Amino acids are building blocks for protein biosynthesis in each living cell. However, due to their reactivity and the similarity between several amino acids, they may also be involved in harmful reactions or in noncognate interactions and thus may be toxic. Bacillus subtilis can deal with otherwise toxic histidine by overexpressing the bipartite amino acid exporter AzlCD.

Model organisms such as the Gram-positive bacterium Bacillus subtilis have been studied intensively for decades. However, even for model organisms, no function has been identified for about one fourth of all proteins. It has recently been realized that such understudied proteins as well as poorly studied functions set a limitation to our understanding of the requirements for cellular life, and the Understudied Proteins Initiative has been launched. Of poorly studied proteins, those that are strongly expressed are likely to be important to the cell and should therefore be considered high priority in further studies. Since the functional analysis of unknown proteins can be extremely laborious, a minimal knowledge is required prior to targeted functional studies. In this review, we discuss strategies to obtain such a minimal annotation, for example, from global interaction, expression, or localization studies. We present a set of 41 highly expressed and poorly studied proteins of B. subtilis. Several of these proteins are thought or known to bind RNA and/or the ribosome, some may control the metabolism of B. subtilis, and another subset of particularly small proteins may act as regulatory elements to control the expression of downstream genes. Moreover, we discuss the challenges of poorly studied functions with a focus on RNA-binding proteins, amino acid transport, and the control of metabolic homeostasis. The identification of the functions of the selected proteins not only will strongly advance our knowledge on B. subtilis, but also on other organisms since many of the proteins are conserved in many groups of bacteria.

Summary Roundup® is the brand name for herbicide solutions containing glyphosate, which specifically inhibits the 5‐enolpyruvyl‐shikimate‐3‐phosphate (EPSP) synthase of the shikimate pathway. The inhibition of the EPSP synthase causes plant death because EPSP is required for biosynthesis of aromatic amino acids. Glyphosate also inhibits the growth of archaea, bacteria, Apicomplexa, algae and fungi possessing an EPSP synthase. Here, we have characterized two glyphosate‐resistant bacteria from a Roundup solution. Taxonomic classification revealed that the isolates 1CH1 and 2CH1 are Burkholderia anthina and Burkholderia cenocepacia strains respectively. Both isolates cannot utilize glyphosate as a source of phosphorus and synthesize glyphosate‐sensitive EPSP synthase variants. Burkholderia. anthina 1CH1 and B. cenocepacia 2CH1 tolerate high levels of glyphosate because the herbicide is not taken up by the bacteria. Previously, it has been observed that the exposure of soil bacteria to herbicides like glyphosate promotes the development of antibiotic resistances. Antibiotic sensitivity testing revealed that the only the B. cenocepacia 2CH1 isolate showed increased resistance to a variety of antibiotics. Thus, the adaptation of B. anthina 1CH1 and B. cenocepacia 2CH1 to glyphosate did not generally increase the antibiotic resistance of both bacteria. However, our study confirms the genomic adaptability of bacteria belonging to the genus Burkholderia.

Glyphosate (GS) specifically inhibits the 5‐enolpyruvyl‐shikimate‐3‐phosphate (EPSP) synthase that converts phosphoenolpyruvate (PEP) and shikimate‐3‐phosphate to EPSP in the shikimate pathway of bacteria and other organisms. The inhibition of the EPSP synthase depletes the cell of the EPSP‐derived aromatic amino acids as well as of folate and quinones. A variety of mechanisms (e.g., EPSP synthase modification) has been described that confer GS resistance to bacteria. Here, we show that the Burkholderia anthina strain DSM 16086 quickly evolves GS resistance by the acquisition of mutations in the ppsR gene. ppsR codes for the pyruvate/ortho‐Pi dikinase PpsR that physically interacts and regulates the activity of the PEP synthetase PpsA. The mutational inactivation of ppsR causes an increase in the cellular PEP concentration, thereby abolishing the inhibition of the EPSP synthase by GS that competes with PEP for binding to the enzyme. Since the overexpression of the Escherichia coli ppsA gene in Bacillus subtilis and E. coli did not increase GS resistance in these organisms, the mutational inactivation of the ppsR gene resulting in PpsA overactivity is a GS resistance mechanism that is probably unique to B. anthina.

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.