Biology subjects

Link, H.

Publications and source records attributed to Link, H..

At least 19 recordsLinked to original sources

bioRxiv2026

A host-directed virulence factor of Clostridium perfringens is modulated by gut commensal strains

In a healthy host, the residential microbes help regulate the growth of pathobionts, which are common members of the human gut microbiome, preventing them from causing diseases, including infections, under certain conditions. In cases of dysbiosis, this protection may be compromised. Targeted microbiome modulation offers a promising approach to restore healthy conditions in a disrupted community and consequently prevent infections using the natural colonization resistance of the microbiome. Elucidating the interaction mechanisms between microbial species within a microbiome is crucial for understanding how a microbiome can be modulated precisely and effectively to benefit the hosts well-being. Here, we investigated the interactions between the pathobiont C. perfringens and human gut commensals on physiological and molecular levels. We found that commensal strains affect C. perfringens growth by competing for substrates such as amino acids or a carbon source other than glucose. We further observed that Bacteroidaceae strains altered the levels of C. perfringens proteins, among others, the host-directed {theta}-toxin. Our findings reinforce the notion that modulating the composition of the gut microbiome is an effective strategy to prevent infections.

microbiology↗

bioRxiv2026

Development of a continuous bioreactor to maintain stable nasal microbiomes from swab specimens and synthetic communities

BackgroundThe nasal microbiome is a collection of diverse microbial populations that inhabit the nose. Staphylococcus aureus is the most common opportunistic pathogen that colonizes the nasal mucosa, increasing the risk of invasive infections in immunocompromised and hospitalized patients. Clinicians usually prescribe antibiotics to decolonize the nasal cavities of at-risk patients from S. aureus. However, their broad antimicrobial activity can damage the resident nasal microbiome. Instead, naturally occurring compounds or resident bacteria in nasal microbiomes can effectively and safely exclude S. aureus from the nose. Cell culture and animal models have been used for nasal microbiome studies. However, their unstable microbiomes reduce the accuracy and reliability of the results. Recently, continuous bioreactors have been proposed as alternatives to these models. ResultsWe designed and operated a continuous bioreactor system to maintain stable nasal microbiomes. Next, we inoculated the bioreactor with nasal-swab specimens that we had collected from healthy volunteers. We operated the bioreactors under varying conditions (i.e., operating mode, dilution rate, temperature, pH, and medium composition), and determined the optimal conditions (continuous mode, 1 d-1, 30xlink:href=" pH 6.5, and synthetic nasal medium 3), resulting in stable microbiomes consisting of the main nasal bacterial species. The nasal microbiomes in the optimized bioreactors showed high reproducibility and resilience during a pH perturbation. Moreover, all microbiomes in the bioreactor, which were inoculated with six different nasal-swab specimens, maintained stable bacterial and metabolite compositions. In addition, we applied a synthetic microbial community (SynCom), which was derived from one of the volunteers, to demonstrate a S. aureus decolonization strategy. The bioreactor, inoculated with this SynCom, maintained a stable nasal microbiome for more than one month. Finally, different S. aureus strains that we inoculated in the SynCom showed distinct growth patterns within the otherwise stable community. ConclusionsThe continuous bioreactor enables the cultivation of stable nasal microbiomes for longer than one month by mimicking the environmental conditions of the human nose. The bioreactor is a valuable model for understanding the functions of the nasal microbiome and devising new decolonization strategies against S. aureus.

bioengineering↗

bioRxiv2026

Community composition and strain identity drive metabolic competition and Staphylococcus aureus colonization resistance in Synthetic Nasal Communities

The human nasal microbiome is a low-diversity ecosystem whose assembly principles and mechanisms of colonization resistance remain poorly understood. Staphylococcus aureus is a member of the nasal microbiome of some individuals with variable abundance. We hypothesized that nutritional competition, strain-level diversity, and nutrient availability shape community stability and the ability of commensal species to inhibit S. aureus. To test this, we constructed 50 defined synthetic communities composed of representative human nasal bacteria differing in strain and species composition and tracked their temporal dynamics, S. aureus growth, metabolic profiles, and nutritional interactions. The composition of synthetic communities with 5-10 species showed robust and reproducible dynamics and converged in one of three stable states. Synthetic communities dominated by a specific strain of Corynebacterium propinquum were highly stable and consistently excluded S. aureus. Growth curves and coculture assays showed that C. propinquum outcompetes S. aureus under poor nutritional conditions resembling the nasal environment, whereas S. aureus dominates in nutrient-rich conditions. Metabolomics analyses revealed that nutritional competition, including siderophores utilization and amino acid limitation, likely underlies this colonization resistance. These results establish a tractable synthetic community model for the human nasal microbiome and identify nutrient-dependent competition and microbial metabolite production as key drivers of community structure and pathogen exclusion.

microbiology↗

bioRxiv2025

Synergy between Enterobacteriaceae and dietary components shapes the competition between the dominant anaerobes of the human gut

The human gut microbiota is frequently dominated by the Bacteroidota phylum, with Bacteroidaceae and Prevotellaceae families prevailing in modern and traditional lifestyles, respectively. While these distinct profiles have been well-documented across cohorts, the underlying mechanisms remain poorly understood. Here, we combine defined gut community models and genetic tools to experimentally dissect the ecological principles that favor Prevotellaceae or Bacteroidaceae. Testing 94 dietary components in the community model shows these two families compete for overlapping nutritional niches. Notably, Segatella copri, a key Prevotellaceae representative, outcompetes Bacteroidaceae strains when preferred polysaccharides are available. Interestingly, this competitive advantage depends on the presence of Enterobacteriaceae, which synergize with dietary components to promote Segatella dominance. Accordingly, we observe that global Segatella-rich non-Westernized microbiomes correlate with higher Enterobacteriaceae content. Our results suggest a revised hypothesis in which the ecosystem-level dynamic interplay between dietary components and key commensals within the community contributes to distinct Bacteroidota family dominance.

microbiology↗

bioRxiv2025

Tyrosine availability shapes Staphylococcus aureus nasal colonization and interactions with commensal communities

Only certain individuals are nasally colonized by Staphylococcus aureus, making them more susceptible to developing endogenous infections. The role of the resident microbiota in modulating S. aureus nasal colonization remains largely unclear. Using a newly assembled nasal strain collection, we demonstrate that nasal commensals can either promote or inhibit S. aureus proliferation through strain- and community-specific effects in vitro and within gnotobiotic animals. We identified nutrient-dependent interactions as a key driver of these dynamics and found that S. aureus strains auxotrophic to tyrosine rely on amino acid-providing nasal communities to thrive in the nutrient-limited environment of the nose. Large-scale screenings revealed tyrosine dependency to affect 8% of nasal S. aureus strains and to impair S. aureus colonization. Our findings uncover a crucial factor in S. aureus physiology and its interactions as a commensal nasal species, paving the way for new decolonization strategies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/651429v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@17b6289org.highwire.dtl.DTLVardef@182249forg.highwire.dtl.DTLVardef@1324dc1org.highwire.dtl.DTLVardef@18ec3bf_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Native metabolomics identifies pteridines as CutA ligands and modulators of copper binding

CutA, a conserved protein across all domains of life, has long been linked to copper tolerance in Escherichia coli, though recent studies question this association. To clarify its function, we studied cutA knockout mutants from two phylogenetically distant species, E. coli and Synechococcus elongatus PCC 7942, using phenotyping combined with targeted and untargeted metabolomics. Native metabolomics of cell extracts revealed the lumazine dehydroxyxanthopterin B2, a previously uncharacterized pteridine, to bind CutA in both species. Based on these results, we identified other pteridines, including the essential cofactor tetrahydrobiopterin, as ligands of CutA proteins. In the presence of pterins, we observed higher affinity of CutA to copper ions. These findings, alongside the known role of pteridines as redox shuttles, suggest a previously unrecognized role for CutA in coordinating copper homeostasis and redox balance via pteridine metabolism. SignificanceWe identified the molecular class of pteridines as natural ligands of CutA, including the so far unknown lumazine dehydroxyxanthopterin B2. Pteridines are known redox shuttles involved in various cellular processes such as cofactors for redox enzymes. Our data showed increased copper binding to CutA in the presence of pteridines. Together, these results suggest that pteridines are physiological ligands of CutA that may modulate copper binding and redox homeostasis.

microbiology↗

bioRxiv2025

Deciphering the metabolic details of L-lysine toxicity in cyanobacteria

L-lysine (Lys) has been explored as a potential cyanobactericide due to its inhibitory effects on cyanobacterial growth at micromolar concentrations, comparable to many antibiotics. Here, we investigated the early metabolic and physiological responses of the model cyanobacterium Synechocystis sp. PCC 6803 to Lys exposure. Physiological analyses revealed cell enlargement, oxidative stress, and photosynthesis inhibition, leading to growth arrest. Metabolomic profiling indicated disruptions in peptidoglycan biosynthesis, evidenced by the accumulation of L-/D-alanine, meso-diaminopimelate, and D-Ala-D-Ala, suggesting interference with cell wall integrity. Furthermore, levels of energy metabolites and other amino acids including tyrosine, tryptophan, valine, and iso-/leucine were significantly altered, implying broader metabolic impacts of Lys toxicity. To explore potential resistance mechanisms, we used a CRISPRi-based genetic screen to identify key genes involved in relieving Lys toxicity. The Bgt permease system, responsible for basic amino acid uptake, was essential for acquiring Lys-resistance, as a bgtA mutant exhibited a normal growth on elevated Lys concentrations, thereby validating our CRISPRi-screen. Additionally, UirR, a DNA-binding response regulator, and genes linked to c-di-AMP signaling, seemed implicated in Lys metabolism. Deletion of c-di-AMP synthase gene increased Lys sensitivity, supporting a role for c-di-AMP in cell wall homeostasis and osmotic stress regulation. Altogether, our findings explored the early metabolic responses and physiological consequences of Lys exposure in Synechocystis, demonstrating its effects on peptidoglycan biosynthesis, amino acid metabolism, and nucleotide biosynthesis. The identification of key genetic factors contributing to Lys resistance provides new insights into cyanobacterial physiology and the potential application of Lys in bloom control strategies.

microbiology↗

bioRxiv2024

Intact protein barcoding enables one-shot identification of CRISPRi strains and their metabolic state

Simultaneous measurements of library barcodes and metabolites can facilitate the screening of metabolically engineered bacterial libraries. Here, we introduce a scalable method for the parallel measurement of protein-based library barcodes and metabolites using flow-injection mass spectrometry (FI-MS). Protein barcodes are based on ubiquitin, a small 76-amino-acid protein, which we labeled with N-terminal tags of six amino acids to identify bacterial strains. We demonstrate that FI-MS effectively detects intact ubiquitin proteins and identifies the N-terminal barcodes. In the same analysis, we semi-quantify relative concentrations of primary metabolites. As a proof of concept, we engineered six ubiquitin-barcoded CRISPRi strains targeting metabolic enzymes, and analyzed their metabolic profiles and ubiquitin barcodes using FI-MS. Our results show distinct metabolome changes within metabolic pathways targeted by CRISPRi. This method enables fast and simultaneously detection of library barcodes and intracellular metabolites, opening up new possibilities to measure metabolomes of engineered bacteria at scale.

synthetic biology↗

bioRxiv2024

The SPFH complex HflK-HflC regulates aerobic respiration in bacteria

The bacterial HflK-HflC membrane complex is a member of the highly conserved SPFH protein family, which is found throughout all domains of life and includes eukaryotic stomatins, flotillins, and prohibitins. These proteins organize cell membranes and are involved in various processes. However, the exact physiological functions of most bacterial SPFH proteins remain unclear. Here, we report that the HflK-HflC complex in Escherichia coli is required for growth under high aeration. The absence of this complex causes an aerobic growth defect due to a reduced abundance of IspG, a crucial enzyme in the isoprenoid biosynthetic pathway. This reduction leads to lower levels of ubiquinone, reduced respiration, lower ATP levels, and misregulated expression of respiratory genes. The regulation of aerobic respiration by the HflK-HflC complex resembles the mitochondrial respiratory defects caused by prohibitin mutations in mammalian and yeast cells, suggesting a functional commonality between these bacterial and eukaryotic SPFH proteins.

microbiology↗

bioRxiv2024

(p)ppGpp-mediated GTP homeostasis ensures the survival and antibiotic tolerance of Staphylococcus aureus during starvation by preserving the proton motive force

Upon nutrient limitation bacteria enter a nongrowing state, which allow bacterial survival and antibiotic tolerance. The mechanisms whether and how the messenger molecule (p)ppGpp contributes to the transition in Firmicutes is debated. Here we show for Staphylococcus aureus that (p)ppGpp-dependent restriction of the GTP pool is essential for the culturability of starved cells and for antibiotic tolerance. Elevated GTP levels in a starving (p)ppGpp-deficient mutant lead to a division-incompetent, dormant state characterized by reduced metabolic activity and alterations in membrane function and architecture. GTP level control of nucleotide sensitive promoters result in transcriptional downregulation of gene of the TCA cycle and electron transport chain. Increasing transcription of qoxABCD, a terminal oxidase of the respiratory chain, through mutation of the transcriptional start site partially restored the culturability of the (p)ppGpp-deficient mutant. Furthermore, we showed that the maintenance of proton motive force under nutritional stress contributes to antibiotic tolerance, supporting the idea of applying (p)ppGpp or PMF inhibitors to combat antibiotic-tolerant bacteria.

microbiology↗

bioRxiv2023

Metabolic rewiring compensates for the loss of glutamate and aspartate biosynthesis in Bacillus subtilis

Glutamate serves as the major cellular amino group donor. In Bacillus subtilis, glutamate is synthesized by the combined action of the glutamine synthetase and the glutamate synthase (GOGAT). The glutamate dehydrogenases are devoted to glutamate degradation in vivo. To keep the cellular glutamate concentration high, the genes and the encoded enzymes involved in glutamate biosynthesis and degradation need to be tightly regulated depending on the available carbon and nitrogen sources. Serendipitously, we found that the inactivation of the ansR and citG genes encoding the repressor of the ansAB genes and the fumarase, respectively, enables the GOGAT-deficient B. subtilis mutant to synthesize glutamate via a non-canonical fumarate-based ammonium assimilation pathway. We also show that the de-repression of the ansAB genes is sufficient to restore aspartate prototrophy of an aspB aspartate transaminase mutant. Moreover, with excess nitrogen, B. subtilis mutants lacking fumarase activity show a growth defect that can be relieved by aspB overexpression, by reducing arginine uptake and by decreasing the metabolic flux through the TCA cycle. It will be interesting to investigate whether the B. subtilis strain using the alternative glutamate biosynthesis route can evolve in such a way that it robustly grows during nitrogen limitation and excess.

microbiology↗

bioRxiv2023

Purine nucleotide limitation undermines antibiotic action in clinical Escherichia coli

Metabolic variation across pathogenic bacterial strains can impact their susceptibility to antibiotics1-4 and promote evolution of antimicrobial resistance (AMR)5,6. However, little is known about which metabolic pathways contribute to AMR, and the underlying mechanisms. Here, we measured antibiotic resistance of 15,120 Escherichia coli mutants, each with a single amino acid change in one of 346 essential proteins. Most of the mutant strains that showed resistance to either of the two tested antibiotics carried mutations in metabolic genes. Resistance mutations against a {beta}-lactam antibiotic (carbenicillin) were associated with purine nucleotide biosynthesis and limited the supply of ATP. We show that ATP limitation confers both resistance and tolerance against {beta}-lactam antibiotics by upregulating the purine nucleoside transporter PunC. These results are clinically relevant, because an E. coli strain isolated from a clinical specimen had a purine nucleotide limitation, which reduced its susceptibility to antibiotics.

microbiology↗

bioRxiv2023

Autotrophic growth of E. coli is achieved by a small number of genetic changes

Synthetic autotrophy is a promising avenue to sustainable bioproduction from CO2. Here, we use iterative laboratory evolution to generate several distinct autotrophic strains. Utilising this genetic diversity, we identify that just three mutations are sufficient for E. coli to grow autotrophically, when introduced alongside non-native energy (formate dehydrogenase) and carbon-fixing (RuBisCO, phosphoribulokinase, carbonic anhydrase) modules. The mutated genes are involved in glycolysis (pgi), central-carbon regulation (crp), and RNA transcription (rpoB). The pgi mutation reduces the enzymes activity, thereby stabilising the carbon-fixing cycle by capping a major branching flux. For the other two mutations, we observe down-regulation of several metabolic pathways and increased expression of native genes associated with the carbon-fixing module (rpiB) and the energy module (fdoGH), as well as an increased ratio of NADH/NAD+ - the cycles electron-donor. This study demonstrates the malleability of metabolism and its capacity to switch trophic modes using only a small number of genetic changes and could facilitate transforming other heterotrophic organisms into autotrophs.

synthetic biology↗

bioRxiv2023

Mapping temperature-sensitive mutations at a genome-scale to engineer growth-switches in E. coli

Temperature-sensitive (TS) mutants are a unique tool to perturb and engineer cellular systems. Here, we constructed a CRISPR library with 15,120 Escherichia coli mutants, each with a single amino acid change in one of 346 essential proteins. 1,269 of these mutants showed temperature-sensitive growth in a time-resolved competition assay. We reconstructed 94 TS mutants and measured their metabolism under growth arrest at 42{degrees}C using metabolomics. Metabolome changes were strong and mutant-specific, showing that metabolism of non-growing E. coli is perturbation-dependent. For example, 24 TS mutants of metabolic enzymes overproduced the direct substrate-metabolite due to a bottleneck in their associated pathway. A strain with TS homoserine kinase (ThrBF267D) produced homoserine for 24 hours, and production was tunable by temperature. Finally, we used a TS subunit of DNA polymerase III (DnaXL289Q) to decouple growth from arginine overproduction in engineered E. coli. These results provide a strategy to identify TS mutants en masse and demonstrate their large potential to produce bacterial metabolites with non-growing cells.

systems biology↗

bioRxiv2023

Helicobacter pylori modulates heptose metabolite biosynthesis and heptose-dependent innate immune host cell activation by multiple mechanisms

Heptose metabolites including ADP-heptose are involved in bacterial lipopolysaccharide and cell envelope biosynthesis. Recently, heptoses were also identified to have potent pro-inflammatory activity on human cells as novel microbe-associated molecular patterns. The gastric pathogenic bacterium Helicobacter pylori produces heptose metabolites which it transports into human cells through its Cag type 4 secretion system. Using H. pylori as a model, we have addressed the question, how pro-inflammatory ADP-heptose biosynthesis can be regulated by the bacteria. We have characterized the inter-strain variability and regulation of heptose biosynthesis genes and the modulation of heptose metabolite production by H. pylori, which impact cell-autonomous pro-inflammatory human cell activation. HldE, a central enzyme of heptose metabolite biosynthesis, showed strong sequence variability between strains, and was also strain-variably expressed. Transcript amounts of genes in the hldE gene cluster displayed intra-strain and inter-strain differences, were modulated by host cell contact and the presence of the cag pathogenicity island, and were affected by carbon starvation regulator A (CsrA). We reconstituted four steps of the H. pylori LPS heptose biosynthetic pathway in vitro using recombinant purified GmhA, HldE and GmhB proteins. On the basis of one- and two-dimensional NMR spectroscopy and mass spectrometry, the structures of major reaction products were identified as {beta}-D-ADP-heptose and {beta}-heptose-1-monophosphate. A pro-inflammatory heptose-monophosphate variant was also identified for the first time as a novel cell-active product in H. pylori bacteria. Separate purified HldE subdomains and variant HldE allowed to uncover additional strain variation in generating heptose metabolites.

microbiology↗

bioRxiv2022

Systematic analysis of in-source modifications of primary metabolites during flow-injection time-of-flight mass spectrometry

Flow-injection mass spectrometry (FI-MS) enables metabolomics studies with a very high sample-throughput. However, FI-MS is prone to in-source modifications of analytes because samples are directly injected into the electrospray ionization source of a mass spectrometer without prior chromatographic separation. Here, we spiked authentic standards of 160 primary metabolites individually into an Escherichia coli metabolite extract and measured the thus derived 160 spike-in samples by FI-MS. Our results demonstrate that FI-MS can capture a wide range of chemically divers analytes within 30 seconds measurement time. However, the data also revealed extensive in-source modifications. Across all 160 spike-in samples, we identified significant increases of 11,013 ion peaks in positive and negative mode combined. To explain these unknown m/z features, we connected them to the m/z feature of the (de-)protonated metabolite using information about mass differences and MS2 spectra. This resulted in networks that explained on average 49 % of all significant features. The networks showed that a single metabolite undergoes compound specific and often sequential in-source modifications like adductions, chemical reactions, and fragmentations. Our results show that FI-MS generates complex MS1 spectra, which leads to an overestimation of significant features, but neutral losses and MS2 spectra explain many of these features. HighlightsO_LIFI-MS enables measurements of chemically divers metabolites. C_LIO_LIExtensive in-source modifications during electrospray ionization are detected by FI-MS. C_LIO_LIA network approach explains 49 % of all recorded in-source modifications. C_LI

biochemistry↗

bioRxiv2022

Horizontal transfer of bacteriocin biosynthesis genes requires metabolic adaptation to improve compound production and cellular fitness

Biosynthetic gene clusters (BGCs) encoding the production of bacteriocins are widespread amongst bacterial isolates and are important genetic determinants of competitive fitness within a given habitat. Staphylococci produce a tremendous diversity of compounds and the corresponding BGCs are frequently associated with mobile genetic elements, suggesting gain and loss of biosynthetic capacity. Pharmaceutical biology has shown that compound production in heterologous hosts is often challenging and many BGC recipients produce initially low compound amounts or show reduced growth rates. To assess whether transfer of BGCs between closely related S. aureus strains can be instantly effective or requires elaborate metabolic adaptation, we investigated the intra species transfer of a BGC encoding the ribosomally synthesized and post-translationally modified peptide (RiPP) micrococcin P1 (MP1). We found that acquisition of the BGC by S. aureus RN4220 enabled immediate MP1 production but also imposed a metabolic burden, which was relieved after prolonged cultivation by adaptive mutation. We used a multiomics approach to study this phenomenon and found adaptive evolution to select for strains with increased activity of the tricarboxylic acid cycle (TCA), which enhanced metabolic fitness and levels of compound production. Metabolome analysis revealed increases of central metabolites including citrate and -ketoglutarate in the adapted strain, suggesting metabolic adaptation to overcome the BGC-associated growth defects. Our results indicate that BCG acquisition requires genetic and metabolic predispositions allowing the integration of bacteriocin production into the cellular metabolism. Inappropriate metabolic characteristics of recipients can entail physiological burdens, negatively impacting the competitive fitness of recipients within natural bacterial communities. ImportanceHuman microbiomes are critically associated with human health and disease. Importantly, pathogenic bacteria can hide in human associated communities and can cause disease when the composition of the community becomes dysbalanced. Bacteriocin producing commensals are able to displace pathogens from microbial communities, suggesting that their targeted introduction in human microbiomes might prevent pathogen colonisation and infection. However, in view of future probiotic approaches, strains are needed that produce high levels of bioactive compounds and retain cellular fitness within mixed bacterial communities. Our work offers insights into the metabolic burdens associated with the production of the bacteriocin micrococcin P1 and highlights evolutionary strategies that increase cellular fitness in the context of production. Most likely metabolic adaptations are broadly relevant for bacteriocin producers and need to be considered for the future development of effective microbiome editing strategies.

microbiology↗

bioRxiv2022

How to deal with toxic amino acids: the bipartite AzlCD complex exports histidine in Bacillus subtilis

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. 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 inactivation of AzlB. IMPORTANCEAmino acids are building blocks for protein biosynthesis in each living cell. However, due to their reactivity as well as the similarity between several amino amino acids, they may also be involved in harmful reactions or in non-cognate interactions and thus be toxic. Bacillus subtilis can deal with otherwise toxic histidine by overexpressing a bipartite amino acid exporter AzlCD. Although encoded in an operon that also contains a gene for an amino acid importer, the corresponding genes are not expressed, irrespective of the availability or not of amino acids in the medium. This suggests that the azl operon is a last resort to deal with histidine stress that can be expressed due to mutational inactivation of the cognate repressor, AzlB.

microbiology↗