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The trial of amateur submarine builder Peter Madsen for the murder of the Swedish journalist Kim Wall was one of the most publicised trials in recent Danish history. Through in-depth interviews with ten prominent Danish reporters who covered the trial, this study examines how court reporters negotiate and struggle with ethical dilemmas related to objectivity as both an institutional ideal and an ethical rule under the Media Liability Act. I demonstrate how reporters negotiate and strategise to maintain objectivity in relation to facts, relevance, the telling of both sides, and the avoidance of prejudging. I further highlight the dispute between fact-based reporters and a minor group endorsing interpretive and narrative reporting and advocating for a more pragmatic approach to objectivity. A core finding is how technological advancements and massive public interest have paved the way for new ethical practices, referred to here as “strategic ritual 2.0”.

Many bacteria metabolize ethanolamine as a nutrient source through cytoplasmic organelles named bacterial microcompartments (BMCs). Here we investigated the molecular assembly, regulation, and function of BMCs in Fusobacterium nucleatum- a Gram-negative oral pathobiont that is associated with adverse pregnancy outcomes. The F. nucleatum genome harbors a conserved ethanolamine utilization ( eut ) locus with 21 genes that encode several putative BMC shell proteins and a two-component signal transduction system (TCS), in addition to the enzymes for ethanolamine transport and catabolism. We show that the expression of most of these genes and BMC formation are highly increased in wild-type fusobacteria when cultured in the presence of ethanolamine as a nutrient source. Deletion of the response regulator EutV eliminated this induction of eut mRNAs and BMCs, thus demonstrating that BMC formation is transcriptionally regulated by the TCS EutV-EutW in response to ethanolamine. Mass spectrometry of isolated BMCs unveiled the identity of the constituent proteins EutL, EutM 1 , EutM 2 , and EutN. Consistent with the role of these proteins in BMC assembly and metabolism, deletion of eutN , eutL / eutM 1 / eutM 2 , or eutL / eutM 1 / eutM 2 / eutN not only affected BMC formation but also ethanolamine utilization, causing cell growth defects with ethanolamine as a nutrient. BMCs are also assembled in fusobacteria cultured with placental cells or the culture media, a process that is dependent on the BMC shell proteins. Significantly, we show that the eutN mutant is defective in inducing preterm birth in a mouse model. Together, these results establish that the BMC-mediated metabolism of ethanolamine is critical for fusobacterial virulence., Importance: The oral anaerobe Fusobacterium nucleatum can spread to distal internal organs, such as the colon and placenta, thereby promoting the development of colorectal cancer and inducing preterm birth, respectively. Yet, how this opportunistic pathogen adapts to the various metabolically distinct host cellular niches remains poorly understood. We demonstrated here that this microbe assembles specialized metabolic organelles, termed bacterial microcompartments (BMCs), to utilize environmental ethanolamine (EA) as a key environmental nutrient source. The formation of F. nucleatum BMCs, containing BMC shell proteins EutLM1M2N, is controlled by a two-component system, EutV-EutW, responsive to EA. Significantly, this ability of F. nucleatum to form BMCs in response to EA is crucial for its pathogenicity evidenced by the fact that the genetic disruption of BMC formation reduces fusobacterial virulence in a mouse model of preterm birth.

Staphylococcus aureus (Sa) and Acinetobacter baumannii (Ab) are frequently co-isolated from polymicrobial infections that are severe and refractory to therapy. Here, we apply a combination of wet-lab experiments and in silico modeling to unveil the intricate nature of the Ab / Sa interaction using both, representative laboratory strains and strains co-isolated from clinical samples. This comprehensive methodology allowed uncovering Sa's capability to exert a partial interference on Ab by the expression of phenol-soluble modulins. In addition, we observed a cross-feeding mechanism by which Sa supports the growth of Ab by providing acetoin as an alternative carbon source. This study is the first to dissect the Ab / Sa interaction dynamics wherein competitive and cooperative strategies can intertwine. Through our findings, we illuminate the ecological mechanisms supporting their coexistence in the context of polymicrobial infections. Our research not only enriches our understanding but also opens doors to potential therapeutic avenues in managing these challenging infections., Competing Interests: None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Background: One of the most complex prokaryotic organelles are magnetosomes, which are formed by magnetotactic bacteria as sensors for navigation in the Earth's magnetic field. In the alphaproteobacterium Magnetospirillum gryphiswaldense magnetosomes consist of chains of magnetite crystals (Fe 3 O 4 ) that under microoxic to anoxic conditions are biomineralized within membrane vesicles. To form such an intricate structure, the transcription of > 30 specific structural genes clustered within the genomic magnetosome island (MAI) has to be coordinated with the expression of an as-yet unknown number of auxiliary genes encoding several generic metabolic functions. However, their global regulation and transcriptional organization in response to anoxic conditions most favorable for magnetite biomineralization are still unclear., Results: Here, we compared transcriptional profiles of anaerobically grown magnetosome forming cells with those in which magnetosome biosynthesis has been suppressed by aerobic condition. Using whole transcriptome shotgun sequencing, we found that transcription of about 300 of the > 4300 genes was significantly enhanced during magnetosome formation. About 40 of the top upregulated genes are directly or indirectly linked to aerobic and anaerobic respiration (denitrification) or unknown functions. The mam and mms gene clusters, specifically controlling magnetosome biosynthesis, were highly transcribed, but constitutively expressed irrespective of the growth condition. By Cappable-sequencing, we show that the transcriptional complexity of both the MAI and the entire genome decreased under anaerobic conditions optimal for magnetosome formation. In addition, predominant promoter structures were highly similar to sigma factor σ 70 dependent promoters in other Alphaproteobacteria., Conclusions: Our transcriptome-wide analysis revealed that magnetite biomineralization relies on a complex interplay between generic metabolic processes such as aerobic and anaerobic respiration, cellular redox control, and the biosynthesis of specific magnetosome structures. In addition, we provide insights into global regulatory features that have remained uncharacterized in the widely studied model organism M. gryphiswaldense, including a comprehensive dataset of newly annotated transcription start sites and genome-wide operon detection as a community resource (GEO Series accession number GSE197098)., (© 2022. The Author(s).)

Fusobacterium nucleatum, an anaerobic Gram-negative bacterium frequently found in the human oral cavity and some extra-oral sites, is implicated in several important diseases: periodontitis, adverse pregnancy outcomes, and colorectal cancer. To date, how this obligate anaerobe copes with oxidative stress and host immunity within multiple human tissues remains unknown. Here, we uncovered a critical role in this process of a multigene locus encoding a single, fused methionine sulfoxide reductase (MsrAB), a two-component signal transduction system (ModRS), and thioredoxin (Trx)- and cytochrome c (CcdA)-like proteins, which are induced when fusobacterial cells are exposed to hydrogen peroxide. Comparative transcriptome analysis revealed that the response regulator ModR regulates a large regulon that includes trx , ccdA , and many metabolic genes. Significantly, specific mutants of the msrAB locus, including msrAB , are sensitive to reactive oxygen species and defective in adherence/invasion of colorectal epithelial cells. Strikingly, the msrAB mutant is also defective in survival in macrophages, and it is severely attenuated in virulence in a mouse model of preterm birth, consistent with its failure to spread to the amniotic fluid and colonize the placenta. Clearly, the MsrAB system regulated by the two-component system ModRS represents a major oxidative stress defense pathway that protects fusobacteria against oxidative damage in immune cells and confers virulence by enabling attachment and invasion of multiple target tissues. IMPORTANCE F. nucleatum colonizes various human tissues, including oral cavity, placenta, and colon. How this obligate anaerobe withstands oxidative stress in host immune cells has not been described. We report here that F. nucleatum possesses a five-gene locus encoding a fused methionine sulfoxide reductase (MsrAB), a two-component signal transduction system (ModRS), and thioredoxin- and cytochrome c -like proteins. Regulated by ModRS, MsrAB is essential for resistance to reactive oxygen species, adherence/invasion of colorectal epithelial cells, and survival in macrophage. Unable to colonize placenta and spread to amniotic fluid, the msrAB mutant failed to induce preterm birth in a murine model.

Magnetosomes are complex membrane organelles synthesized by magnetotactic bacteria (MTB) for navigation in the Earth's magnetic field. In the alphaproteobacterium Magnetospirillum gryphiswaldense, all steps of magnetosome formation are tightly controlled by >30 specific genes arranged in several gene clusters. However, the transcriptional organization of the magnetosome gene clusters has remained poorly understood. Here, by applying Cappable-seq and whole-transcriptome shotgun RNA sequencing, we show that mamGFDCop and feoAB1op are transcribed as single transcriptional units, whereas multiple transcription start sites (TSS) are present in mms6op , mamXYop , and the long (>16 kb) mamABop . Using a bioluminescence reporter assay and promoter knockouts, we demonstrate that most of the identified TSS originate from biologically meaningful promoters which mediate production of multiple transcripts and are functionally relevant for proper magnetosome biosynthesis. In addition, we identified a strong promoter in a large intergenic region within mamXYop , which likely drives transcription of a noncoding RNA important for gene expression in this operon. In summary, our data suggest a more complex transcriptional architecture of the magnetosome operons than previously recognized, which is largely conserved in other magnetotactic Magnetospirillum species and, thus, is likely fundamental for magnetosome biosynthesis in these organisms. IMPORTANCE Magnetosomes have emerged as a model system to study prokaryotic organelles and a source of biocompatible magnetic nanoparticles for various biomedical applications. However, the lack of knowledge about the transcriptional organization of magnetosome gene clusters has severely impeded the engineering, manipulation, and transfer of this highly complex biosynthetic pathway into other organisms. Here, we provide a high-resolution image of the previously unappreciated transcriptional landscape of the magnetosome operons. Our findings are important for further unraveling the complex genetic framework of magnetosome biosynthesis. In addition, they will facilitate the rational reengineering of magnetic bacteria for improved bioproduction of tunable magnetic nanoparticles, as well as transplantation of magnetosome biosynthesis into foreign hosts by synthetic biology approaches. Overall, our study exemplifies how a genetically complex pathway is orchestrated at the transcriptional level to ensure the balanced expression of the numerous constituents required for the proper assembly of one of the most intricate prokaryotic organelles.

A gram-negative colonizer of the oral cavity, Fusobacterium nucleatum not only interacts with many pathogens in the oral microbiome but also has the ability to spread to extraoral sites including placenta and amniotic fluid, promoting preterm birth. To date, however, the molecular mechanism of interspecies interactions-termed coaggregation-by F. nucleatum and how coaggregation affects bacterial virulence remain poorly defined. Here, we employed genome-wide transposon mutagenesis to uncover fusobacterial coaggregation factors, revealing the intertwined function of a two-component signal transduction system (TCS), named CarRS, and a lysine metabolic pathway in regulating the critical coaggregation factor RadD. Transcriptome analysis shows that CarR modulates a large regulon including radD and lysine metabolic genes, such as kamA and kamD , the expression of which are highly up-regulated in the Δ carR mutant. Significantly, the native culture medium of Δ kamA or Δ kamD mutants builds up abundant amounts of free lysine, which blocks fusobacterial coaggregation with streptococci. Our demonstration that lysine-conjugated beads trap RadD from the membrane lysates suggests that lysine utilizes RadD as its receptor to act as a metabolic inhibitor of coaggregation. Lastly, using a mouse model of preterm birth, we show that fusobacterial virulence is significantly attenuated with the Δ kamA and Δ carR mutants, in contrast to the enhanced virulence phenotype observed upon diminishing RadD (Δ radD or Δ carS mutant). Evidently, F. nucleatum employs the TCS CarRS and environmental lysine to modulate RadD-mediated interspecies interaction, virulence, and nutrient acquisition to thrive in the adverse environment of oral biofilms and extraoral sites., Competing Interests: The authors declare no competing interest.

Controlled RNA degradation is a crucial process in bacterial cell biology for maintaining proper transcriptome homeostasis and adaptation to changing environments. mRNA turnover in many Gram-positive bacteria involves a specialized ribonuclease called RNase J (RnJ). To date, however, nothing is known about this process in the diphtheria-causative pathogen Corynebacterium diphtheriae , nor is known the identity of this ribonuclease in this organism. Here, we report that C. diphtheriae DIP1463 encodes a predicted RnJ homolog, comprised of a conserved N-terminal β-lactamase domain, followed by β-CASP and C-terminal domains. A recombinant protein encompassing the β-lactamase domain alone displays 5'-exoribonuclease activity, which is abolished by alanine-substitution of the conserved catalytic residues His 186 and His 188 . Intriguingly, deletion of DIP1463/ rnj in C. diphtheriae reduces bacterial growth and generates cell shape abnormality with markedly augmented cell width. Comparative RNA-seq analysis revealed that RnJ controls a large regulon encoding many factors predicted to be involved in biosynthesis, regulation, transport, and iron acquisition. One upregulated gene in the ∆ rnj mutant is ftsH , coding for a membrane protease (FtsH) involved in cell division, whose overexpression in the wild-type strain also caused cell-width augmentation. Critically, the ∆ rnj mutant is severely attenuated in virulence in a Caenorhabditis elegans model of infection, while the FtsH-overexpressing and toxin-less strains exhibit full virulence as the wild-type strain. Evidently, RNase J is a key ribonuclease in C. diphtheriae that post-transcriptionally influences the expression of numerous factors vital to corynebacterial cell physiology and virulence. Our findings have significant implications for basic biological processes and mechanisms of corynebacterial pathogenesis.

Biosensors have emerged as a valuable tool with high specificity and sensitivity for fast and reliable detection of hazardous substances in drinking water. Numerous substances have been addressed using synthetic biology approaches. However, many proposed biosensors are based on living, genetically modified organisms and are therefore limited in shelf life, usability and biosafety. We addressed these issues by the construction of an extensible, cell-free biosensor. Storage is possible through freeze drying on paper. Following the addition of an aqueous sample, a highly efficient cell-free protein synthesis (CFPS) reaction is initiated. Specific allosteric transcription factors modulate the expression of 'superfolder' green fluorescent protein (sfGFP) depending on the presence of the substance of interest. The resulting fluorescence intensities are analyzed with a conventional smartphone accompanied by simple and cheap light filters. An ordinary differential equitation (ODE) model of the biosensors was developed, which enabled prediction and optimization of performance. With an optimized cell-free biosensor based on the Shigella flexneri MerR transcriptional activator, detection of 6 μg/L Hg(II) ions in water was achieved. Furthermore, a completely new biosensor for the detection of gamma-hydroxybutyrate (GHB), a substance used as date-rape drug, was established by employing the naturally occurring transcriptional repressor BlcR from Agrobacterium tumefaciens., Competing Interests: The authors have declared that no competing interests exist.

Background: The human pathogen Corynebacterium diphtheriae is the causative agent of diphtheria. In the 1990s a large diphtheria outbreak in Eastern Europe was caused by the strain C. diphtheriae NCTC 13129. Although the genome was sequenced more than a decade ago, not much is known about its transcriptome. Our aim was to use transcriptome sequencing (RNA-Seq) to close this knowledge gap and gain insights into the transcriptional landscape of a C. diphtheriae tox + strain., Results: We applied two different RNA-Seq techniques, one to retrieve 5'-ends of primary transcripts and the other to characterize the whole transcriptional landscape in order to gain insights into various features of the C. diphtheriae NCTC 13129 transcriptome. By examining the data we identified 1656 transcription start sites (TSS), of which 1202 were assigned to genes and 454 to putative novel transcripts. By using the TSS data promoter regions recognized by the housekeeping sigma factor σ A and its motifs were analyzed in detail, revealing a well conserved -10 but an only weakly conserved -35 motif, respectively. Furthermore, with the TSS data 5'-UTR lengths were explored. The observed 5'-UTRs range from zero length (leaderless transcripts), which make up 20% of all genes, up to over 450 nt long leaders, which may harbor regulatory functions. The C. diphtheriae transcriptome consists of 471 operons which are further divided into 167 sub-operon structures. In a differential expression analysis approach, we discovered that genetic disruption of the iron-sensing transcription regulator DtxR, which controls expression of diphtheria toxin (DT), causes a strong influence on general gene expression. Nearly 15% of the genome is differentially transcribed, indicating that DtxR might have other regulatory functions in addition to regulation of iron metabolism and DT. Furthermore, our findings shed light on the transcriptional landscape of the DT encoding gene tox and present evidence for two tox antisense RNAs, which point to a new way of transcriptional regulation of toxin production., Conclusions: This study presents extensive insights into the transcriptome of C. diphtheriae and provides a basis for future studies regarding gene characterization, transcriptional regulatory networks, and regulation of the tox gene in particular.