Biology subjects

Warneke, R.

Publications and source records attributed to Warneke, R..

5 recordsLinked to original sources

bioRxiv2024

Coenzyme A biosynthesis in Bacillus subtilis: Discovery of a novel precursor metabolite for salvage and its uptake system

The Gram-positive model bacterium Bacillus subtilis is used for many biotechnological applications, including the large-scale production of vitamins. For vitamin B5, a precursor for coenzyme A synthesis, there is so far no established fermentation process available, partly due to the incomplete knowledge on the metabolic pathways that involve this vitamin. In this study, we have elucidated the complete pathways for the biosynthesis pantothenate and coenzyme A in B. subtilis. We have identified the enzymes involved in the pathway and have identified a salvage pathway for coenzyme A acquisition that acts on complex medium even in the absence of pantothenate synthesis. This pathway requires rewiring of sulfur metabolism resulting in the expression of a cysteine transporter. In the salvage pathway, the bacteria import cysteinopantetheine, a novel naturally occurring metabolite, using the cystine transport system TcyJKLMN. This work lays the foundation for the development of effective processes for vitamin B5 production.

microbiology↗

bioRxiv2023

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

The Gram-positive model bacterium Bacillus subtilis can acquire amino acids by import, de novo biosynthesis, or by degradation of proteins and peptides. The accumulation of several amino acids inhibits growth of B. subtilis, probably due to misincorporation into cellular macromolecules such as proteins or peptidoglycan or due to interference with other amino acid biosynthetic pathways. Here, we studied the adaptation of B. subtilis to toxic concentrations of the three-carbon amino acids L-alanine, {beta}-alanine, and 2,3-diaminopropionic acid as well as glycine. Resistance to the non-proteinogenic amino acid {beta}-alanine, which is a precursor for the vitamin B5 and thus for coenzyme A biosynthesis is achieved by mutations that either activate a cryptic amino acid exporter, AexA (previously YdeD), or inactivate the amino acid importers AimA, AimB (previously YbxG), and BcaP. The aexA gene is very poorly expressed under most conditions studied. However, mutations afecting the transcription factor AerA (previously YdeC), can result in strong constitutive aexA expression. AexA is the founding member of a conserved family of amino acid exporters in B. subtilis, which are all very poorly expressed. Therefore, we suggest to call this family "sleeping beauty family of amino acid exporters". 2,3-Diaminopropionic acid can also be exported by AexA, and this amino acid also seems to be a natural substrate of AerA/ AexA, as it can cause a slight but significant induction of aexA expression, and AexA also provides some natural resistance towards 2,3-diaminopropionic acid. Moreover, our work shows how low specificity amino acid transporters contribute to amino acid homeostasis in B. subtilis. IMPORTANCEEven though B. subtilis is of of the most-studied bacteria, amino acid homeostasis in this organism is not fully understood. We have identified import and export systems for the C2 and C3 amino acids. Our work demonstrates that the responsible amino acid permeases contribute in a rather promiscuitive way to amino acid uptake. In addition, we have discovered AexA, the first member of a family of very poorly expressed amino acid exporters, that we call "sleeping beauty amino acid exporters". The expression of these transporters is typically triggered by mutations in corresponding regulator genes that are acquired upon exposure to toxic amino acids. These exporters are ubiquitous in all domains of life. It is tempting to speculate that many of them are not expressed until the cells experience a selective pressure by toxic compounds and that the protect the cells from rare but potentially dangerous accounters with such compounds.

microbiology↗

bioRxiv2023

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

Amino acids are the main building block for proteins. 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. IMPORTANCETransport 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 other bacteria as well.

microbiology↗

bioRxiv2023

Control of iron homeostasis by a regulatory protein-protein interaction in Bacillus subtilis: The FurA (YlaN) acts as an antirepressor to the ferric uptake regulator Fur

Iron is essential for most organisms. However, two problems are associated with the use of iron for aerobically growing organisms: (i) its accumulation leads to the formation of toxic reactive oxygen species and (ii) it is present mainly as the highly insoluble ferric iron which makes the access to iron difficult. As a consequence, a tight regulation of iron homeostasis is required. This regulation is achieved in many bacteria by the ferric uptake repressor Fur. The way how the activity of Fur is controlled, has so far remained elusive. Here, we have identified the Fur antirepressor FurA (previously YlaN) in the model bacterium Bacillus subtilis and describe its function to release Fur from the DNA under conditions of iron limitation. The FurA protein physically interacts with Fur, and this interaction prevents Fur from binding to its target sites due to a complete re-orientation of the protein. Both in vivo and in vitro experiments using a reporter fusion and Fur-DNA binding assays, respectively, demonstrate that the Fur-FurA interaction prevents Fur from binding DNA and thus from repressing the genes required for iron uptake. Accordingly, the lack of FurA results in the inability of the cell to express the genes for iron uptake under iron-limiting conditions. This explains why the furA gene was identified as being essential under standard growth conditions in B. subtilis. Phylogenetic analysis suggests that the control of Fur activity by the antirepressor FurA is confined to, but very widespread in bacteria of the class Bacilli. IMPORTANCEIron is essential for most bacteria since it is required for many redox reactions. Under aerobic conditions, iron is both essential and toxic due to radical formation. Thus, iron homeostasis must be faithfully controlled. The transcription factor Fur is responsible for this regulation in many bacteria; however, the control of Fur activity has remained open. Here we describe the FurA protein, a so far unknown protein which acts as an antirepressor to Fur in Bacillus subtilis. This mechanism seems to be widespread in B. subtilis and several important pathogens and might be a promising target for drug development.

microbiology↗

bioRxiv2023

Ornithine is the central intermediate in the arginine degradative pathway and its regulation in Bacillus subtilis

ABSTRACTThe Gram-positive model bacterium Bacillus subtilis is able to utilize a variety of proteinogenic and non proteinogenic amino acids as sources of carbon, energy and nitrogen. The utilization of the amino acids arginine, citrulline and ornithine is catalyzed by enzymes that are encoded in the rocABC and rocDEF operons and by the rocG gene. Expression of these genes is under control of the alternative sigma factor SigL. RNA polymerase associated to this sigma factor depends on an ATP-hydrolyzing transcription activator to initiate transcription. The RocR protein acts as transcription activator for the roc genes. In this work, we have studied the contributions of all enzymes of the Roc pathway to the degradation of arginine, citrulline and ornithine. This identified the previously uncharacterized RocB protein as responsible for the conversion of citrulline to ornithine. In vitro assays with the purified enzyme suggest that it acts as a manganese-dependent N-carbamoyl-L-ornithine hydrolase that cleaves citrulline to ornithine and carbamate. So far, the molecular effector that triggers transcription activation by RocR has not been unequivocally identified. Using a combination of transcription reporter assays and biochemical experiments we demonstrate that ornithine is the molecular inducer for RocR activity. Our work suggests that binding of ATP to RocR triggers its hexamerization, and binding of ornithine then allows ATP hydrolysis and activation of roc gene transcription. Thus, ornithine is the central molecule of the roc degradative pathway as it is the common intermediate of arginine and citrulline degradation and the molecular effector for the transcription regulator RocR. IMPORTANCEAmino acids serve as building blocks for protein biosynthesis in each living cell but can also be used as sources of carbon, energy and nitrogen. In this work we have identified ornithine as the central player in the utilization of arginine, citrulline and ornithine in the Gram-positive bacterium B. subtilis. Ornithine is the common intermediate after the first steps of arginine and citrulline degradation. We have identified the so far uncharacterized protein RocB as the enzyme responsible for the cleavage of citrulline to ornithine and carbamate. Moreover, we demonstrate that ornithine is the molecular effector that triggers ATPase activity of the transcription factor RocR. Binding of ornithine to RocR and the subsequent hydrolysis of ATP allow a functional interaction with the alternative sigma factor SigL and subsequent transcription activation of all genes of the degradative pathway.

microbiology↗