Bacteria are under constant attack from bacteriophages (phages), bacterial parasites that are the most abundant biological entity on earth. To resist phage infection, bacteria have evolved an ...impressive arsenal of anti-phage systems. Recent advances have significantly broadened and deepened our understanding of how bacteria battle phages, spearheaded by new systems like CRISPR-Cas. This review aims to summarize bacterial anti-phage mechanisms, with an emphasis on the most recent developments in the field.
Bacteria are threatened by viral attack in every environment they colonize. For more than 100 years, microbiologists have investigated the mechanisms in place to contain these attacks. Rostøl and Marraffini review the vast and sophisticated arsenal of bacterial defense mechanisms, with an emphasis on recent discoveries in the field.
In the type III CRISPR-Cas immune response of prokaryotes, infection triggers the production of cyclic oligoadenylates that bind and activate proteins that contain a CARF domain
. Many type III loci ...are associated with proteins in which the CRISPR-associated Rossman fold (CARF) domain is fused to a restriction endonuclease-like domain
. However, with the exception of the well-characterized Csm6 and Csx1 ribonucleases
, whether and how these inducible effectors provide defence is not known. Here we investigated a type III CRISPR accessory protein, which we name cyclic-oligoadenylate-activated single-stranded ribonuclease and single-stranded deoxyribonuclease 1 (Card1). Card1 forms a symmetrical dimer that has a large central cavity between its CRISPR-associated Rossmann fold and restriction endonuclease domains that binds cyclic tetra-adenylate. The binding of ligand results in a conformational change comprising the rotation of individual monomers relative to each other to form a more compact dimeric scaffold, in which a manganese cation coordinates the catalytic residues and activates the cleavage of single-stranded-but not double-stranded-nucleic acids (both DNA and RNA). In vivo, activation of Card1 induces dormancy of the infected hosts to provide immunity against phage infection and plasmids. Our results highlight the diversity of strategies used in CRISPR systems to provide immunity.
In many prokaryotes, type III clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated (Cas) systems detect and degrade invasive genetic elements by an RNA-guided, ...RNA-targeting multisubunit interference complex. The CRISPR-associated protein Csm6 additionally contributes to interference by functioning as a standalone RNase that degrades invader RNA transcripts, but the mechanism linking invader sensing to Csm6 activity is not understood. Here we show that Csm6 proteins are activated through a second messenger generated by the type III interference complex. Upon target RNA binding by the interference complex, its Cas10 subunit converts ATP into a cyclic oligoadenylate product, which allosterically activates Csm6 by binding to its CRISPR-associated Rossmann fold (CARF) domain. CARF domain mutations that abolish allosteric activation inhibit Csm6 activity in vivo, and mutations in the Cas10 Palm domain phenocopy loss of Csm6. Together, these results point to an unprecedented mechanism for regulation of CRISPR interference that bears striking conceptual similarity to oligoadenylate signalling in mammalian innate immunity.
Extended producer responsibility (EPR) for packaging was introduced to achieve closed‐loop end‐of‐life management and design for recyclability and is an integral part of the European Union's (EU) ...environmental policy mix. Despite common objectives, EPR systems differ across EU member states in design and implementation. Existing cross‐country comparisons often fall short of systematizing EPR and reflecting its complexity. Understanding the components and interdependencies of EPR is crucial for adjusting policy design. This article links previously isolated components of EPR and studies the design, implementation, and transfer of EPR systems in 25 European countries through a developmental approach. It extends EPR theory by systematizing EPR design features at three hierarchical levels: system scope, responsibility allocation, and instrument type. It then tests the approach by systematically examining similarities and differences, including a uniform coding process and cluster analysis. My results indicate that EPR for plastic packaging is becoming increasingly fine‐grained and diverse. I show that ‘‘path‐dependencies’’ emerge between certain features at the responsibility allocation level. I also find common implementation patterns across countries revolving around, for example, EPR market structure or producer responsibility type. System scope converges across countries, while instrument types are becoming increasingly multi‐faceted. I find that policy transfer mechanisms, such as first‐mover dynamics and geographic proximity, have likely influenced existing landscapes of EPR design across the EU. These insights provide an intriguing first step toward deeper analysis of EPR design and, if further extended, can be operationalized for future ex ante or ex post policy analysis of EPR system setups.
Horizontal gene transfer and mutation are the two major drivers of microbial evolution that enable bacteria to adapt to fluctuating environmental stressors
. Clustered, regularly interspaced, short ...palindromic repeats (CRISPR) systems use RNA-guided nucleases to direct sequence-specific destruction of the genomes of mobile genetic elements that mediate horizontal gene transfer, such as conjugative plasmids
and bacteriophages
, thus limiting the extent to which bacteria can evolve by this mechanism. A subset of CRISPR systems also exhibit non-specific degradation of DNA
; however, whether and how this feature affects the host has not yet been examined. Here we show that the non-specific DNase activity of the staphylococcal type III-A CRISPR-Cas system increases mutations in the host and accelerates the generation of antibiotic resistance in Staphylococcus aureus and Staphylococcus epidermidis. These mutations require the induction of the SOS response to DNA damage and display a distinct pattern. Our results demonstrate that by differentially affecting both mechanisms that generate genetic diversity, type III-A CRISPR systems can modulate the evolution of the bacterial host.
Bacterial and archaeal CRISPR-Cas systems provide RNA-guided immunity against genetic invaders such as bacteriophages and plasmids. Upon target RNA recognition, type III CRISPR-Cas systems produce ...cyclic-oligoadenylate second messengers that activate downstream effectors, including Csm6 ribonucleases, via their CARF domains. Here, we show that Enteroccocus italicus Csm6 (EiCsm6) degrades its cognate cyclic hexa-AMP (cA6) activator, and report the crystal structure of EiCsm6 bound to a cA6 mimic. Our structural, biochemical, and in vivo functional assays reveal how cA6 recognition by the CARF domain activates the Csm6 HEPN domains for collateral RNA degradation, and how CARF domain-mediated cA6 cleavage provides an intrinsic off-switch to limit Csm6 activity in the absence of ring nucleases. These mechanisms facilitate rapid invader clearance and ensure termination of CRISPR interference to limit self-toxicity.
Bacteria encode sophisticated anti-phage systems that are diverse and versatile and display high genetic mobility. How this variability and mobility occurs remains largely unknown. Here, we ...demonstrate that a widespread family of pathogenicity islands, the phage-inducible chromosomal islands (PICIs), carry an impressive arsenal of defense mechanisms, which can be disseminated intra- and inter-generically by helper phages. These defense systems provide broad immunity, blocking not only phage reproduction, but also plasmid and non-cognate PICI transfer. Our results demonstrate that phages can mobilize PICI-encoded immunity systems to use them against other mobile genetic elements, which compete with the phages for the same bacterial hosts. Therefore, despite the cost, mobilization of PICIs may be beneficial for phages, PICIs, and bacteria in nature. Our results suggest that PICIs are important players controlling horizontal gene transfer and that PICIs and phages establish mutualistic interactions that drive bacterial ecology and evolution.
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•PICIs carry an impressive arsenal of defense systems•PICI-encoded defense systems confer resistance to various mobile genetic elements•These defense systems block horizontal gene transfer•Phages benefit from mobilizing PICIs to target competing mobile genetic elements
Phage-inducible chromosomal islands (PICIs) are highly mobile genetic elements that not only provide a diversity of defense systems in gram-positive and gram-negative bacteria, but can also be induced by a helper phage to protect that helper phage and the bacterial host from competing phages and other mobile genetic elements. This reveals a complex mutualistic relationship between bacteria, PICIs, and their helper phages that may broadly affect horizontal gene transfer.
Disorder in proteins is vital for biological function, yet it is challenging to characterize. Therefore, methods for predicting protein disorder from sequence are fundamental. Currently, predictors ...are trained and evaluated using data from X-ray structures or from various biochemical or spectroscopic data. However, the prediction accuracy of disordered predictors is not calibrated, nor is it established whether predictors are intrinsically biased towards one of the extremes of the order-disorder axis. We therefore generated and validated a comprehensive experimental benchmarking set of site-specific and continuous disorder, using deposited NMR chemical shift data. This novel experimental data collection is fully appropriate and represents the full spectrum of disorder. We subsequently analyzed the performance of 26 widely-used disorder prediction methods and found that these vary noticeably. At the same time, a distinct bias for over-predicting order was identified for some algorithms. Our analysis has important implications for the validity and the interpretation of protein disorder, as utilized, for example, in assessing the content of disorder in proteomes.
Type III-A CRISPR-Cas systems employ the Cas10-Csm complex to destroy bacteriophages and plasmids, using a guide RNA to locate complementary RNA molecules from the invader and trigger an immune ...response that eliminates the infecting DNA. In addition, these systems possess the non-specific RNase Csm6, which provides further protection for the host. While the role of Csm6 in immunity during phage infection has been determined, how this RNase is used against plasmids is unclear. Here, we show that Staphylococcus epidermidis Csm6 is required for immunity when transcription across the plasmid target is infrequent, leading to impaired target recognition and inefficient DNA degradation by the Cas10-Csm complex. In these conditions, Csm6 causes growth arrest in the host and prevents further plasmid replication through the indiscriminate degradation of host and plasmid transcripts. In contrast, when plasmid target sequences are efficiently transcribed, Csm6 is dispensable and DNA degradation by Cas10 is sufficient for anti-plasmid immunity. Csm6 therefore provides robustness to the type III-A CRISPR-Cas immune response against difficult targets for the Cas10-Csm complex.
Resources have received significant attention in recent years resulting in development of a wide range of resource depletion indicators within life cycle assessment (LCA). Understanding the ...differences in assessment principles used to derive these indicators and the effects on the impact assessment results is critical for indicator selection and interpretation of the results. Eleven resource depletion methods were evaluated quantitatively with respect to resource coverage, characterization factors (CF), impact contributions from individual resources, and total impact scores. We included 2247 individual market inventory data sets covering a wide range of societal activities (ecoinvent database v3.0). Log–linear regression analysis was carried out for all pairwise combinations of the 11 methods for identification of correlations in CFs (resources) and total impacts (inventory data sets) between methods. Significant differences in resource coverage were observed (9–73 resources) revealing a trade-off between resource coverage and model complexity. High correlation in CFs between methods did not necessarily manifest in high correlation in total impacts. This indicates that also resource coverage may be critical for impact assessment results. Although no consistent correlations between methods applying similar assessment models could be observed, all methods showed relatively high correlation regarding the assessment of energy resources. Finally, we classify the existing methods into three groups, according to method focus and modeling approach, to aid method selection within LCA.
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