Isolation and Characterisation of the Bundooravirus Genus and Phylogenetic Investigation of the Salasmaviridae Bacteriophages

Bacillus is a highly diverse genus containing over 200 species that can be problematic in both industrial and medical settings. This is mainly attributed to Bacillus sp. being intrinsically resistant to an array of antimicrobial compounds, hence alternative treatment options are needed. In this study, two bacteriophages, PumA1 and PumA2 were isolated and characterized. Genome nucleotide analysis identified the two phages as novel at the DNA sequence level but contained proteins similar to phi29 and other related phages. Whole genome phylogenetic investigation of 34 phi29-like phages resulted in the formation of seven clusters that aligned with recent ICTV classifications. PumA1 and PumA2 share high genetic mosaicism and form a genus with another phage named WhyPhy, more recently isolated from the United States of America. The three phages within this cluster are the only candidates to infect B. pumilus. Sequence analysis of B. pumilus phage resistant mutants revealed that PumA1 and PumA2 require polymerized and peptidoglycan bound wall teichoic acid (WTA) for their infection. Bacteriophage classification is continuously evolving with the increasing phages’ sequences in public databases. Understanding phage evolution by utilizing a combination of phylogenetic approaches provides invaluable information as phages become legitimate alternatives in both human health and industrial processes.

Resolving the structure of phage-bacteria interactions in the context of natural diversity

Microbial communities are shaped by viral predators. Yet, resolving which viruses (phages) and bacteria are interacting is a major challenge in the context of natural levels of microbial diversity. Thus, fundamental features of how phage-bacteria interactions are structured and evolve in "the wild" remain poorly resolved. Here we use large-scale isolation of environmental marine Vibrio bacteria and their phages to obtain quantitative estimates of strain-level phage predator loads, and use all-by-all host range assays to discover how phage and host genomic diversity shape interactions. We show that killing in environmental interaction networks is sparse - with phage predator loads low for most bacterial strains and phages host-strain-specific in their killing. Paradoxically, we also find that although overlap in killing is generally rare between phages, recombination is common. Together, these results indicate that the number of hosts that phages infect is often larger than the number that they kill and suggest that recombination during cryptic co-infections is an important mode of phage evolution in microbial communities. In the development of phages for bioengineering and therapeutics it will be important to consider that nucleic acids of introduced phages may spread into local phage populations through recombination, and that the likelihood of transfer is not predictable based on killing host range.

Epidemiological and evolutionary consequences of CRISPR-Cas reactivity

Adaptive immune systems face a control challenge: they should react with enough strength to clear an infection while avoiding to harm their organism. CRISPR-Cas systems are adaptive immune systems of prokaryotes that defend against fast evolving viruses. Here, we explore the CRISPR-Cas control challenge and look how its reactivity, i.e. its probability to acquire a new resistance, impacts the epidemiological outcome of a phage outbreak and the prokaryote’s fitness. We show that in the absence of phage evolution, phage extinction is driven by the probability to acquire at least one resistance. However, when phage evolution is fast, phage extinction is driven by an epidemiological critical threshold: any reactivity below this critical threshold leads to phage survival whereas any reactivity above it leads to phage extinction. We also show that in the absence of autoimmunity, high levels of reactivity evolve. However, when CRISPR-Cas systems are prone to autoimmune reactions, intermediate levels of reactivity are evolutionarily optimal. These results help explaining why natural CRISPR-Cas systems do not show high levels of reactivity.

Genome Sequence and Characterization of Five Bacteriophages Infecting Streptomyces coelicolor and Streptomyces venezuelae: Alderaan, Coruscant, Dagobah, Endor1 and Endor2

Streptomyces are well-known antibiotic producers, also characterized by a complex morphological differentiation. Streptomyces, like all bacteria, are confronted with the constant threat of phage predation, which in turn shapes bacterial evolution. However, despite significant sequencing efforts recently, relatively few phages infecting Streptomyces have been characterized compared to other genera. Here, we present the isolation and characterization of five novel Streptomyces phages. All five phages belong to the Siphoviridae family, based on their morphology as determined by transmission electron microscopy. Genome sequencing and life style predictions suggested that four of them were temperate phages, while one had a lytic lifestyle. Moreover, one of the newly sequenced phages shows very little homology to already described phages, highlighting the still largely untapped viral diversity. Altogether, this study expands the number of characterized phages of Streptomyces and sheds light on phage evolution and phage-host dynamics in Streptomyces.

Genome sequence and characterization of five bacteriophages infecting Streptomyces coelicolor and Streptomyces venezuelae: Alderaan, Coruscant, Dagobah, Endor1 and Endor2

Streptomyces are well-known antibiotic producers, and are also characterized by a complex morphological differentiation. Streptomyces, like all bacteria, are confronted with the constant threat of phage predation, which in turn shapes bacterial evolution. However, despite significant sequencing efforts recently, relatively few phages infecting Streptomyces have been characterized compared to other genera. Here, we present the isolation and characterization of five novel Streptomyces phages. All five phages belong to the Siphoviridae family, based on their morphology as determined by transmission electron microscopy. Genome sequencing revealed that four of them were temperate phages, while one had a lytic lifestyle. Moreover, one of the newly sequenced phages shows very little homology to already described phages, highlighting the still largely untapped viral diversity. Altogether, this study expands the number of characterized phages of Streptomyces and sheds light on phage evolution and phage-host dynamics in Streptomyces.

Pervasive prophage recombination occurs during evolution of spore-forming Bacilli

Phages are the main source of within-species bacterial diversity and drivers of horizontal gene transfer, but we know little about the mechanisms that drive genetic diversity of these mobile genetic elements (MGEs). Recently, we showed that a sporulation selection regime promotes evolutionary changes within SPβ prophage of Bacillus subtilis, leading to direct antagonistic interactions within the population. Herein, we reveal that under a sporulation selection regime, SPβ recombines with low copy number phi3Ts phage DNA present within the B. subtilis population. Recombination results in a new prophage occupying a different integration site, as well as the spontaneous release of virulent phage hybrids. Analysis of Bacillus sp. strains suggests that SPβ and phi3T belong to a distinct cluster of unusually large phages inserted into sporulation-related genes that are equipped with a spore-related genetic arsenal. Comparison of Bacillus sp. genomes indicates that similar diversification of SPβ-like phages takes place in nature. Our work is a stepping stone toward empirical studies on phage evolution, and understanding the eco-evolutionary relationships between bacteria and their phages. By capturing the first steps of new phage evolution, we reveal striking relationship between survival strategy of bacteria and evolution of their phages.