Biology subjects

Frunzke, J.

Publications and source records attributed to Frunzke, J..

At least 19 recordsLinked to original sources

bioRxiv2026

CyranoPS, a novel compact temperate corynephage infecting Corynebacterium glutamicum MB001

Here, we describe CyranoPS, a temperate phage isolated from the river Yvette in France, using the prophage-free strain Corynebacterium glutamicum MB001. Negative-stain electron microscopy revealed virions with siphovirus morphology. Its 14,910-bp genome has a GC content of 53.0% and 14-nt 3' overhangs at both ends. It carries 26 predicted genes, 10 without predicted functions, and no tRNAs. CyranoPS belongs to the newly established actinophage cluster ER, whose members have the smallest known genomes among Corynebacterium-infecting phages. Deletion of genes encoding the porin PorAH and the protein mycoloyltransferase MytC abolished infection, while genes encoding the mycomembrane proteins PorBC and ProtX were dispensable. CyranoPS established lysogeny in C. glutamicum through chromosomal integration probably mediated by its predicted tyrosine integrase. Integration truncated the immunity repressor gene Cyr_18, resulting in a protein devoid of its 15 C-terminal residues. The truncated repressor conferred stronger protection against superinfection than the full-length repressor. CyranoPS lysogens exhibited high levels of spontaneous prophage induction, reaching 109 PFU/mL under standard conditions, with no further increase upon treatment with mitomycin C. These findings suggest a potentially SOS-independent lytic-lysogenic switch and establish CyranoPS as a compact model to study infection and lysogeny regulation in Mycobacteriales.

microbiology↗

bioRxiv2026

Systematic evaluation of Cyanidioschyzon merolae across photobioreactor systems: Linking reactor design to biomass production and biochemical composition

Extremophilic red microalgae are promising platforms for sustainable biotechnology, combining robust growth under selective thermoacidophilic conditions with production of thermostable phycobiliproteins and carbon-rich biomass. However, reactor-dependent effects on growth, product formation and biomass composition remain insufficiently resolved. Here, we systematically evaluated the extremophilic red microalga Cyanidioschyzon merolae across cultivation scales and reactor formats and benchmarked its performance against the well-established Galdieria javensis and Limnospira platensis. In small-scale multi-cultivator photobioreactors and microfluidic growth chambers, C. merolae showed superior growth, reaching a maximum growth rate of 0.034 {+/-} 0.001 h-1 and 8.3 {+/-} 0.3 g l-1 cell dry weight. Microfluidic cultivation enabled growth analysis at single-cell resolution and matched growth rates obtained in photobioreactors. To identify scalable production strategies, C. merolae was further cultivated in a flat-panel photobioreactor and a custom-designed internally illuminated photobioreactor. The custom-designed photobioreactor delivered the highest biomass concentration and productivity, yielding 11.5 {+/-} 0.6 g l-1 cell dry weight and 1.07 {+/-} 0.06 g l-1 d-1, and comparable yields with regard to R-phycocyanin and R-allophycocyanin. Biomass analysis revealed substantial carbon and nitrogen contents, starch accumulation up to > 20 % of cell dry weight, and fatty acids dominated by palmitic, linoleic and oleic acids. Despite its reduced cell wall fraction, C. merolae contained structurally diverse, cultivation-dependent polysaccharides. These results establish C. merolae as a versatile chassis for thermostable pigment production and renewable feedstock generation, highlighting photobioreactor design as a key determinant of productivity and biomass quality.

microbiology↗

bioRxiv2026

Context-dependent siderophore exploitability shapes microbial community structure

1)Siderophores are classically viewed as shared iron-scavenging public goods, yet their ecological roles in multispecies communities remain poorly defined. Here, we establish a synthetic microbial community to dissect how different siderophores, their uptake compatibility and spatial structure shape iron competition. Using Corynebacterium glutamicum as a model, we show that this siderophore non-producer accesses diverse xenosiderophores, including enterobactin secreted by Escherichia coli. However, exploitation was constrained and co-cultures converged to stable compositions. Dose-response experiments combined with mathematical modelling indicated that the producer retains more effective access to enterobactin than the exploiter. Presence of Pseudomonas putida altered this interaction, as it exploited enterobactin while producing pyoverdine, a siderophore inaccessible to the other community members that restricted their iron access. Across different cultivation scales, community dynamics was strongly influenced by spatial organization and initial composition. These findings identify siderophores as context-dependent iron-allocation agents that can promote microbial coexistence or exclusion.

microbiology↗

bioRxiv2026

DNA-intercalating antiphage molecules trigger abortive infection through mutual destruction and synergize with bacterial immunity

Bacteria deploy diverse antiphage defense systems, including small bioactive molecules providing protection at the multicellular level. DNA-intercalating anthracyclines, such as daunorubicin, exhibit broad antiphage activity, but the underlying mechanism has remained elusive. Here, we systematically screened the Escherichia coli BASEL phage collection to elucidate the mode of action of DNA-intercalating antiphage molecules. We identified taxonomically distinct clusters of susceptible viral groups and show that, in T5-like phages (Markadamsvirinae), daunorubicin blocks infection after first-step transfer (FST). In the presence of daunorubicin, continued expression of pre-early genes leads to abortive infection via mutual destruction, where both phage and host succumb. Analogous abortive-infection phenotypes occur across taxonomically diverse phages exposed to chemically distinct DNA-intercalating molecules. Notably, we show that daunorubicin synergizes with downstream nucleic acid-targeting defenses underscoring context-dependent outcomes. Together, these findings reveal how chemical defense contributes to the multilayered antiviral immunity and highlight the intricate interplay between mechanistic inhibition and infection outcome.

microbiology↗

bioRxiv2025

A novel biosensor for ferrous iron developed via CoBiSe:A computational method for rapid biosensor design

Genetically encoded biosensors enable monitoring of metabolite dynamics in living organisms. We present CoBiSe, a computational approach using Constraint Network Analysis to identify optimal insertion sites for reporter modules in molecular recognition elements (MREs). Applied to the iron-binding protein DtxR from Corynebacterium glutamicum, CoBiSe identified a flexible connective loop (residues 138-150) for inserting the reporter module, resulting in IronSenseR, a novel ratiometric biosensor for ferrous iron (Fe{superscript 2}). IronSenseR demonstrates high specificity for Fe{superscript 2} with dissociation constants of 1.55 {+/-} 0.08 {micro}M (FeSO4) and 2.44 {+/-} 0.28 {micro}M (FeCl2), while showing no binding to Fe3 and other divalent cations. In vivo assessment in Escherichia coli, Pseudomonas putida and Corynebacterium glutamicum confirmed IronSenseRs capability to detect changes in the intracellular iron pool. The creation of IronSenseR underlines that, by reducing search space and eliminating labor-intensive screening, CoBiSe streamlines biosensor development and enables precise creation of next-generation biosensors for diverse metabolites.

bioengineering↗

bioRxiv2025

Phage biocontrol reduces the burden on plant immunity through suppression of bacterial virulence

Bacteriophages are increasingly recognized as key players in modulating plant-microbe interactions, including their potential in the biocontrol of plant pathogenic bacteria. In this study, we investigated the tripartite interaction between Arabidopsis thaliana, the bacterial plant pathogen Xanthomonas campestris pv. campestris (Xcc), and the lytic phage Seregon. Using parallel transcriptomic profiling, we characterized host and pathogen responses during infection and phage treatment. While treatment with phage Seregon did not lead to the eradication of Xcc, it significantly mitigated Xcc-induced disease symptoms, restoring leaf area to levels comparable to the uninfected control within 14 days post-inoculation. Our data revealed that phage-mediated protection is associated with early bacterial recognition, and suppression of Jasmonate (JA)-related responses in the host. Analysis of nuclear localized reporter plant lines further confirmed a significant reduction in ROS levels and JA biosynthesis in phage-treated plants. Concurrently, Xcc exhibited significant transcriptional downregulation of key virulence factors in the presence of the phage, including the genes encoding the type III secretion system, its associated effectors, and components involved in flagella biosynthesis. Remarkably, phage treatment did not lead to a significant increase in bacterial resistance to phage infection, which is in stark contrast to in vitro conditions. Taken together, this study provides first mechanistic insight into how phages can be harnessed to shape plant-pathogen interactions and highlights their potential role in enhancing plant resilience through targeted modulation of both host immunity and pathogen behavior.

microbiology↗

bioRxiv2025

Genome-wide analysis of DtxR and HrrA regulons reveals novel targets and a high level of interconnectivity between iron and heme regulatory networks in Corynebacterium glutamicum

Iron is an essential trace element required by nearly all organisms as a cofactor in enzymes, regulatory proteins, and cytochromes of the respiratory chain. Maintaining iron homeostasis is crucial, since elevated levels cause oxidative stress through the formation of reactive oxygen species. In Corynebacterium glutamicum, iron and heme homeostasis are tightly interconnected and controlled by the global regulators DtxR and HrrA. While DtxR senses intracellular Fe2+, the two-component system HrrSA is activated by heme, functioning as a global regulator of heme homeostasis. This study provides the first genome-wide analysis of DtxR and HrrA binding dynamics under varying iron and heme conditions using chromatin affinity purification sequencing (ChAP-seq). Our conditional ChAP- Seq approach revealed 25 novel DtxR targets and 210 previously unrecognized HrrA targets. Among these, metH, encoding homocysteine methyltransferase, and xerC, encoding a tyrosine recombinase, were bound by DtxR exclusively under heme conditions, underscoring condition-dependent variation in DtxR binding. Activation of metH by DtxR links iron metabolism to methionine synthesis, potentially relevant for the mitigation of oxidative stress. Beyond novel targets, this study highlights the interconnected nature of the DtxR and HrrA regulons, identifying 16 shared targets with in some cases overlapping operator sequences. Strikingly, we provide several examples for weak ChAP-Seq peaks, often disregarded in global approaches, that feature a significant impact of the regulator on differential gene expression. These findings emphasize the importance of genome-wide profiling under different conditions to uncover novel targets and shed light on the complexity and dynamic nature of bacterial regulatory networks. ImportanceThe trace element iron is essential for life, but elevated levels can rapidly cause cellular damage through oxidative stress. Bacteria, like Corynebacterium glutamicum, tightly regulate iron and heme homeostasis via the global regulators DtxR and HrrA. This study provides the first analysis of the genome-wide binding patterns of these two regulators demonstrating significant differences in binding dependent on the tested iron regimes. Overall, we identified 25 new DtxR targets and 210 previously unknown HrrA targets, including genes with crucial roles in central metabolism and DNA repair. Notably, DtxR was shown to link iron metabolism to methionine synthesis, which might be important to protect the cell from oxidative stress. Our findings highlight the interconnected nature of DtxR and HrrA networks and underscore the value of condition-specific analysis to deepen the understanding of how bacteria adapt to environmental changes.

microbiology↗

bioRxiv2024

CRISPR/dCas-mediated counter-silencing: Reprogramming dCas proteins into antagonists of xenogeneic silencers

Lsr2-like nucleoid-associated proteins function as xenogeneic silencers (XSs) inhibiting expression of horizontally acquired, AT-rich DNA in actinobacteria. Interference by transcription factors can lead to counter-silencing of XS target promoters, but relief of this repression typically requires promoter engineering. In this study, we developed a novel CRISPR/dCas-mediated counter-silencing (CRISPRcosi) approach by using nuclease-deficient dCas enzymes to counteract the Lsr2-like XS protein CgpS in Corynebacterium glutamicum or Lsr2 in Streptomyces venezuelae. Systematic in vivo reporter studies with dCas9 and dCas12a and various guide RNAs revealed effective counter-silencing of different CgpS target promoters in response to guide RNA/dCas DNA binding - independent of promoter sequence modifications. The most prominent CRISPRcosi effect was observed when targeting the CgpS nucleation site, an effect that was also seen in S. venezuelae when targeting a known Lsr2 nucleation site within the chloramphenicol biosynthesis gene cluster. Analyzing the system in C. glutamicum strains lacking the XS protein CgpS revealed varying strengths of counteracting CRISPR interference (CRISPRi) effects based on the target position and strand. Genome-wide transcriptome profiling in sgRNA/dCas9 co-expressing C. glutamicum wild-type strains revealed high counter-silencing specificity with minimal off-target effects. Thus, CRISPRcosi provides a promising strategy for the precise upregulation of XS target genes with significant potential for studying gene networks as well as for developing applications in biotechnology and synthetic biology. IMPORTANCELsr2-like nucleoid-associated proteins act as xenogeneic silencers (XSs), repressing the expression of horizontally acquired, AT-rich DNA in actinobacteria. The targets of Lsr2-like proteins are very diverse, including prophage elements, virulence gene clusters and biosynthetic gene clusters. Consequently, the targeted activation of XS target genes is of interest for fundamental research and biotechnological applications. Traditional methods for counter-silencing typically require promoter modifications. In this study, we developed a novel CRISPR/dCas-mediated counter-silencing (CRISPRcosi) approach, utilizing nuclease-deficient dCas enzymes to counteract repression by Lsr2-like proteins in Corynebacterium glutamicum and Streptomyces venezuelae. The strongest effect was observed when targeting the Lsr2 nucleation site. Genome-wide transcriptome profiling revealed high specificity with minimal off-target effects. Overall, CRISPRcosi emerges as a powerful tool for the precise activation of genes silenced by xenogeneic silencers, offering new opportunities for exploring gene networks and advancing biotechnological applications.

microbiology↗

bioRxiv2024

Decoupling the molecular versatility of aminoglycosides via drug or target modification enables community-wide antiphage defense

The ongoing arms race between bacteria and phages has forced bacteria to evolve a sophisticated set of antiphage defense mechanisms that constitute the bacterial immune system. In our previous study, we highlighted the antiphage properties of aminoglycoside antibiotics, which are naturally secreted by Streptomyces. Successful inhibition of phage infection was achieved by addition of pure compounds and supernatants from a natural producer strain highlighting the potential for community-wide antiphage defense. However, given the dual functionality of these compounds, neighboring bacterial cells require resistance to the antibacterial activity of aminoglycosides to benefit from the protection they confer against phages. In this study, we demonstrated the successful uncoupling of antiphage and antibacterial properties via different aminoglycoside-resistance mechanisms encompassing drug and target site modifications. Furthermore, we confirmed the antiphage impact of aminoglycosides in a community context by co-culturing phage-susceptible, apramycin-resistant S. venezuelae with the apramycin-producing strain Streptoalloteichus tenebrarius. Given the prevalence of aminoglycoside resistance among natural bacterial isolates that allow functional uncoupling of these compounds, this study highlights the ecological relevance of chemical defense via aminoglycosides at the community level.

microbiology↗

bioRxiv2024

Isolation and characterization of the new Streptomyces phages Kamino, Geonosis, Abafar and Scarif infecting a broad range of host species

Streptomyces, a multifaceted genus of soil-dwelling bacteria within the phylum Actinobacteria, features intricate phage-host interactions shaped by its complex life cycle and the synthesis of a diverse array of specialized metabolites. Here, we describe the isolation and characterization of four novel Streptomyces phages infecting a variety of different host species. While phage Kamino, isolated on Streptomyces kasugaensis, is predicted to be temperate and encodes a serine integrase in its genome, phages Geonosis (isolated on S. griseus) and phages Abafar and Scarif, isolated on S. albidoflavus, are virulent phages. Phages Kamino and Geonosis were shown to amplify well in liquid culture leading to a pronounced culture collapse already at low titres. Determination of the host range by testing >40 different Streptomyces species identified phages Kamino, Abafar and Scarif as broad host range phages, with Kamino showing productive infections on 15 different species. Isolated phages were further tested regarding their sensitivity to antiphage molecules. Here, the strongest effects were observed for the DNA-intercalating molecule daunorubicin. Overall, the phages described in this study expand the publicly available portfolio of phages infecting Streptomyces and will be instrumental for advancing the mechanistic understanding of the intricate antiviral strategies employed by these multicellular bacteria.

microbiology↗

bioRxiv2024

Expanding the phage galaxy: Isolation and characterization of five novel Streptomyces siphoviruses Ankus, Byblos, DekoNeimoidia, Mandalore, and Naboo

Key features of the actinobacterial genus Streptomyces are multicellular, filamentous growth and production of a broad portfolio of bioactive molecules. These characteristics appear to play an important role in phage-host interactions and are modulated by phages during infection. To accelerate research of such interactions and the investigation of novel immune systems in multicellular bacteria, phage isolation, sequencing, and characterization are needed. This is a prerequisite for establishing systematic collections that appropriately cover phage diversity for comparative analyses. As part of a public outreach programme within the priority programme SPP 2330, involving local schools, we describe the isolation and characterization of five novel Streptomyces siphoviruses infecting S. griseus, S. venezuelae, and S. olivaceus. All isolates are virulent members of two existing genera and, additionally, establish a new genus in the Stanwilliamsviridae family. In addition to an extensive set of tRNAs and proteins involved in phage replication, about 80% of phage genes encode hypothetical proteins, underlining the yet underexplored phage diversity and genomic dark matter still found in bacteriophages infecting actinobacteria. Taken together, phages Ankus, Byblos, DekoNeimoidia, Mandalore, and Naboo expand the phage diversity and contribute to ongoing research in the field of Streptomyces phage-host interactions.

microbiology↗

bioRxiv2024

Seed coating with phages for sustainable plant biocontrol of plant pathogens and influence of the seed coat mucilage

Pathogens resistant to classical control strategies are on the rise and cause significant damage in crop yield production with seeds as one major transmission route. Bacteriophages are specialized viruses of bacteria and their interaction with seeds holds great potential as targeted and sustainable solution to this problem. In this study, we isolated and characterized two novel phages, Athelas and Alfirin, infecting Pseudomonas syringae and Agrobacterium tumefaciens, respectively, and included the recently published phage Pfeifenkraut infecting Xanthomonas translucens. The three phages were tested for their interaction with the seed coat mucilage. Phage binding on Arabidopsis seeds, which exude the mucilage as a polysaccharide-polymer-matrix, was assessed by comparison to seeds with removed mucilage. Two of the three phages were dependant on mucilage for seed binding, and podophage Athelas showed the highest dependency. Further podoviruses of the Autographiviridae obtained from the systematic E. coli (BASEL) phage collection were tested and showed a similar dependency on the mucilage for seed adhesion. Comparative analysis using a set of Arabidopsis seed coat mutants revealed the diffusible cellulose fraction as important component for phage binding. Long-term activity tests revealed a high stability of phages on seed surfaces and phage coating effectively increased the survival rate of plant seedling in the presence of the pathogen. Utilization of non-virulent host strains was further successfully applied to boost the presence of infectious phage particles on seed surfaces. Altogether, our study highlights the high potential of phage-based applications as sustainable biocontrol strategy on the seed level.

microbiology↗

bioRxiv2021

Aminoglycoside antibiotics inhibit phage infection by blocking an early step of the phage infection cycle

In response to viral predation, bacteria have evolved a wide range of defense mechanisms, which rely mostly on proteins acting at the cellular level. Here, we show that aminoglycosides, a well-known class of antibiotics produced by Streptomyces, are potent inhibitors of phage infection in widely divergent bacterial hosts. We demonstrate that aminoglycosides block an early step of the viral life cycle, prior to genome replication. Phage inhibition was also achieved using supernatants from natural aminoglycoside producers, hinting at a broad physiological significance of the antiviral properties of aminoglycosides. Strikingly, we show that acetylation of the aminoglycoside antibiotic apramycin abolishes its antibacterial effect, but retains its antiviral properties. Altogether, this study expands the knowledge of potential aminoglycoside functions in bacterial communities suggesting that aminoglycosides are not only used by their producers as toxic molecules against their bacterial competitors, but could also provide protection against the threat of phage predation at the community level.

microbiology↗

bioRxiv2021

Genome sequencing of the bacteriophage CL31 and interaction with the host strain Corynebacterium glutamicum ATCC 13032

In this study, we provide a comprehensive analysis of the genomic features of the phage CL31 and the infection dynamics with the biotechnologically relevant host strain Corynebacterium glutamicum ATCC 13032. Genome sequencing and annotation of CL31 revealed a 45-kbp genome composed of 72 open reading frames, mimicking the GC content of its host strain (54.4 %). An ANI-based distance matrix showed the highest similarity of CL31 to the temperate corynephage {Phi}16. While the C. glutamicum ATCC 13032 wild type strain showed only mild propagation of CL31, a strain lacking the cglIR-cglIIR-cglIM restriction-modification system was efficiently infected by this phage. Interestingly, the prophage-free strain C. glutamicum MB001 featured an even accelerated amplification of CL31 compared to the {Delta}resmod strain suggesting a role of cryptic prophage elements in phage defense. Proteome analysis of purified phage particles and transcriptome analysis provide important insights into structural components of the phage and the response of C. glutamicum to CL31 infection. Isolation and sequencing of CL31-resistant strains revealed SNPs in genes involved in mycolic acid biosynthesis suggesting a role of this cell envelope component in phage adsorption. Altogether, these results provide an important basis for further investigation of phage-host interactions in this important biotechnological model organism.

microbiology↗

bioRxiv2021

Identification of Gip as a novel phage-encoded gyrase inhibitor protein featuring a broad activity profile

Bacteriophages represent a powerful source for the identification of novel antimicrobial proteins. In this study, a screening of small cytoplasmic proteins encoded by the CGP3 prophage of Corynebacterium glutamicum, resulted in the identification of the novel gyrase-inhibiting protein Cg1978 (Gip), which shows a direct interaction with the gyrase subunit A (GyrA). In vitro supercoiling assays further suggest a stabilization of the cleavage complex by Gip. Overproduction of Gip in C. glutamicum resulted in a severe growth defect as well as an induction of the SOS response. The cells adapted to gip overexpression by increasing expression levels of gyrAB and by reducing topA expression reflecting the homeostatic control of DNA topology. Interestingly, Gip features a similar activity profile towards gyrases of Escherichia coli, Mycobacterium tuberculosis and C. glutamicum. Therefore, the detailed elucidation of the mechanism of Gip action may provide novel directions for the design of drugs targeting DNA gyrase.

microbiology↗

bioRxiv2020

Automated Rational Strain Construction Based on High-Throughput Conjugation

Molecular cloning is the core of Synthetic Biology, as it comprises the assembly of DNA and its expression in target hosts. At present, however, cloning is most often a manual, time-consuming and repetitive process that highly benefits from automation. The automation of a complete rational cloning procedure, i.e., from DNA part creation to expression in the target host, involves the integration of different operations and machines. Examples of such workflows are sparse, especially when the design is rational (i.e., the DNA sequence design is fixed, and not based on randomized libraries) and the target host is less genetically tractable (e.g., not sensitive to heat-shock transformation). In this study, an automated workflow for the rational construction of plasmids and their subsequent conjugative transfer into the biotechnological platform organism Corynebacterium glutamicum is presented. The whole workflow is accompanied by a custom-made software tool. As an application example, a rationally designed library of transcription factor biosensors based on the regulator Lrp was constructed and characterized. A sensor with an improved dynamic range was obtained, and insights from the screening provided evidence for a dual regulator function of C. glutamicum Lrp.

synthetic biology↗

bioRxiv2020

Genome sequence and characterization of five bacteriophages infecting Streptomyces coelicolor and Streptomyces venezuelae: Alderaan, Coruscant, Dagobah, Endor1 and Endor2

Streptomyces are well-known antibiotic producers, and are also characterized by a complex morphological differentiation. Streptomyces, like all bacteria, are confronted with the constant threat of phage predation, which in turn shapes bacterial evolution. However, despite significant sequencing efforts recently, relatively few phages infecting Streptomyces have been characterized compared to other genera. Here, we present the isolation and characterization of five novel Streptomyces phages. All five phages belong to the Siphoviridae family, based on their morphology as determined by transmission electron microscopy. Genome sequencing revealed that four of them were temperate phages, while one had a lytic lifestyle. Moreover, one of the newly sequenced phages shows very little homology to already described phages, highlighting the still largely untapped viral diversity. Altogether, this study expands the number of characterized phages of Streptomyces and sheds light on phage evolution and phage-host dynamics in Streptomyces.

microbiology↗

bioRxiv2019

Deciphering the rules underlying xenogeneic silencing and counter-silencing of Lsr2-like proteins

Lsr2-like nucleoid-associated proteins play an important role as xenogeneic silencers (XS) of horizontally acquired genomic regions in actinobacteria. In this study, we systematically analyzed the in vivo constraints underlying silencing and counter-silencing of the Lsr2-like protein CgpS in Corynebacterium glutamicum. Genome-wide analysis revealed binding of CgpS to regions featuring a distinct drop in GC-profile close to the transcription start site (TSS), but also identified an overrepresented motif with multiple A/T steps at the nucleation site of the nucleoprotein complex. Binding of specific transcription factors (TFs) may oppose XS activity leading to counter-silencing. Following a synthetic counter-silencing approach, target gene activation was realized by inserting operator sites of an effector-responsive TF within various CgpS target promoters resulting in an increased promoter activity upon TF binding. Analysis of reporter constructs revealed maximal counter-silencing when the TF operator site was inserted at the position of maximal CgpS coverage. This principle was implemented in a synthetic toggle switch, which features a robust and reversible response to effector availability highlighting the potential for biotechnological applications. Altogether, our results provide comprehensive insights into how Lsr2 silencing and counter-silencing shapes evolutionary network expansion in this medically- and biotechnologically-relevant bacterial phylum.\n\nIMPORTANCEIn actinobacteria, Lsr2-like nucleoid-associated proteins function as xenogeneic silencers (XS) of horizontally acquired genomic regions, including viral elements, virulence gene clusters in Mycobacterium tuberculosis, and genes involved in cryptic specialized metabolism in Streptomyces species. Consequently, a detailed mechanistic understanding of Lsr2 binding in vivo is relevant as a potential drug target and for the identification novel bioactive compounds. Here, we followed an in vivo approach to investigate the rules underlying xenogeneic silencing and counter-silencing of the Lsr2-like XS CgpS from Corynebacterium glutamicum. Our results demonstrated that CgpS distinguishes between self and foreign by recognizing a distinct drop in GC-profile in combination with a short, sequence specific motif at the nucleation site. Following a synthetic counter-silencer approach, we studied the potential and constraints of transcription factors to counteract CgpS silencing thereby facilitating the integration of new genetic traits into host regulatory networks.

microbiology↗