Abstract
Over the past two decades, small noncoding RNAs (sRNAs) that regulate mRNAs by short base pairing have gone from a curiosity to a major class of post-transcriptional regulators in bacteria. ...They are integral to many stress responses and regulatory circuits, affecting almost all aspects of bacterial life. Following pioneering sRNA searches in the early 2000s, the field quickly focused on conserved sRNA genes in the intergenic regions of bacterial chromosomes. Yet, it soon emerged that there might be another rich source of bacterial sRNAs—processed 3′ end fragments of mRNAs. Several such 3′ end-derived sRNAs have now been characterized, often revealing unexpected, conserved functions in diverse cellular processes. Here, we review our current knowledge of these 3′ end-derived sRNAs—their biogenesis through ribonucleases, their molecular mechanisms, their interactions with RNA-binding proteins such as Hfq or ProQ and their functional scope, which ranges from acting as specialized regulators of single metabolic genes to constituting entire noncoding arms in global stress responses. Recent global RNA interactome studies suggest that the importance of functional 3′ end-derived sRNAs has been vastly underestimated and that this type of cross-regulation between genes at the mRNA level is more pervasive in bacteria than currently appreciated.
There are a growing number of examples of bacterial mRNAs that produce small RNAs cleaved from their own 3′ ends to post-transcriptionally control other genes.
Over the past few years, numerous anti-phage defense systems have been discovered in bacteria. Although the mechanism of defense for some of these systems is understood, a major unanswered question ...is how these systems sense phage infection. To systematically address this question, we isolated 177 phage mutants that escape 15 different defense systems. In many cases, these escaper phages were mutated in the gene sensed by the defense system, enabling us to map the phage determinants that confer sensitivity to bacterial immunity. Our data identify specificity determinants of diverse retron systems and reveal phage-encoded triggers for multiple abortive infection systems. We find general themes in phage sensing and demonstrate that mechanistically diverse systems have converged to sense either the core replication machinery of the phage, phage structural components, or host takeover mechanisms. Combining our data with previous findings, we formulate key principles on how bacterial immune systems sense phage invaders.
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•Phage mutants escaping bacterial immunity reveal how infection is detected•Diverse defense systems have converged to sense the same phage components•Discovery of the phage triggers for multiple retron defense systems•Discovery of 5 abortive infection systems and the phage proteins that trigger them
A tour de force analysis provides fresh insights into the ways in which bacterial immune systems sense phage invaders and how the latter escape the defense mechanism.
Bacterial anti-phage systems are frequently clustered in microbial genomes, forming defense islands. This property enabled the recent discovery of multiple defense systems based on their genomic ...co-localization with known systems, but the full arsenal of anti-phage mechanisms remains unknown. We report the discovery of 21 defense systems that protect bacteria from phages, based on computational genomic analyses and phage-infection experiments. We identified multiple systems with domains involved in eukaryotic antiviral immunity, including those homologous to the ubiquitin-like ISG15 protein, dynamin-like domains, and SEFIR domains, and show their participation in bacterial defenses. Additional systems include domains predicted to manipulate DNA and RNA molecules, alongside toxin-antitoxin systems shown here to function in anti-phage defense. These systems are widely distributed in microbial genomes, and in some bacteria, they form a considerable fraction of the immune arsenal. Our data substantially expand the inventory of defense systems utilized by bacteria to counteract phage infection.
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•Discovery of 21 anti-phage systems based on their abundance in defense islands•Some of the discovered defense systems show homology to human immune proteins•Other systems include diverse protein domains and predicted functionalities•Systems are found in 47% of genomes and make up 12% of their known defensive arsenal
Millman et al. discover 21 defense systems that protect bacteria from phages based on their genomic co-localization with known systems. These defense systems, some of which show homology to human immune genes, are widely distributed in microbial genomes and substantially expand the known arsenal of anti-phage defense systems.
While bacteria were long thought to rely primarily on transcriptional control, it is now well established that they also use numerous small RNAs to regulate mRNA translation and stability. There has ...recently been a surge in studies, including one by Waters et al () in this issue of The EMBO Journal, that have used clever variations of the RNA‐seq technique to comprehensively map small RNA–target networks.
Several recent studies have used clever variations of RNA‐seq techniques to comprehensively map small RNA–target networks involved in controlling bacterial gene expression.
Plant growth can be affected by soil bacteria. In turn, plants are known to influence soil bacteria through rhizodeposits and changes in abiotic conditions. We aimed to quantify the phylotype ...richness and relative abundance of rhizosphere bacteria that are actually influenced in a plant species-specific manner and to determine the role of the disproportionately large diversity of low-abundance bacteria belonging to the rare biosphere (<0.1 relative abundance) in this process. In addition, we aimed to determine whether plant phylogeny has an influence on the plant species-specific rhizosphere bacterial community. For this purpose, 19 herbaceous plant species from five different plant orders were grown in a common soil substrate. Bacterial communities in the initial soil substrate and the established rhizosphere soils were compared by 16S rRNA gene amplicon sequencing. Only a small number of bacterial operational taxonomic units (OTUs, 97% sequence identity) responded either positively (ca. 1%) or negatively (ca. 1%) to a specific plant species. On average, 91% of plant-specific positive response OTUs comprised bacteria belonging to the rare biosphere, highlighting that low-abundance populations are metabolically active in the rhizosphere. In addition, low-abundance OTUs were in terms of their summed relative abundance major drivers of the bacterial phyla composition across the rhizosphere of all tested plant species. However, no effect of plant phylogeny could be observed on the established rhizosphere bacterial communities, neither when considering differences in the overall established rhizosphere communities nor when considering plant species-specific responders only. Our study provides a quantitative assessment of the effect of plants on their rhizosphere bacteria across multiple plant orders. Plant species-specific effects on soil bacterial communities involved only 18-111 bacterial OTUs out of several 1000s; this minority may potentially impact plant growth in plant-bacteria interactions.
The identification of new RNA functions and the functional annotation of transcripts in genomes represent exciting yet challenging endeavours of modern biology. Crucial insights into the biological ...roles of RNA molecules can be gained from the identification of the proteins with which they form specific complexes. Modern interactome techniques permit to profile RNA–protein interactions in a genome-wide manner and identify new RNA classes associated with globally acting RNA-binding proteins. Applied to a variety of organisms, these methods are already revolutionising our understanding of RNA-mediated biological processes. Here, we focus on one such approach—Gradient sequencing or Grad-seq—which has recently guided the discovery of protein ProQ and its associated small RNAs as a new domain of post-transcriptional control in bacteria.
Bacteria are an exceedingly diverse group of organisms whose molecular exploration is experiencing a renaissance. While the classical view of bacterial gene expression was relatively simple, the ...emerging view is more complex, encompassing extensive post-transcriptional control involving riboswitches, RNA thermometers, and regulatory small RNAs (sRNAs) associated with the RNA-binding proteins CsrA, Hfq, and ProQ, as well as CRISPR/Cas systems that are programmed by RNAs. Moreover, increasing interest in members of the human microbiota and environmental microbial communities has highlighted the importance of understudied bacterial species with largely unknown transcriptome structures and RNA-based control mechanisms. Collectively, this creates a need for global RNA biology approaches that can rapidly and comprehensively analyze the RNA composition of a bacterium of interest. We review such approaches with a focus on RNA-seq as a versatile tool to investigate the different layers of gene expression in which RNA is made, processed, regulated, modified, translated, and turned over.
RNA-seq-based approaches are revolutionizing how bacterial RNA biology can be studied. Hör, Gorski, and Vogel review the available global methods that can be used to chart the increasingly diverse number of RNA species and functions in any microbe of interest.
The last few decades have led to an explosion in our understanding of the major roles that small regulatory RNAs (sRNAs) play in regulatory circuits and the responses to stress in many bacterial ...species. Much of the foundational work was carried out with
and
serovar Typhimurium. The studies of these organisms provided an overview of how the sRNAs function and their impact on bacterial physiology, serving as a blueprint for sRNA biology in many other prokaryotes. They also led to the development of new technologies. In this chapter, we first summarize how these sRNAs were identified, defining them in the process. We discuss how they are regulated and how they act and provide selected examples of their roles in regulatory circuits and the consequences of this regulation. Throughout, we summarize the methodologies that were developed to identify and study the regulatory RNAs, most of which are applicable to other bacteria. Newly updated databases of the known sRNAs in
K-12 and
Typhimurium SL1344 serve as a reference point for much of the discussion and, hopefully, as a resource for readers and for future experiments to address open questions raised in this review.
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.
Ribosome profiling (Ribo-seq) is a powerful method for the transcriptome-wide assessment of protein synthesis rates and the study of translational control mechanisms. Yet, Ribo-seq also has ...limitations. These include difficulties with the analysis of translation-modulating molecules such as antibiotics, which are often toxic or challenging to deliver into living cells. Here, we have developed in vitro Ribo-seq (INRI-seq), a cell-free method to analyze the translational landscape of a fully customizable synthetic transcriptome. Using Escherichia coli as an example, we show how INRI-seq can be used to analyze the translation initiation sites of a transcriptome of interest. We also study the global impact of direct translation inhibition by antisense peptide nucleic acid (PNA) to analyze PNA off-target effects. Overall, INRI-seq presents a scalable, sensitive method to study translation initiation in a transcriptome-wide manner without the potentially confounding effects of extracting ribosomes from living cells.
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