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CRISPRi-ART enables functional genomics of diverse bacteriophages using RNA-binding dCas13d

Bacteriophages constitute one of the largest reservoirs of genes of unknown function in the biosphere. Even in well-characterized phages, the functions of most genes remain unknown. Experimental approaches to study phage gene fitness and function at genome scale are lacking, partly because phages subvert many modern functional genomics tools. Here we leverage RNA-targeting dCas13d to selectively interfere with protein translation and to measure phage gene fitness at a transcriptome-wide scale. We find CRISPR Interference through Antisense RNA-Targeting (CRISPRi-ART) to be effective across phage phylogeny, from model ssRNA, ssDNA and dsDNA phages to nucleus-forming jumbo phages. Using CRISPRi-ART, we determine a conserved role of diverse rII homologues in subverting phage Lambda RexAB-mediated immunity to superinfection and identify genes critical for phage fitness. CRISPRi-ART establishes a broad-spectrum phage functional genomics platform, revealing more than 90 previously unknown genes important for phage fitness.

Coevolution between marine Aeromonas and phages reveals temporal trade-off patterns of phage resistance and host population fitness

Coevolution of bacteria and phages is an important host and parasite dynamic in marine ecosystems, contributing to the understanding of bacterial community diversity. On the time scale, questions remain concerning what is the difference between phage resistance patterns in marine bacteria and how advantageous mutations gradually accumulate during coevolution. In this study, marine Aeromonas was co-cultured with its phage for 180 days and their genetic and phenotypic dynamics were measured every 30 days. We identified 11 phage resistance genes and classified them into three categories: lipopolysaccharide (LPS), outer membrane protein (OMP), and two-component system (TCS). LPS shortening and OMP mutations are two distinct modes of complete phage resistance, while TCS mutants mediate incomplete resistance by repressing the transcription of phage genes. The co-mutation of LPS and OMP was a major mode for bacterial resistance at a low cost. The mutations led to significant reductions in the growth and virulence of bacterial populations during the first 60 days of coevolution, with subsequent leveling off. Our findings reveal the marine bacterial community dynamics and evolutionary trade-offs of phage resistance during coevolution, thus granting further understanding of the interaction of marine microbes.

Contrasting drivers of abundant phage and prokaryotic communities revealed in diverse coastal ecosystems

Phages (viruses of bacteria and archaea) are a ubiquitous top-down control on microbial communities by selectively infecting and killing cells. As obligate parasites, phages are inherently linked to processes that impact their hosts’ distribution and physiology, but phages can also be impacted by external, environmental factors, such as UV radiation degrading their virions. To better understand these complex links of phages to their hosts and the environment, we leverage the unique ecological context of the Isthmus of Panama, which narrowly disconnects the productive Tropical Eastern Pacific (EP) and nutrient-poor Tropical Western Atlantic (WA) provinces. We could thus compare patterns of phage and prokaryotic communities at both global scales (between oceans) and local-scales (between habitats within an ocean). Although both phage and prokaryotic communities differed sharply between the oceans, phage community composition did not significantly differ between mangroves and reefs of the WA, while prokaryotic communities were distinct. These results suggest phages are more shaped by dispersal processes than local conditions regardless of spatial scale, while prokaryotes tend to be shaped by local conditions at smaller spatial scales. Collectively, we provide a framework for addressing the co-variability between phages and prokaryotes in marine systems and identifying factors that drive consistent versus disparate trends in community shifts, essential to informing models of biogeochemical cycles that include these interactions.

Simultaneous entry as an adaptation to virulence in a novel satellite-helper system infecting Streptomyces species

Satellites are mobile genetic elements that are dependent upon the replication machinery of their helper viruses. Bacteriophages have provided many examples of satellite nucleic acids that utilize their helper morphogenic genes for propagation. Here we describe two novel satellite-helper phage systems, Mulch and Flayer, that infect Streptomyces species. The satellites in these systems encode for encapsidation machinery but have an absence of key replication genes, thus providing the first example of bacteriophage satellite viruses. We also show that codon usage of the satellites matches the tRNA gene content of the helpers. The satellite in one of these systems, Flayer, does not appear to integrate into the host genome, which represents the first example of a virulent satellite phage. The Flayer satellite has a unique tail adaptation that allows it to attach to its helper for simultaneous co-infection. These findings demonstrate an ever-increasing array of satellite strategies for genetic dependence on their helpers in the evolutionary arms race between satellite and helper phages.

Filamentation activates bacterial Avs5 antiviral protein

Bacterial antiviral STANDs (Avs) are evolutionarily related to the nucleotide-binding oligomerization domain (NOD)-like receptors widely distributed in immune systems across animals and plants. EfAvs5, a type 5 Avs from Escherichia fergusonii, contains an N-terminal SIR2 effector domain, a NOD, and a C-terminal sensor domain, conferring protection against diverse phage invasions. Despite the established roles of SIR2 and STAND in prokaryotic and eukaryotic immunity, the mechanism underlying their collaboration remains unclear. Here we present cryo-EM structures of EfAvs5 filaments, elucidating the mechanisms of dimerization, filamentation, filament bundling, ATP binding, and NAD+ hydrolysis, all of which are crucial for anti-phage defense. The SIR2 and NOD domains engage in intra- and inter-dimer interaction to form an individual filament, while the outward C-terminal sensor domains contribute to bundle formation. Filamentation potentially stabilizes the dimeric SIR2 configuration, thereby activating the NADase activity of EfAvs5. Furthermore, we identify the nucleotide kinase gp1.7 of phage T7 as an activator of EfAvs5, demonstrating its ability to induce filamentation and NADase activity. Together, we uncover the filament assembly of Avs5 as a unique mechanism to switch enzyme activities and perform anti-phage defenses.

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