Biology subjects

Warneke, R.

Publications and source records attributed to Warneke, R..

10 recordsLinked to original sources

bioRxiv2026

House fly proto-Y chromosomes differentially affect courtship success regardless of social context and female type

Genetic variation exists for many male reproductive traits despite apparent strong sexual selection in favor of particular males. Understanding how that variation is maintained is an unresolved problem in evolutionary biology. One proposed solution to this problem is genotype-by-environment interactions (GEIs), where no male genotype is universally superior across environments. Another proposed answer is variation in female preference, such that different females prefer different male genotypes. Additionally, male genotypes may differ in their reproductive output as they age. The house fly Musca domestica is a well-suited system to evaluate these explanations for genetic variation in male reproductive traits because two male genotypes (YM and IIIM) differ in mating performance. Specifically, IIIM males outcompete YM males in mating assays because IIIM males have a shorter copulation latency. To determine the relative importance of GEIs, female variation, and male age on the maintenance of genetic variation in male reproductive behaviors, we performed non-competitive (one male to one female) and competitive (one IIIM male and one YM male to one female) mating assays while considering multiple ages and different female strains. IIIM males attempted more matings regardless of competition level, female genotype or age, but mated faster than YM males only with females of their own strain and only performed more matings in non-competitive trials. However, female strain did not improve the mating success of YM males. We also did not observe evidence for genotype-by-age interactions on male mating success. Importantly, we did not identify any conditions where YM males outcompeted IIIM males, and therefore there was no evidence for interactions or trade-offs that could explain the maintenance of genetic variation for male mating success. To explain the maintenance of IIIM and YM males, other factors such as additional social environments or varying temperature should be investigated, as the genotypes are found in natural environments with contrasting temperatures.

evolutionary biology↗

bioRxiv2026

KlinkPPI: Single Point of Access to Protein-Protein Interactions Across Databases

Protein-protein interaction (PPI) information is distributed across resources that differ in organism coverage, identifier systems, evidence models, confidence scores and access mechanisms, so assembling and comparing evidence for a protein requires source-specific queries, identifier conversion and extensive post-processing. We present KlinkPPI, a web server that retrieves, compares and exports PPI evidence from STRING, BioGRID, IntAct, CORUM, HuRI and Predictomes from a single query. KlinkPPI accepts UniProtKB accessions, NCBI Gene and Ensembl identifiers and gene names, and performs taxonomy-aware mapping to a common identifier space. Users can query individual proteins across all resources available for an organism, or retrieve organism-wide interaction sets. Results are presented per source so that database-specific evidence, annotations and confidence values are retained, while an integrated view exposes coverage and agreement between resources. KlinkPPI deliberately does not merge heterogeneous confidence scores, nor collapse functional associations, complex co-membership, binary interactions and structural predictions into a single consensus network. Results can be exported in PSI-MI TAB 2.8-compatible or Apache Parquet format with user-selected evidence fields. KlinkPPI is freely available at https://rappsilberlab.org/KlinkPPI/ and the source code at https://github.com/Rappsilber-Laboratory/KlinkPPI. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/742057v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@12dcaceorg.highwire.dtl.DTLVardef@15d9d0aorg.highwire.dtl.DTLVardef@f0fc39org.highwire.dtl.DTLVardef@13f172e_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2026

RapCluster: Bridging the Reproducibility Gap in Clustering Analysis

Clustering is ubiquitous across science, yet a text-mining audit of 736,399 open-access articles identified as using clustering (2000-2025) reveals common practice leaves key parameters undocumented or untuned, contributing to the reproducibility crisis in science. We developed an interactive web platform featuring 11 widely adopted clustering algorithms to enable transparent clustering analysis and reporting, aligning practical use with best practices in computational research. Code availabilityThe browser-based clustering analysis platform RapCluster is available for download at https://github.com/lutfia95/RapCluster under MIT License and accessible at https://rappsilberlab.org/rapcluster/ The web version is capacity-limited to 8 GB of memory (ca. 12,000 candidates with 130 features). All source codes used for the analysis are included with the publication.

bioinformatics↗

bioRxiv2026

The evolution of a Na+-sensitive Vibrio cholerae mutant unmasks the moonlighting aminopeptidase PepA as a regulator of nhaB Na+/H+ antiporter gene expression

The pathogenic bacterium Vibrio cholerae is native to seawater and can therefore be cultivated in nutrient media with increased salt concentration. To maintain osmotic balance, V. cholerae uses Na+/H+ antiporters and an Na+-translocating NADH:quinone oxidoreductase (Na+-NQR). The exact contribution of the various Na+/H+ antiporters to maintain Na+ and H+ homeostasis in V. cholerae is unclear. However, genetic studies indicate the major Na+/H+ antiporter NhaA and the Na+-NQR are required by the bacteria for Na+ resistance at alkaline conditions. Here we show that the growth defect of a V. cholerae nhaA nqr mutant at increased Na+ concentrations and alkaline pH is relieved by the rapid acquisition of suppressor mutations. The suppressor mutants could be assigned to two classes. We identified (i) mutations in the promoter of the nhaB gene encoding a Na+/H+ antiporter and (ii) mutations that either affect the expression level of the pepA gene or DNA binding activity of the encoded multifunctional aminopeptidase PepA. The characterization of the PnhaB and PpepA promoters of the suppressor mutants, membrane potential measurements and comparative proteome analyses identified PepA as a novel factor controlling Na+ homeostasis in V. cholerae. IMPORTANCEThe emergence and spread of multi-resistant bacteria are a major problem for humans and animals. Therefore, the identification of novel targets for the development of novel antibiotics to combat pathogenic bacteria is extremely important. Since sodium ion homeostasis is an essential process in many pathogenic bacteria, especially those living in marine environments, it represents an interesting target for antibiotics. The perturbation of sodium ion homeostasis does indeed impair the fitness of the human pathogenic bacterium Vibrio cholerae but can be restored by the rapid evolution of the bacteria. This suggests that inhibiting multiple targets with different antibiotics might be more effective in preventing the development of resistant bacteria.

microbiology↗

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

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↗