Biology subjects

Gerovac, M.

Publications and source records attributed to Gerovac, M..

9 recordsLinked to original sources

bioRxiv2024

Non-genetic messenger RNA silencing reveals essential genes in phage-host interplay

Bacteriophages are the most abundant entities on earth and exhibit vast genetic and phenotypic diversity. Exploitation of this largely unexplored molecular space requires identification and functional characterisation of genes that act at the phage-host interface. To date, this is restricted to few model phage-host systems that are amenable to genetic manipulation. To overcome this limitation, we introduce a non-genetic mRNA targeting approach using exogenous delivery of programmable antisense oligomers (ASOs) to silence genes of both DNA and RNA phages. A systematic knockdown screen of core and accessory genes of the nucleus-forming jumbo phage {Phi}KZ, coupled to RNA-sequencing and microscopy analyses, reveals previously unrecognised proteins that are essential for phage replication and that, upon silencing, elicit distinct phenotypes at the level of the phage and host response. One of these factors is a {Phi}KZ-encoded nuclease that acts at a major decision point during infection, linking the formation of the protective phage nucleus to phage genome amplification. The non-genetic ASO-based gene silencing used here promises to be a versatile tool for molecular discovery in phage biology, will help elucidate defence and anti-defence mechanisms in non-model phage-host pairs, and offers potential for optimising phage therapy and biotechnological procedures.

molecular biology↗

bioRxiv2024

A phage nucleus-associated protein from the jumbophage Churi inhibits bacterial growth through protein translation interference

Antibacterial proteins inhibiting Pseudomonas aeruginosa have been identified in various phages and explored as antibiotic alternatives. Here, we isolated a phiKZ-like phage, Churi, which encodes 364 open reading frames. We examined 15 early-expressed phage proteins for their ability to inhibit bacterial growth, and found that gp335, closely related to phiKZ-gp14, exhibits antibacterial activity. Similar to phiKZ-gp14, recently shown to form a complex with the P. aeruginosa ribosome, we predict experimentally that gp335 interacts with ribosomal proteins, suggesting its involvement in protein translation. GFP-tagged gp335 clusters around the phage nucleus as early as 15 minutes post-infection and remains associated with it throughout the infection, suggesting its role in protein expression in the cell cytoplasm. CRISPR-Cas13-mediated deletion of gp355 reveals that the mutant phage has a prolonged latent period. Altogether, we demonstrate that gp335 is an antibacterial protein of nucleus-forming phages that associates with the ribosomes at the phage nucleus.

molecular biology↗

bioRxiv2023

Immediate targeting of host ribosomes by jumbo phage encoded proteins

Bacteriophages must seize control of the host gene expression machinery to promote their own protein synthesis. Since the bacterial hosts are armed with numerous anti-phage defence systems, it is essential that mechanisms of host take-over act immediately upon infection. Although individual proteins that modulate components of the bacterial gene expression apparatus have been described in several different phages, systematic approaches which capture the phages arsenal for immediate targeting of host transcription and translation processes have been lacking. In particular, there are no known phage factors that associate directly with host ribosomes to modulate protein synthesis. Here, we take an integrative high-throughput approach to uncover numerous new proteins encoded by the jumbo phage {Phi}KZ that target the gene expression machinery of the Gram-negative human pathogen Pseudomonas aeruginosa immediately upon infection. By integrating biochemical and structural analyses, we identify a conserved phage factor that associates with the large ribosomal subunit by binding the 5S ribosomal RNA. This highly abundant factor is amongst the earliest {Phi}KZ proteins expressed after infection and stays bound to ribosomes during the entire translation cycle. Our study provides a general strategy to decipher molecular components of phage-mediated host take-over and argues that phage genomes represent a large discovery space for proteins that modulate the host gene expression machinery.

microbiology↗

bioRxiv2022

Grad-seq analysis of Enterococcus faecalis and Enterococcus faecium provides a global view of RNA and protein complexes in these two opportunistic pathogens

Enterococcus faecalis and Enterococcus faecium are major nosocomial pathogens. Despite their relevance to public health and their role in the development of bacterial antibiotic resistance, relatively little is known about gene regulation in these species. RNA-protein complexes serve crucial functions in all cellular processes associated with gene expression, including post-transcriptional control mediated by small regulatory RNAs (sRNAs). Here, we present a new resource for the study of enterococcal RNA biology, employing the Grad-seq technique to comprehensively predict complexes formed by RNA and proteins in E. faecalis V583 and E. faecium AUS0004. Analysis of the generated global RNA and protein sedimentation profiles led to the identification of RNA-protein complexes and putative novel sRNAs. Validating our data sets, we observe well-established cellular RNA-protein complexes such as the 6S RNA-RNA polymerase complex, suggesting that 6S RNA-mediated global control of transcription is conserved in enterococci. Focusing on the largely uncharacterized RNA-binding protein KhpB, we use the RIP-seq technique to predict that KhpB interacts with sRNAs, tRNAs, and untranslated regions of mRNAs, and might be involved in the processing of specific tRNAs. Collectively, these datasets provide departure points for in-depth studies of the cellular interactome of enterococci that should facilitate functional discovery in these and related Gram-positive species. Our data are available to the community through a user-friendly Grad-seq browser that allows interactive searches of the sedimentation profiles (https://resources.helmholtz-hiri.de/gradseqef/).

microbiology↗

bioRxiv2022

A network of small RNAs regulates sporulation initiation in C. difficile

The obligate anaerobic, enteric pathogen Clostridioides difficile persists in the intestinal tract by forming antibiotic resistant endospores that contribute to relapsing and recurrent infections. Despite the importance of sporulation for C. difficile pathogenesis, environmental cues, and molecular mechanisms regulating sporulation initiation remain ill defined. Here, using RIL-seq to capture the Hfq-dependent RNA-RNA interactome, we discovered a network of small RNAs that bind to mRNAs encoding sporulation-related genes. We show that two of these small RNAs, SpoX and SpoY, regulate translation of the master regulator of sporulation, Spo0A, in an opposing manner, which ultimately leads to altered sporulation rates. Infection of antibiotic-treated mice with SpoX and SpoY deletion mutants revealed a global effect on gut colonization and intestinal sporulation. Our work uncovers an elaborate RNA-RNA interactome controlling the physiology and virulence of C. difficile and identifies a complex post-transcriptional layer in the regulation of spore formation in this important human pathogen.

molecular biology↗

bioRxiv2022

Global profiling of the RNA and protein complexes of Escherichia coli by size exclusion chromatography followed by RNA sequencing and mass spectrometry (SEC-seq)

New methods for the global identification of RNA-protein interactions have led to greater recognition of the abundance and importance of RNA-binding proteins (RBPs) in bacteria. Here, we expand this tool kit by developing SEC-seq, a method based on a similar concept as the established Grad-seq approach. In Grad-seq, cellular RNA and protein complexes of a bacterium of interest are separated in a glycerol gradient, followed by high-throughput RNA-sequencing and mass spectrometry analyses of individual gradient fractions. New RNA-protein complexes are predicted based on the similarity of their elution profiles. In SEC-seq, we have replaced the glycerol gradient with separation by size exclusion chromatography, which shortens operation times and offers greater potential for automation. Applying SEC-seq to Escherichia coli, we find that the method provides a higher resolution than Grad-seq in the lower molecular weight range up to [~]500 kDa. This is illustrated by the ability of SEC-seq to resolve two distinct, but similarly sized complexes of the global translational repressor CsrA with either of its antagonistic small RNAs, CsrB and CsrC. We also characterized changes in the SEC-seq profiles of the small RNA MicA upon deletion of its RNA chaperones Hfq and ProQ and investigated the redistribution of these two proteins upon RNase treatment. Overall, we demonstrate that SEC-seq is a tractable and reproducible method for the global profiling of bacterial RNA-protein complexes that offers the potential to discover yet-unrecognized associations between bacterial RNAs and proteins.

molecular biology↗

bioRxiv2020

A Grad-seq view of RNA and protein complexes in Pseudomonas aeruginosa under standard and bacteriophage predation conditions

The Gram-negative rod-shaped bacterium Pseudomonas aeruginosa is not only a major cause of nosocomial infections but also serves as a model species of bacterial RNA biology. While its transcriptome architecture and post-transcriptional regulation through the RNA-binding proteins Hfq, RsmA and RsmN have been studied in detail, global information about stable RNA-protein complexes is currently lacking in this human pathogen. Here, we implement Gradient profiling by sequencing (Grad-seq) in exponentially growing P. aeruginosa cells to comprehensively predict RNA and protein complexes, based on glycerol gradient sedimentation profiles of >73% of all transcripts and [~]40% of all proteins. As to benchmarking, our global profiles readily reported complexes of stable RNAs of P. aeruginosa, including 6S RNA with RNA polymerase and associated pRNAs. We observe specific clusters of non-coding RNAs, which correlate with Hfq and RsmA/N, and provide a first hint that P. aeruginosa expresses a ProQ-like FinO domain containing RNA-binding protein. To understand how biological stress may perturb cellular RNA/protein complexes, we performed Grad-seq after infection by the bacteriophage {Phi}KZ. This model phage, which has a well-defined transcription profile during host takeover, displayed efficient translational utilization of phage mRNAs and tRNAs, as evident from their increased co-sedimentation with ribosomal subunits. Additionally, Grad-seq experimentally determines previously overlooked phage-encoded non-coding RNAs. Taken together, the Pseudomonas protein and RNA complex data provided here will pave the way to a better understanding of RNA-protein interactions during viral predation of the bacterial cell. IMPORTANCEStable complexes by cellular proteins and RNA molecules lie at the heart of gene regulation and physiology in any bacterium of interest. It is therefore crucial to globally determine these complexes in order to identify and characterize new molecular players and regulation mechanisms. Pseudomonads harbour some of the largest genomes known in bacteria, encoding [~]5,500 different proteins. Here, we provide a first glimpse on which proteins and cellular transcripts form stable complexes in the human pathogen Pseudomonas aeruginosa. We additionally performed this analysis with bacteria subjected to the important and frequently encountered biological stress of a bacteriophage infection. We identified several molecules with established roles in a variety of cellular pathways, which were affected by the phage and can now be explored for their role during phage infection. Most importantly, we observed strong co-localization of phage transcripts and host ribosomes, indicating the existence of specialized translation mechanisms during phage infection. All data are publicly available in an interactive and easy to use browser.

molecular biology↗

bioRxiv2020

Grad-seq shines light on unrecognized RNA and protein complexes in the model bacterium Escherichia coli

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 establish an RNA/protein complexome, reconstructing sedimentation profiles in a glycerol gradient for ~85% of all E. coli transcripts and ~49% of the proteins. These include the majority of small noncoding RNAs (sRNAs) detectable in this bacterium as well as the general sRNA-binding proteins, CsrA, Hfq and ProQ. In presenting use cases for utilization of these RNA and protein maps, we show that a stable association of RyeG with 30S ribosomes gives this seemingly noncoding RNA of prophage origin away as an mRNA of a toxic small protein. Similarly, we show that the broadly conserved uncharacterized protein YggL is a 50S subunit factor in assembled 70S ribosomes. Overall, this study crucially extends our knowledge about the cellular interactome of the primary model bacterium E. coli through providing global RNA/protein complexome information and should facilitate functional discovery in this and related species.

microbiology↗

bioRxiv2020

Global discovery of bacterial RNA-binding proteins by RNase-sensitive gradient profiles reports a new FinO domain protein

RNA-binding proteins (RBPs) play important roles in bacterial gene expression and physiology but their true number and functional scope remain little understood even in model microbes. To advance global RBP discovery in bacteria, we here establish glycerol gradient sedimentation with RNase treatment and mass spectrometry (GradR). Applied to Salmonella enterica, GradR confirms many known RBPs such as CsrA, Hfq and ProQ by their RNase-sensitive sedimentation profiles, and discovers the FopA protein as a new member of the emerging family of FinO/ProQ-like RBPs. FopA, encoded on resistance plasmid pCol1B9, primarily targets a small RNA associated with plasmid replication. The target suite of FopA dramatically differs from the related global RBP ProQ, revealing context-dependent selective RNA recognition by FinO-domain RBPs. Numerous other unexpected RNase-induced changes in gradient profiles suggest that cellular RNA helps to organize macromolecular complexes in bacteria. By enabling poly(A)-independent generic RBP discovery, GradR provides an important element in the quest to build a comprehensive catalogue of microbial RBPs.

microbiology↗