Search Results (53)

Background: The intracellular pathogen Brucella requires biotin for survival, yet the role of its de novo synthesis intermediate enzyme, BioA, in virulence remains undefined. This study investigates the contribution of BioA to the pathogenicity of Brucella abortus. Methods: We constructed a ΔBioA mutant in Brucella abortus 104M via homologous recombination and characterized its phenotype using growth assays, electron microscopy, macrophage infection models, and murine splenic colonization. Virulence gene expression was quantified by RT-qPCR. Results: The ΔBioA mutant exhibited severe growth auxotrophy in a biotin-deficient medium and displayed damaged outer membrane integrity. Furthermore, intracellular survival in macrophages was reduced by approximately 95% compared to the wild-type strain at 48 h post-infection. Notably, mice infected with the mutant showed a significant decrease in both splenic bacterial loads and spleen weight at 3 weeks, concomitant with a marked downregulation of VirB type IV secretion system (T4SS) genes. Conclusions: This study is the first to identify BioA as a critical nexus linking biotin metabolism to Brucella virulence. We demonstrate that BioA deficiency attenuates pathogenicity by impairing both structural integrity and the transcription of key virulence-related genes (VirB operon), thereby nominating BioA as a novel and promising target for anti-brucellosis interventions. Full article

Growth, reproduction, and survival are fundamental biological priorities that animals balance by evaluating dietary cues. Cholesterol occupies a unique position among nutrients, serving both as a structural component of cellular membranes and as a precursor for steroid hormones, yet its regulation differs fundamentally across taxa. In mammals, cholesterol availability is buffered by endogenous synthesis and post-ingestive metabolic control. In contrast, insects have evolutionarily lost sterol biosynthesis and are therefore sterol auxotrophs that rely entirely on dietary sources. Here, we synthesize current understanding of cholesterol biology in Drosophila melanogaster, with a focus on sterol auxotrophy, life-stage–specific allocation, and endocrine and lifespan outcomes in a comparative framework. We highlight cholesterol not only as a metabolic substrate but also as a sensory-encoded nutrient that shapes feeding behavior. We further examine how lipophorin (Lpp)-mediated transport, Niemann–Pick type C proteins, ATP-binding cassette transporters, and the nuclear receptor DHR96 coordinate systemic sterol distribution and endocrine output in the absence of endogenous synthesis. By integrating these mechanisms across development, we illustrate how cholesterol availability governs larval growth, ecdysteroid production, adult reproduction, neural function, and lifespan through coupled endocrine and nutrient-signaling networks. This review positions cholesterol as a multifunctional signal linking sensory perception, metabolic regulation, and life-history strategy in sterol-auxotrophic insects, offering a framework for understanding how evolutionary loss of biosynthetic capacity reshapes nutrient sensing and homeostatic control. Full article

Background/Objectives: Cancer is predicted to become the leading cause of premature mortality worldwide within this century. Among the hallmarks of cancer, metabolic reprogramming has received growing attention, and arginine deprivation therapy (ADT) represents a potential treatment strategy for tumors exhibiting arginine auxotrophy. Colorectal cancer cells frequently suppress the expression of argininosuccinate synthetase 1 (ASS1), rendering them dependent on extracellular arginine. However, how CRC cells adapt to and resist ADT remains largely unknown. Methods: We combined ATAC-seq and RNA-seq analyses with multiple functional assays—including CCK-8 viability, apoptosis detection, wound-healing, and transwell migration tests—to investigate the molecular basis of ADT response in cancer cells. Results: ADT markedly inhibited cancer cell proliferation (p < 0.001) and motility (p < 0.05) across three cell lines. Integrative multi-omics analyses revealed substantial chromatin remodeling and transcriptional reprogramming under ADT, with differentially expressed genes enriched in autophagy and cell-growth-related pathways. Among these, the motif CGTTTCCGGT was identified as an arginine deficiency-responsive DNA element in cancer cells, and C11orf54 showed pronounced downregulation accompanied by reduced chromatin accessibility at its genomic locus. Conclusions: These findings suggest that ADT restricts cancer cell proliferation and migration through chromatin remodeling mediated by the motif CGTTTCCGGT and the downregulation of C11orf54, identifying C11orf54 as a potential target for enhancing the efficacy of arginine deprivation therapy in cancer cells. Full article

Simultaneous conversion of syngas and sugars is a promising approach to overcome limitations of syngas fermentation. Clostridium autoethanogenum LAbrini, obtained by adaptive laboratory evolution, is known to show improved autotrophic process performance. Under purely autotrophic conditions, C. autoethanogenum LAbrini exhibits substantially faster growth and biomass formation compared to the wild-type in fully controlled, stirred-tank bioreactors with a continuous gas supply. In mixotrophic processes, the pre-culture strategy has a significant impact on the growth and metabolic activity of C. autoethanogenum LAbrini. C. autoethanogenum LAbrini can metabolize sugars (D-fructose, D-xylose, or L-arabinose) and CO simultaneously. All mixotrophic batch processes showed increased growth and product formation compared to the autotrophic process. The mixotrophic batch process with D-fructose enabled superior production of alcohols (10.7 g L⁻¹ ethanol and 3.2 g L⁻¹ D-2,3-butanediol) with a heterotrophic pre-culture. Using an autotrophic pre-culture and L-arabinose resulted in a total alcohol formation of more than 13 g L⁻¹. The formation of meso-2,3-butanediol (>0.50 g L⁻¹) occurred exclusively under mixotrophic conditions. Thus, C. autoethanogenum LAbrini, clearly representing notable improvements over the wild-type strain in mixotrophic batch processes, offers a good basis for further strain improvements to shift the product range even further towards more reduced products. Full article

Background/Objectives: Despite the introduction of targeted therapies such as Nivolumab, survival outcomes for patients with esophageal adenocarcinoma remain poor. During tumorigenesis, some tumors develop auxotrophy by downregulation of Argininosuccinate Synthetase-1 (ASS1), making them reliant on external arginine supply and thus potentially susceptible to arginine deprivation therapy. Arginine deprivation therapy with agents such as pegargiminase has shown improved survival in patients with pleural mesothelioma exhibiting ASS1 loss in tumor cells. Therefore, we investigated the prevalence of ASS1 loss in esophageal adenocarcinoma. Methods: First, we compared the staining patterns of three antibodies for ASS1 with RNA in situ Scope analysis results to identify the most reliable antibody for ASS1 immunohistochemical staining in esophageal adenocarcinoma. Subsequently, we performed ASS1 immunohistochemical staining on samples from 97 patients who underwent curative resection. The staining results were classified into three categories based on expression levels: negative, low-positive, and positive. Results: Among all included patients, 6.2% exhibited an ASS1 loss, and 6.2% showed low ASS1 expression. Notably, patients with an ASS1 loss did not demonstrate a response to neoadjuvant therapy. Patients with ASS1 loss or low expression were significantly younger. Conclusions: Our findings indicate that approximately 12.4% of patients with esophageal adenocarcinoma may be eligible and could potentially benefit from arginine deprivation therapy. This underscores the urgent need for clinical trials evaluating the efficacy of pegargiminase in this patient population. Additionally, incorporating ASS1 immunohistochemical staining into pre-neoadjuvant biopsy assessments should be considered to optimize neoadjuvant treatment strategies and advance the implementation of personalized cancer therapy. Full article

All animals have outsourced about half of the 20 proteinogenic amino acids (AAs). We recently demonstrated that the loss of biosynthetic pathways for these outsourced AAs is driven by energy-saving selection. Paradoxically, these metabolic simplifications enabled animals to use costly AAs more frequently in their proteomes, allowing them to explore sequence space more freely. Based on these findings, we proposed that environmental AA availability and cellular respiration mode are the two primary factors determining the evolution of AA auxotrophies in animals. Remarkably, our recent analysis showed that bacterial AA auxotrophies are also governed by energy-related selection, thereby roughly converging with animals. However, bacterial AA auxotrophies are highly heterogeneous and scattered across the bacterial phylogeny, making direct ecological and physiological comparisons with the animal AA outsourcing model challenging. To better test the universality of our model, we focused on Bdellovibrionota and Myxococcota—two closely related bacterial phyla that, through aerobic respiration and a predatory lifestyle, best parallel animals. Here, we show that Bdellovibrionota, driven by energy-related selection, outsourced a highly similar set of AAs to those in animals. This sharply contrasts with Myxococcota, which exhibit far fewer AA auxotrophies and rarely show signatures of energy-driven selection. These differences are also reflected in Bdellovibrionota proteomes, which are substantially more expensive than those of Myxococcota. Finally, we found evidence that the expression of costly proteins plays a crucial role in the predatory phase of the Bdellovibrio life cycle. Together, our findings suggest that Bdellovibrionota, through their obligate predatory lifestyle, exhibit the closest analogy to the AA auxotrophy phenotype observed in animals. In contrast, facultative predation, as seen in Myxococcota, appears to substantially limit the evolution of AA auxotrophies. These cross-domain convergences strongly support the general validity of our AA outsourcing model. Full article

The outsourcing of amino acid (AA) production to the environment is relatively common across the tree of life. We recently showed that the massive loss of AA synthesis capabilities in animals is governed by selective pressure linked to the energy costs of AA production. Paradoxically, these AA auxotrophies facilitated the evolution of costlier proteomes in animals by enabling the increased use of energetically expensive AAs. Experiments in bacteria have shown that AA auxotrophies can provide a fitness advantage in competition with prototrophic strains. However, it remains unclear whether energy-related selection also drives the evolution of bacterial AA auxotrophies and whether this affects the usage of expensive AAs in bacterial proteomes. To investigate these questions, we computationally determined AA auxotrophy odds across 980 bacterial genomes representing diverse taxa and calculated the energy costs of all their proteins. Here, we show that auxotrophic AAs are generally more expensive to synthesize than prototrophic AAs in bacteria. Moreover, we found that the cost of auxotrophic AAs significantly correlates with the cost of their respective proteomes. Interestingly, out of all considered taxa, Mollicutes and Borreliaceae—chronic pathogens highly successful in immune evasion—have the most AA auxotrophies and code for the most expensive proteomes. These findings indicate that AA auxotrophies in bacteria, similar to those in animals, are shaped by selective pressures related to energy management. Our study reveals that bacterial AA auxotrophies act as costly outsourced functions, enabling bacteria to explore protein sequence space more freely. It remains to be investigated whether this relaxed use of expensive AAs also enabled auxotrophic bacteria to evolve proteins with improved or novel functionality. Full article

N⁴-methylcytosine is a modified heterocyclic base present both in RNA and DNA. The biosynthesis and function of this derivative are widely investigated. However, how the demethylation of this base occurs is not known. Here, we have investigated the growth of an Escherichia coli uracil auxotroph strain in minimal M9 medium supplemented with N⁴-methylcytosine. We have found that this compound, but not the related N⁴,N⁴-dimethylcytosine, well supports growth with a generation time of the bacterium being 3 h compared to 1.5 h for media supplemented with cytosine or uracil. Using high-performance liquid chromatography (HPLC), we have demonstrated that the concentration of N⁴-methylcytosine in the growth medium decreases by 12% after 24 h of growth. We have shown that N⁴-methylcytosine is not directly converted into uracil by E. coli CodA cytosine deaminase. Instead, we propose the enzymatic pathway in which N⁴-methylcytosine is converted into cytosine by yet unknown demethylase, whereas CodA converts the resulting cytosine to uracil, thereby supporting the growth. Full article

The apple is a significant global fruit cultivated extensively worldwide. Fire blight, caused by Erwinia amylovora (Ea), poses a significant threat to global apple production. To control this disease, characterizing the virulence mechanisms/factors is imperative. Carbamoyl phosphate synthase is an essential enzyme in the biosynthesis of arginine and pyrimidine. However, the functions of this protein in Ea remains poorly understood. This study aimed to investigate the functions of the carbamoyl phosphate synthase large subunit in Ea (CarBEa). In a virulence assay using fruitlets, an Ea strain lacking CarBEa exhibited significantly reduced virulence on fruitlets. In the auxotrophy assay, this mutant failed to grow in minimal media lacking both arginine and pyrimidine, but growth was restored when both compounds were supplemented. The comparative proteomic analysis suggests that CarBEa is involved in diverse biological processes, including amino acid and nucleotide metabolism, and inorganic ion transport. Finally, we demonstrated that CarBEa is related to siderophore secretion/production by the chrome azurol S agar plate assay. This report provides valuable insights into the functions of carbamoyl phosphate synthase large subunit, which serves as a potential target for developing efficient anti-virulence substances to control fire blight. Full article

Background: Elongation factor protein (EF-P) in bacteria helps ribosomes to incorporate contiguous proline residues (xPro) into proteins. In this way, EF-P rescues ribosomes from stalling at these xPro motifs. Whereas EF-P deficiency is lethal for some species, others show reduced virulence or generally lower growth rates, such as Escherichia coli (E. coli). EF-P needs to be post-translationally modified to gain full functionality. Methods: We constructed E. coli K-12 mutant strains with deletion of the serA gene leading to an auxotrophy for L-serine. Then, we engineered a 6xPro motif in the recombinant serA gene, which was then chromosomally inserted under its native promoter. Furthermore, mutant strains which were deleted for efp and/or epmA (encoding the EF-P modification protein EpmA) were engineered. Results: Δefp, ΔepmA, and Δefp/ΔepmA double mutants showed already significantly reduced growth rates in minimal media. ΔserA derivatives of these strains were complemented by the wt serA gene but not by 6xPro-serA. ΔserA mutants with intact efp were complemented by all serA-constructs. Chromosomal expression of the recombinant efp gene from E. coli or from the pathogen, Staphylococcus aureus (S. aureus), restored growth, even without epmA expression. Conclusions: We provide a novel synthetic reporter system for in vivo evaluation of EF-P deficiency. In addition, we demonstrated that both EF-P-E. coli and EF-P-S. aureus restored the growth of a 6xPro-serA: Δefp, ΔepmA strain, which is evidence that modification of EF-P might be dispensable for rescuing of ribosomes stalled during translation of proline repeats. Full article

Since the development of recombinant DNA technologies, the need to establish biosafety and biosecurity measures to control genetically modified organisms has been clear. Auxotrophies, or conditional suicide switches, have been used as firewalls to avoid horizontal or vertical gene transfer, but their efficacy has important limitations. The use of xenobiological systems has been proposed as the ultimate biosafety tool to circumvent biosafety problems in genetically modified organisms. Xenobiology is a subfield of Synthetic Biology that aims to construct orthogonal biological systems based on alternative biochemistries. Establishing true orthogonality in cell-based or cell-free systems promises to improve and assure that we can progress in synthetic biology safely. Although a wide array of strategies for orthogonal genetic systems have been tested, the construction of a host harboring fully orthogonal genetic system, with all parts operating in an orchestrated, integrated, and controlled manner, still poses an extraordinary challenge for researchers. In this study, we have performed a thorough review of the current literature to present the main advances in the use of xenobiology as a strategy for biocontainment, expanding on the opportunities and challenges of this field of research. Full article

Recently, arginine has been proven to play an important role in ADPKD physiopathology. Arginine auxotrophy in ADPKD induces cell hyperproliferation, blocking the normal differentiation of renal tube cells and causing cyst formation. We explored the L-arginine (Arg)–nitric oxide (NO) molecular pathway in ADPKD, a multisystemic arginine auxotrophe disease. We developed a prospective case–control study that included a group of 62 ADPKD subjects with an estimated filtration rate over 60 mL/min/1.73 mp, 26 subjects with chronic kidney disease with an eGFR > 60 mL/min/1.73 mp, and a group of 37 healthy subjects. The laboratory determinations were the serum level of arginine, the enzymatic activity of arginase 2 and inducible nitric oxide synthase, the serum levels of the stable metabolites of nitric oxide (nitrate, direct nitrite, and total nitrite), and the endogenous inhibitors of nitric oxide synthesis (asymmetric dimethylarginine and symmetric dimethylarginine). In the ADPKD group, the levels of the arginine and nitric oxide metabolites were low, while the levels of the metabolization enzymes were higher compared to the control group. Statistical analysis of the correlations showed a positive association between the serum levels of Arg and the eGFR and a negative association between Arg and albuminuria. ADPKD is a metabolic kidney disease that is auxotrophic for arginine. Exploring arginine reprogramming and L-Arg–NO pathways could be an important element in the understanding of the pathogenesis and progression of ADPKD. Full article

Plasmid shuttle vectors are a common tool used to study yeast physiology. The majority of yeast plasmids have been optimized for Saccharomyces cerevisiae lab strain compatibility, relying on auxotrophic complementation as their selective property. We sought to construct a series of plasmid shuttle vectors to extend functionality beyond strains with auxotrophic requirements, and test compatibility across a diverse panel of yeasts. We constructed 18 plasmids which were successfully maintained by yeasts from several genera. From a panel of 24 yeast strains, these plasmids were maintained by 18 yeasts, spanning 11 species within the genera Lachancea, Metschnikowia, Pichia, Saccharomyces, and Torulaspora. Additionally, an integrated gene expression reporter was assayed for functional compatibility with the 18 strains. Plasmid-derived gene expression was observed for 13 strains, spanning five species within the Saccharomyces genus, in addition to Torulaspora delbrueckii. These results indicate that this plasmid series is broadly useful for advancements and applications within academia, biotechnology, and the food and fermentation industries for research utilizing diverse Saccharomyces and non-Saccharomyces yeasts. Full article

The parasites Trypanosoma brucei (Tb) and Leishmania major (Lm) cause the tropical diseases sleeping sickness, nagana, and cutaneous leishmaniasis. Every year, millions of humans, as well as animals, living in tropical to subtropical climates fall victim to these illnesses’ health threats. The parasites’ frequent drug resistance and widely spread natural reservoirs heavily impede disease prevention and treatment. Due to pteridine auxotrophy, trypanosomatid parasites have developed a peculiar enzyme system consisting of dihydrofolate reductase-thymidylate synthase (DHFR-TS) and pteridine reductase 1 (PTR1) to support cell survival. Extending our previous studies, we conducted a comparative study of the T. brucei (TbDHFR, TbPTR1) and L. major (LmDHFR, LmPTR1) enzymes to identify lead structures with a dual inhibitory effect. A pharmacophore-based in silico screening of three natural product databases (approximately 4880 compounds) was performed to preselect possible inhibitors. Building on the in silico results, the inhibitory potential of promising compounds was verified in vitro against the recombinant DHFR and PTR1 of both parasites using spectrophotometric enzyme assays. Twelve compounds were identified as dual inhibitors against the Tb enzymes (0.2 μM < IC₅₀ < 85.1 μM) and ten against the respective Lm enzymes (0.6 μM < IC₅₀ < 84.5 μM). These highly promising results may represent the starting point for the future development of new leads and drugs utilizing the trypanosomatid pteridine metabolism as a target. Full article

While modern molecular methods have decisively accelerated and improved microbiological diagnostics, phenotypic variants still pose a challenge for their detection, identification and characterization. This particularly applies if they are unstable and hard to detect, which is the case for the small-colony-variant (SCV) phenotype formed by staphylococci. On solid agar media, staphylococcal SCVs are characterized by tiny colonies with deviant colony morphology. Their reduced growth rate and fundamental metabolic changes are the result of their adaptation to an intracellular lifestyle, regularly leading to specific auxotrophies, such as for menadione, hemin or thymidine. These alterations make SCVs difficult to recognize and render physiological, biochemical and other growth-based methods such as antimicrobial susceptibility testing unreliable or unusable. Therefore, diagnostic procedures require prolonged incubation times and, if possible, confirmation by molecular methods. A special approach is needed for auxotrophy testing. However, standardized protocols for SCV diagnostics are missing. If available, SCVs and their putative parental isolates should be genotyped to determine clonality. Since their detection has significant implications for the treatment of the infection, which is usually chronic and relapsing, SCV findings should be specifically reported, commented on, and managed in close collaboration with the microbiological laboratory and the involved clinicians. Full article