The Gram-positive bacterium Bacillus subtilis uses serine not only as a building block for proteins but also as an important precursor in many anabolic reactions. Moreover, a lack of serine results in the initiation of biofilm formation. However, excess serine inhibits the growth of B. subtilis. To unravel the underlying mechanisms, we isolated suppressor mutants that can tolerate toxic serine concentrations by three targeted and non-targeted genome-wide screens. All screens as well as genetic complementation in Escherichia coli identified the so far uncharacterized permease YbeC as the major serine transporter of B. subtilis. In addition to YbeC, the threonine transporters BcaP and YbxG make minor contributions to serine uptake. A strain lacking these three transporters was able to tolerate 100 mM serine whereas the wild type strain was already inhibited by 1 mM of the amino acid. The screen for serine-resistant mutants also identified mutations that result in increased serine degradation and in increased expression of threonine biosynthetic enzymes suggesting that serine toxicity results from interference with threonine biosynthesis.
To better understand cellular life, it is essential to decipher the contribution of individual components and their interactions. Minimal genomes are an important tool to investigate these interactions. Here, we provide a database of 105 fully annotated genomes of a series of strains with sequential deletion steps of the industrially relevant model bacterium Bacillus subtilis starting with the laboratory wild type strain B. subtilis 168 and ending with B. subtilis PG38, which lacks approximately 40% of the original genome. The annotation is supported by sequencing of key intermediate strains as well as integration of literature knowledge for the annotation of the deletion scars and their potential effects. The strain compendium presented here represents a comprehensive genome library of the entire MiniBacillus project. This resource will facilitate the more effective application of the different strains in basic science as well as in biotechnology.
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...
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