Biology subjects

Herzberg, C.

Publications and source records attributed to Herzberg, C..

7 recordsLinked to original sources

bioRxiv2025

Photo-leucine incorporation in Bacillus subtilis: Identification of transporters and of a selectivity determinant in the Leu-tRNA synthetase

The elucidation of the intricate network of proteins is essential for our understanding of cellular function. In vivo crosslinking is a powerful tool for probing these interactions, with photoactivatable amino acids representing a promising next step. Here, we evaluated the application of photo-leucine in the gram-positive model organism Bacillus subtilis. This amino acid analog is incorporated into proteins, but high concentrations inhibit the growth of B. subtilis. We investigated photo-leucine homeostasis and identified the branched-chain amino acid importers BcaP and BraB as well as the bipartite exporter AzlCD as key players of its uptake and export, respectively. Additionally, we identified a previously uncharacterized exporter, AexB, a member of the "sleeping beauty" group of EamA amino acid exporters. Expression of the aexB gene is positively regulated by the transcription factor AerB, that is normally normally inactive. Selective pressure by photo-leucine triggered the acqusition of a point mutation in aerB, allowing AerB to activate aexB expression. Mutants highly resistant to photo-leucine carried a single amino acid exchange, Ala-494 to Thr, in the leucine-tRNA synthetase LeuS. Molecular docking analysis revealed that this mutation alters the leucine-binding pocket, in a manner that still allowed binding of leucine, but not photo-leucine. While high photo-leucine incorporation is beneficial for effective crosslinking, our results suggest that B. subtilis intrinsically limits its incorporation to maintain cellular tolerance. Thus, evolutionary processes provide adaptive mechanisms to adjust amino acid analogue incorporation to tolerable levels, making photo amino acids a candidate for life cell investigation of protein struture and interactions.

microbiology↗

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

c-di-AMP determines the hierarchical organization of bacterial RCK proteins

RCK domains or proteins are important regulatory components of cation and K+ channels and transporters both in eukaryotic and prokaryotic organisms. A lasting unanswered question about bacterial RCK proteins relates to the physiological role of the multiple, sometimes closely related, RCK genes encoded in the genome. We explored this question with the Ktr channels of Bacillus subtilis that include two genes encoding RCK proteins (KtrA and KtrC) and two genes encoding their membrane protein partners (KtrB and KtrD). Using a combination of in vivo characterization and in vitro functional analysis, we determined that the two RCK proteins are neither physiologically redundant or functionally equivalent. Instead, KtrC is the physiologically dominant RCK protein due to its ability to mediate K+ transport inactivation by c-di-AMP, a bacterial second messenger that is the master regulator of the K+ machinery in many species, while KtrA assembled channels are very insensitive to the dinucleotide. Moreover, KtrC and KtrA can form heteromeric assemblies that can control the Ktr channel activity and are sensitive to c-di-AMP inhibition. In parallel, we showed that conditions with a large number of Ktr channels assembled with KtrA, or with RCK proteins that do not mediate c-di-AMP inhibition, are toxic to the cell. Altogether, we have demonstrated that c-di-AMP regulation of Ktr channels goes beyond affecting transcription and functional activity, it also determines the hierarchical organization of bacterial RCK proteins.

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↗

bioRxiv2020

Essentiality of c-di-AMP in Bacillus subtilis: Bypassing mutations converge in potassium and glutamate homeostasis

In order to adjust to changing environmental conditions, bacteria use nucleotide second messengers to transduce external signals and translate them into a specific cellular response. Cyclic di-adenosine monophosphate (c-di-AMP) is the only known essential nucleotide second messenger. In addition to the well-established role of this second messenger in the control of potassium homeostasis, we observed that glutamate is as toxic as potassium for a c-di-AMP-free strain of the Gram-positive model bacterium Bacillus subtilis. In this work, we isolated suppressor mutants that allow growth of a c-di-AMP-free strain under these toxic conditions. Characterization of glutamate resistant suppressors revealed that they contain pairs of mutations, in most cases affecting glutamate and potassium homeostasis. Among these mutations, several independent mutations affected a novel glutamate transporter, AimA (Amino acid importer A, formerly YbeC). This protein is the major transporter for glutamate and serine in B. subtilis. Unexpectedly, some of the isolated suppressor mutants could suppress glutamate toxicity by a combination of mutations that affect phospholipid biosynthesis and a specific gain-of-function mutation of a mechanosensitive channel of small conductance (YfkC) suggesting the acquisition of a device for glutamate export. Cultivation of the c-di-AMP-free strain on complex medium was an even greater challenge because the amounts of potassium, glutamate, and other osmolytes are substantially higher than in minimal mediu. Suppressor mutants viable on complex medium could only be isolated under anaerobic conditions if one of the two c-di-AMP receptor proteins, DarA or DarB, was absent. Also on complex medium, potassium and osmolyte toxicity are the major bottlenecks for the growth of B. subtilis in the absence of c-di-AMP. Our results indicate that the essentiality of c-di-AMP in B. subtilis is caused by the global impact of the second messenger nucleotide on different aspects of cellular physiology. AUTHOR SUMMARYBacteria are exposed to constantly changing environmental conditions. In order to respond to these changes, they use nucleotide second messengers to transduce external signals and translate them into a specific cellular response. Among the repertoire of bacterial second messenger nucleotides, cyclic di-AMP (c-di-AMP) stands out as it is the only second messenger that is essential for the bacteria that produce it, including the Gram-positive model organism Bacillus subtilis. C-di-AMP plays a major role in the control of potassium homeostasis, and we found that glutamate is toxic to a B. subtilis strain lacking c-di-AMP to the same extent as potassium. These toxic conditions were the starting point for an extensive suppressor analysis, which led to the identification of a novel glutamate transporter (AimA). If the B. subtilis strain lacking c-di-AMP was cultivated on complex medium, the isolation of suppressor mutants was only possible under anaerobic conditions and if either of the two c-di-AMP-binding signal transduction proteins was absent. This suggests that these proteins are a major burden for the cell on complex medium in their c-di-AMP free state. Our result underline the complexity of c-di-AMP signaling and propose new directions for research.

microbiology↗

bioRxiv2020

A rendezvous of two second messengers: The c-di-AMP receptor protein DarB controls (p)ppGpp synthesis in Bacillus subtilis

Many bacteria use cyclic di-AMP as a second messenger to control potassium and osmotic homeostasis. In Bacillus subtilis, several c-di-AMP binding proteins and RNA molecules have been identified. Most of these targets play a role in controlling potassium uptake and export. In addition, c-di-AMP binds to two conserved target proteins of unknown function, DarA and DarB, that exclusively consist of the c-di-AMP binding domain. Most likely these proteins transduce their signal by regulatory interactions with other proteins. Here, we have investigated the function of the c-di-AMP-binding protein DarB in B. subtilis, a protein consisting of two CBS (cystathionine-beta synthase) domains. We have used an unbiased search for DarB interaction partners and identified the (p)ppGpp synthetase/hydrolase Rel as a major interaction partner of DarB. (p)ppGpp is another second messenger that is formed upon amino acid starvation and under other stress conditions to stop translation and active metabolism. The interaction between DarB and Rel only takes place if the bacteria grow at very low potassium concentrations and intracellular levels of c-di-AMP are low. Indeed, c-di-AMP inhibits the binding of DarB to Rel. The interaction results in the Rel-dependent accumulation of pppGpp. Our results link potassium and c-di-AMP signaling to the stringent response and thus to the global control of cellular physiology.

microbiology↗