acids, amino acid analogs, or structually similar metabolites may act as anti-metabolites that inhibit normal cellular metabolism and thus growth of the bacteria.…”

“…Often, such mutations affect uptake systems and prevent the uptake of the toxic amino acid or its analogues. In this way, transporters for threonine, proline, alanine, serine, and glutamate as well as for the anti-metabolites 4-hydroxy-L-threonine and glyphosate have been identified in B. subtilis (9, 10, 11, 12, 13, 14, 15). A second way to achieve resistance against toxic amino acids is the activation of export mechanisms.…”

“…This has been reported in the cases of 4-azaleucine and glutamate (14, 16). Third, suppressor mutations may facilitate detoxification of the toxic amino acid by degradation or modification to a non-toxic metabolite as observed for glutamate and serine (7, 10, 14, 17). Forth, the protein target of the toxic metabolite may be modified in a way that it becomes resistant (18).…”