Streptomyces development is involved in the efficient containment of viral infections.

The formation of plaques represents the hallmark of phage infection visualizing the clearance of the bacterial lawn in structured environments. In this study, we have addressed the impact of cellular development on phage infection in Streptomyces undergoing a complex developmental life cycle. Analysis of plaque dynamics revealed, after a period of plaque size enlargement, a significant regrowth of transiently phage-resistant Streptomyces mycelium into the lysis zone. Analysis of Streptomyces venezuelae mutant strains defective at different stages of cellular development indicated that this regrowth was dependent on the onset of the formation of aerial hyphae and spores at the infection interface. Mutants restricted to vegetative growth (ΔbldN) featured no significant constriction of plaque area. Fluorescence microscopy further confirmed the emergence of a distinct zone of cells/spores with reduced cell permeability towards propidium iodide staining at the plaque periphery. Mature mycelium was further shown to be significantly less susceptible to phage infection, which is less pronounced in strains defective in cellular development. Transcriptome analysis revealed the repression of cellular development at the early stages of phage infection probably facilitating efficient phage propagation. We further observed an induction of the chloramphenicol biosynthetic gene cluster highlighting phage infection as a trigger of cryptic metabolism in Streptomyces. Altogether, our study emphasizes cellular development and the emergence of transient phage resistance as an important layer of Streptomyces antiviral immunity.

Bacterial multicellular behavior in antiviral defense.

Multicellular behavior benefits seemingly simple organisms such as bacteria, by improving nutrient uptake, resistance to stresses, or by providing advantages in predatory interactions. Several recent studies have shown that this also extends to the defense against bacteriophages, which are omnipresent in almost all habitats. In this review, we summarize strategies conferring protection against phage infection at the multicellular level, covering secretion of small antiphage molecules or membrane vesicles, the role of quorum sensing in phage defense, the development of transient phage resistance, and the impact of biofilm components and architecture. Recent studies focusing on these topics push the boundaries of our understanding of the bacterial immune system and set the ground for an appreciation of bacterial multicellular behavior in antiviral defense.

Systematic analysis of prophage elements in actinobacterial genomes reveals a remarkable phylogenetic diversity.

Actinobacteria represent one of the largest bacterial phyla harboring many species of high medical, biotechnological and ecological relevance. Prophage elements are major contributors to bacterial genome diversity and were shown to significantly shape bacterial fitness and host-microbe interactions. In this study, we performed a systematic analysis of prophage elements in 2406 complete actinobacterial genomes. Overall, 2106 prophage elements were predicted to be present in about 50% (1172/2406) of the analyzed datasets. Interestingly, these identified sequences compose a high prevalence of cryptic prophage elements, indicating genetic decay and domestication. Analysis of the sequence relationship of predicted prophages with known actinobacteriophage genomes revealed an exceptional high phylogenetic diversity of prophage elements. As a trend, we observed a higher prevalence of prophage elements in vicinity to the terminus. Analysis of the prophage-encoded gene functions revealed that prophage sequences significantly contribute to the bacterial antiviral immune system, but no biosynthetic gene clusters involved in the synthesis of known antiphage molecules were identified in prophage genomes. Overall, the current study highlights the remarkable diversity of prophages in actinobacterial genomes, with highly divergent prophages in actinobacterial genomes and thus provides an important basis for further investigation of phage-host interactions in this important bacterial phylum.

Aminoglycoside Antibiotics Inhibit Phage Infection by Blocking an Early Step of the Infection Cycle.

In response to viral predation, bacteria have evolved a wide range of defense mechanisms, which rely mostly on proteins acting at the cellular level. Here, we show that aminoglycosides, a well-known class of antibiotics produced by Streptomyces, are potent inhibitors of phage infection in widely divergent bacterial hosts. We demonstrate that aminoglycosides block an early step of the viral life cycle, prior to genome replication. Phage inhibition was also achieved using supernatants from natural aminoglycoside producers, indicating a broad physiological significance of the antiviral properties of aminoglycosides. Strikingly, we show that acetylation of the aminoglycoside antibiotic apramycin abolishes its antibacterial effect but retains its antiviral properties. Altogether, our study expands the knowledge of aminoglycoside functions, suggesting that aminoglycosides not only are used by their producers as toxic molecules against their bacterial competitors but also could provide protection against the threat of phage predation at the community level. IMPORTANCE Predation by phages is a major driver of bacterial evolution. As a result, elucidating antiphage strategies is crucial from both fundamental and therapeutic standpoints. While protein-mediated defense mechanisms, like restriction-modification systems or CRISPR/Cas, have been extensively studied, much less is known about the potential antiphage activity of small molecules. Focusing on the model bacteria Escherichia coli and Streptomyces venezuelae, our findings revealed significant antiphage properties of aminoglycosides, a major class of translation-targeting antibiotics produced by Streptomyces. Further, we demonstrate that supernatants from natural aminoglycoside producers protect bacteria from phage propagation, highlighting the physiological relevance of this inhibition. Suppression of phage infection by aminoglycosides did not result from the indirect inhibition of bacterial translation, suggesting a direct interaction between aminoglycosides and phage components. This work highlights the molecular versatility of aminoglycosides, which have evolved to efficiently block protein synthesis in bacterial competitors and provide protection against phages.

Phylogenetic Distribution of WhiB- and Lsr2-Type Regulators in Actinobacteriophage Genomes.

Viruses that infect different actinobacterial host species are known as actinobacteriophages. They are composed of highly divergent and mosaic genomes due to frequent gene exchange between their bacterial hosts and related viral species. This is also reflected by the adaptive incorporation of host transcription factors (TFs) into phage regulatory networks. Previous studies discovered Lsr2-type and WhiB-type regulators encoded by actinobacteriophage genomes. However, limited information is available about their distribution, evolution, and impact on host species. In this study, we computationally screened the distribution of known bacterial and phage TFs inside 2951 complete actinobacteriophage genomes and identified 13 different TF domains. Among those, WhiB, Lsr2, MerR, and Cro/CI-like proteins were widespread and found in more than 10% of the analyzed actinobacteriophage genomes. Neighboring genomic context analysis of the whiB and lsr2 loci showed group-specific conservation of gene synteny and potential involvement of these genes in diverse regulatory functions. Both genes were significantly enriched in temperate phages, and the Lsr2-encoding genomes featured an overall lower GC content. Phylogenetic analysis of WhiB and Lsr2 proteins showed the grouping of phage sequences within bacterial clades, suggesting gene acquisition by phages from their bacterial host species or by multiple, independent acquisition events. Overall, our study reports the global distribution of actinobacteriophage regulatory proteins and sheds light on their origin and evolution. IMPORTANCE Actinobacteriophages are viruses that infect bacterial species of the diverse phylum of Actinobacteria. Phages engage in a close relationship with their bacterial host. This is also reflected by the adoption of genetic material from their host and its incorporation into phage regulatory circuits. In this study, we systematically searched the genomes of actinobacteriophages for the presence of transcription factor domains. We show that proteins belonging to the regulator families of WhiB and Lsr2 belong to the most abundant regulatory proteins encoded by actinobacteriophages. Further phylogenetic analysis shed light on their origin and evolution. Altogether, this study provides an important basis for further experimental investigation of their role in the coordination of the phage life cycle and their interaction with the host regulatory network in this important bacterial phylum.