Konrad U. Förstner

ZB MED - Information Centre for Life Sciences | ZBMED

Significance Understanding the functions of cellular transcripts based on their sequence is challenging, in particular for noncoding RNAs, which tend to lack easily recognizable motifs. A more functionally relevant criterion is the association of RNAs with cognate RNA-binding proteins. Here, we describe the gradient profiling by sequencing (Grad-se...

Bacteria express many small RNAs for which the regulatory roles in pathogenesis have remained poorly understood due to a paucity of robust phenotypes in standard virulence assays. Here we use a generic ‘dual RNA-seq’ approach to profile RNA expression simultaneously in pathogen and host during Salmonella enterica serovar Typhimurium infection and r...

Significance To our knowledge, this work describes the first genome-wide annotation of transcriptional start sites in Vibrio cholerae and the discovery and characterization of a regulatory RNA, named VqmR, which controls collective behaviors in this major human pathogen. We show that VqmR is activated by the VqmA transcriptional regulator. VqmR rep...

RNA-Seq has become a potent and widely used method to qualitatively and quantitatively study transcriptomes. To draw biological conclusions based on RNA-Seq data, several steps, some of which are computationally intensive, have to be taken. Our READemption pipeline takes care of these individual tasks and integrates them into an easy-to-use tool wi...

Antibiotic resistance is a key medical concern, with antibiotic use likely being an important cause. However, here we describe an alternative route to clinically relevant antibiotic resistance that occurs solely due to competitive interactions among bacterial cells. We consistently observe that isolates of Methicillin-resistant Staphylococcus aureu...

Streptococcus agalactiae (Group B Streptococcus ) strain COH1 is a representative strain of serotype III, multi-locus sequence type 17, which is disproportionately associated with neonatal meningitis. Here we report the transcriptome of COH1 when interacting with human brain endothelial cells compared with COH1 alone.

The insertion sequence IS200 is widely distributed in Eubacteria. Despite its wide-ranging occurrence, IS200 does not appear to be mobile and as such is considered an ancestral component of bacterial genomes. Previous work in Salmonella enterica revealed that the IS200 tnpA transcript is processed to form a small highly structured RNA ( 5’tnpA ) th...

Bacterial meningitis is a life-threatening infection of the central nervous system (CNS) that occurs when bacteria are able to cross the blood–brain barrier (BBB) or the meningeal–cerebrospinal fluid barrier (mBCSFB). The BBB and mBCSFB comprise highly specialized brain endothelial cells (BECs) that typically restrict pathogen entry. Group B Strept...

The intestinal epithelium is the first line of defense against enteric pathogens. Removal of infected cells by exfoliation prevents mucosal translocation and systemic infection in the adult host, but is less commonly observed in the neonatal intestine. Instead, here, we describe non-professional efferocytosis of Salmonella-infected enterocytes by n...

This study aimed to compare diagnostic sensitivities of a rapid test (Rt) and an ELISA kit for detecting anti-SARS-CoV-2 IgM/IgG in virus-RT-PCR-positive (VPP) and virus-RT-PCR-unchecked (VPU) subjects in an Egyptian cohort during the first wave of SARS-CoV-2 infection. The results revealed higher sensitivity of the Rt for detecting IgM/IgG in the...

Proper RNA localisation is essential for physiological gene expression. Various kinds of genome-wide approaches permit to comprehensively profile subcellular transcriptomes. Among them, cell fractionation methods, that couple RNase treatment of isolated organelles to the sequencing of protected transcripts, remain most widely used, mainly because t...

Methanosarcina spherical virus (MetSV), infecting Methanosarcina species, encodes 22 genes, but their role in the infection process in combination with host genes has remained unknown. To study the infection process in detail, infected and uninfected M. mazei cultures were compared using dual-RNAseq, qRT-PCRs, and transmission electron microscopy (...

Strategically located at mucosal sites, mast cells are instrumental in sensing invading pathogens and modulating the quality of the ensuing immune responses depending on the nature of the infecting microbe. It is believed that mast cells produce type I IFN (IFN-I) in response to viruses, but not to bacterial infections, because of the incapacity of...

A central question concerning natural competence is why orthologs of competence genes are conserved in non-competent bacterial species, suggesting they have a role other than in transformation. Here we show that competence induction in the human pathogen Staphylococcus aureus occurs in response to ROS and host defenses that compromise bacterial res...

Q fever is a zoonotic disease that is a source of active epidemiological concern due to its persistent threat to public health. In this project, we have identified areas in the field of Coxiella research, especially regarding public health and genomic analysis, where there is an inadequacy of resources to monitor, organize, and analyze genomic data...

Noncoding small RNAs (sRNAs) are crucial for posttranscriptional regulation of gene expression in all organisms and are known to be involved in the regulation of bacterial virulence. We have investigated the presence of sRNAs in the obligate human pathogen B. pertussis , using transcriptome sequencing (RNA-seq) and the recently developed prokaryoti...

Many different protein domains are conserved among numerous species, but their function remains obscure. Proteins with DUF1127 domains number >17 000 in current databases, but a biological function has not yet been assigned to any of them. They are mostly found in alpha- and gammaproteobacteria, some of them plant and animal pathogens, symbionts or...

Trans-acting regulatory RNAs have the capacity to base pair with more mRNAs than generally detected under defined conditions, raising the possibility that sRNA target specificities vary depending on the specific metabolic or environmental conditions. In Sinorhizobium meliloti, the sRNA rnTrpL is derived from a tryptophan (Trp) transcription attenua...

Energy conservation via organohalide respiration (OHR) in dehalogenating Sulfurospirillum species is an inducible process. However, the gene products involved in tetrachloroethene (PCE) sensing and signal transduction have not been unambiguously identified. Here, genome sequencing of Sulfurospirillum strains defective in PCE respiration and compara...

Stable protein complexes, including those formed with RNA, are major building blocks of every living cell. Escherichia coli has been the leading bacterial organism with respect to global protein-protein networks. Yet, there has been no global census of RNA/protein complexes in this model species of microbiology. Here, we performed Grad-seq to estab...

Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) loci are found in bacterial and archaeal genomes where they provide the molecular machinery for acquisition of immunity against foreign DNA. In addition to the cas genes fundamentally required for CRISPR activity, a second class of genes is associated with the CRISPR loci, of which m...

Negative feedback regulation, that is the ability of a gene to repress its own synthesis, is the most abundant regulatory motif known to biology. Frequently reported for transcriptional regulators, negative feedback control relies on binding of a transcription factor to its own promoter. Here, we report a novel mechanism for gene autoregulation in...

Negative feedback regulation, that is the ability of a gene to repress its own synthesis, is the most abundant regulatory motif known to biology. Frequently reported for transcriptional regulators, negative feedback control relies on binding of a transcription factor to its own promoter. Here, we report a novel mechanism for gene autoregulation in...

Negative feedback regulation, that is the ability of a gene to repress its own synthesis, is the most abundant regulatory motif known to biology. Frequently reported for transcriptional regulators, negative feedback control relies on binding of a transcription factor to its own promoter. Here, we report a novel mechanism for gene autoregulation in...

Stable protein complexes, including those formed with RNA, are major building blocks of every living cell. Escherichia coli has been the leading bacterial organism with respect to global protein-protein networks. Yet, there has been no global census of RNA/protein complexes in this model species of microbiology. Here, we performed Grad-seq to estab...

Leader peptides encoded by transcription attenuators are widespread small proteins that are considered nonfunctional in trans . We found that the leader peptide peTrpL of the soil-dwelling plant symbiont Sinorhizobium meliloti is required for differential, posttranscriptional regulation of a multidrug resistance operon upon antibiotic exposure. Mul...

The ultraconserved RNA-binding protein YbeY has been associated with ribosomal RNA processing in numerous bacteria. However, mechanistic understanding of its function is still lacking, in particular in the light of the generally poor conservation of rRNA processing pathways across the bacterial kingdom. By leveraging the fact that YbeY homologues a...

RNA-protein interactions are the crucial basis for many steps of bacterial gene expression, including post-transcriptional control by small regulatory RNAs (sRNAs). In stark contrast to recent progress in the analysis of Gram-negative bacteria, knowledge about RNA-protein complexes in Gram-positive species remains scarce. Here, we used the Grad-seq...

Campylobacter spp. are one of the most important food-borne pathogens, which are quite susceptible to environmental or technological stressors compared to other zoonotic bacteria. This might be due to the lack of many stress response mechanisms described in other bacteria. Nevertheless, Campylobacter is able to survive in the environment and food p...

Ribosome biogenesis requires numerous trans-acting factors, some of which are deeply conserved. In Bacteria, the endoribonuclease YbeY is believed to be involved in 16S rRNA 3'-end processing and its loss was associated with ribosomal abnormalities. In Eukarya, YBEY appears to generally localize to mitochondria (or chloroplasts). Here we show that...

Ribosome biogenesis requires numerous trans-acting factors, some of which are deeply conserved. In Bacteria, the endoribonuclease YbeY is believed to be involved in 16S rRNA 3’-end processing and its loss was associated with ribosomal abnormalities. In Eukarya, YBEY appears to generally localize to mitochondria (or chloroplasts). Here we show that...

Small regulatory RNAs (sRNAs) are crucial components of many stress response systems. The envelope stress response (ESR) of Gram-negative bacteria is a paradigm for sRNA-mediated stress management and involves, among other factors, the alternative sigma factor E (σE) and one or more sRNAs. In this study, we identified the MicV sRNA as a new member...

Meningococcal meningitis is a severe central nervous system infection that occurs when Neisseria meningitidis (Nm) penetrates brain endothelial cells (BECs) of the meningeal blood-cerebrospinal fluid barrier. As a human-specific pathogen, in vivo models are greatly limited and pose a significant challenge. In vitro cell models have been developed,...

Riboregulation involving regulatory RNAs, RNA chaperones and ribonucleases is fundamental for the rapid adaptation of gene expression to changing environmental conditions. The gene coding for the RNase YbeY belongs to the minimal prokaryotic genome set and has a profound impact on physiology in a wide range of bacteria. Here, we show that the Agrob...

Haloferax volcanii is a well-established model species for haloarchaea. Small scale RNomics and bioinformatics predictions were used to identify small non-coding RNAs (sRNAs), and deletion mutants revealed that sRNAs have important regulatory functions. A recent dRNA-Seq study was used to characterize the primary transcriptome. Unexpectedly, it was...

Comparison of three codon usage tables: 1) the codon usage table computed from the more than 4000 protein coding genes of H. volcanii, 2) the codon usage table computed from the 1077 small proteins of up to 150 amino acids from H. volcanii, and 3) the codon usage table for E. coli. (DOC)

Oligonucleotides that were used to generate probes for the Northern blot analyses. (DOC)

Detection of an upstream sRNA (rather than a very long 5’-UTR). A. Screenshot from the Integrated Genome Browser. For explanations of panels see Fig 2. B. Northern blot analysis. (PPTX)

Example for one gene that is transcribed into two transcript isoforms of different lengths and example for overlapping 3’-ends of RNAs. A. Screenshot from the Integrated Genome Browser. For explanations of panels see Fig 2. B. Northern blot analysis with probe a. C. Northern blot analysis with probe b. (PPTX)

Example for two isoforms of an asRNA. A. Screenshot from the Integrated Genome Browser. For explanations of panels see Fig 1. B. Northern blot analysis with probe a. C. Northern blot analysis with probe b. (PPTX)

Complete uncropped versions of all Northern blots shown in the Figs 1 and 5–7, and S2–S6. (PPTX)

Results of previous RNA-Seq and dRNA-Seq studies with archaea. (DOC)

Summary of results of the RNA-Seq study. The Table lists the predicted RNAs, their genomic locations and their lengths. It also includes the associated HVO_number (gene identifier), presence of overlapping 3’-UTRs, and number of genes on polycistronic transcripts. For cdRNAs, the identification in the previous dRNA-Seq study is flagged [19]. (XLSX)

Examples of RNA-Seq results and the comparison with the genome annotation and dRNA-Seq results. Screenshots from the Integrated Genome Browser are shown. The upper half shows results from the top strand, the lower half from the bottom strand. The middle line represents the genome sequence, genome positions are indicated. The following data are show...

Detection of a very long 5’-UTR (rather than an upstream sRNA). A. Screenshot from the Integrated Genome Browser. For explanations of panels see Fig 2. B. Northern blot analysis. (PPTX)

Detection of a very long 3’-UTR. A. Screenshot from the Integrated Genome Browser. For explanations of panels see Fig 2. B. Northern blot analysis. (PPTX)

Polysaccharide intercellular adhesin (PIA)‐associated biofilm formation is mediated by the intercellular adhesin (ica) locus and represents a major pathomechanism of Staphylococcus epidermidis. Here we report on a novel long non‐coding (nc)RNA, named IcaZ, which is approximately 400 nucleotides in size. icaZ is located downstream of the ica repress...

RsaE is a conserved small regulatory RNA (sRNA) which was previously reported to represent a riboregulator of central carbon flow and other metabolic pathways in Staphylococcus aureus and Bacillus subtilis. Here we show that RsaE contributes to extracellular (e)DNA release and biofilm-matrix switching towards polysaccharide intercellular adhesin (P...

dRNA-seq transcription profiling data of S. epidermidis PS2 and PS10. Complete list of (differentially expressed) genes. (XLSX)

EMSAs using increasing amounts of sucD target RNA and radioactively labeled (*) full-length (RsaE, left panel) or processed RsaE (RsaEp, right panel) as binding partners. Filled triangles indicate labeled unbound RsaE species and open triangles mark RsaE/target complexes. (PDF)

List of oligonucleotides used in this study. (PDF)

Differentially transcribed metabolic genes in S. epidermidis PS2 (early) and PS10 (late) isolates based on RNAseq data analysis. The scheme was generated by employing the Interactive Pathway Explorer (https://pathways.embl.de). Genes and pathways highlighted in blue are upregulated in PS2 (and downregulated in PS10), while features downregulated in...

RsaE expression and correlation with biofilm production and eDNA release in different S. epidermidis strains. (A) Quantification of rsaE transcript by qRT-PCR at the time point indicated. The graph displays relative mRNA amounts using gyrB expression as reference. (B) Analysis of biofilm production by static 96-well microtiter plate biofilm assays....

rsaE expression in planktonic culture. CLSM images of S. epidermidis PS10 p_(PrsaECFP) during growth in liquid culture. At the indicated time points, cells were taken and subjected to CLSM imaging. Shown is an overlay of total bacterial cell mass, visualized by transmission microscopy, and rsaE-expressing bacterial cells, highlighted by cerulean ex...

Time lapse video of CLSM live cell imaging monitoring rsaE promoter activity (light blue) and eDNA release (red) in an S. epidermidis PS10 biofilm during 20 hours of growth in chamber slides. Same video as S1 Video, but without transmission microscopy. Instead, eDNA is visualized in red by Ethidium Homodimer II staining with light blue cells repres...

List of RsaE target mRNA predictions by IntaRNA. (PDF)

Supporting information. Methods. (DOCX)

Analysis of biofilm production of S. epidermidis O-47 (pCG248_rsaE) by static 96-well microtiter plate biofilm assays. Plasmid pCG248_rsaE harbours the rsaE gene under the control of an anhydrotetracycline (ATc)-inducible promoter. Expression of rsaE was induced by increasing ATc concentrations (25 to 75 ng/ml). Total biofilm (BF) mass as well as P...

lrgA/RsaE EMSA upon competition with unlabeled RsaE (A) or with an antisense-RsaE RNA oligonucleotide (B) during complex formation. (A) Increasing amounts of unlabeled competitor RsaE (lanes 3–7) or a 500-fold and 1000-fold excess of yeast tRNAs (lane 8+9) were mixed with 200 nM radioactively (*) labeled RsaE before addition to 200 nM lrgA target R...

(A) Secondary structures of full-length and processed RsaE species according to MFold-4.7-based predictions [16]. Red boxes highlight positions of the C-rich motifs within the RsaE molecules. Position of the antisense-RsaE RNA oligonucleotide is underlined. (B) IntaRNA predictions of full-length (top) and processed (bottom) RsaE interaction with su...

Time lapse video of CLSM live cell imaging monitoring rsaE promoter activity in an S. epidermidis PS10 biofilm during 20 hours of growth in chamber slides. The strain carries plasmid p_(PrsaEcfp) in which the rsaE promoter is fused to the blue-fluorescent cerulean protein gene cfp as a reporter. Light blue cells represent bacteria with an active rs...

The protein ProQ has recently been discovered as the centerpiece of a previously overlooked “third domain” of small RNA-mediated control of gene expression in bacteria. As in vitro work continues to reveal molecular mechanisms, it is also important to understand how ProQ affects the life cycle of bacterial pathogens as these pathogens infect eukary...

To understand the gene regulation of an organism of interest, a comprehensive genome annotation is essential. While some features, such as coding sequences, can be computationally predicted with high accuracy based purely on the genomic sequence, others, such as promoter elements or non-coding RNAs are harder to detect. RNA-Seq has proven to be an...