Bacteriophage self-counting in the presence of viral replication

When host cells are in low abundance, temperate bacteriophages opt for dormant (lysogenic) infection. Phage lambda implements this strategy by increasing the frequency of lysogeny at higher multiplicity of infection (MOI). However, it remains unclear how the phage reliably counts infecting viral genomes even as their intracellular number increases because of replication. By combining theoretical modeling with single-cell measurements of viral copy number and gene expression, we find that instead of hindering lambda’s decision, replication facilitates it. In a nonreplicating mutant, viral gene expression simply scales with MOI rather than diverging into lytic (virulent) and lysogenic trajectories. A similar pattern is followed during early infection by wild-type phage. However, later in the infection, the modulation of viral replication by the decision genes amplifies the initially modest gene expression differences into divergent trajectories. Replication thus ensures the optimal decision—lysis upon single-phage infection and lysogeny at higher MOI.

Characterization and genomic analysis of the first Oceanospirillum phage, vB_OliS_GJ44, representing a novel siphoviral cluster

Background Marine bacteriophages play key roles in the community structure of microorganisms, biogeochemical cycles, and the mediation of genetic diversity through horizontal gene transfer. Recently, traditional isolation methods, complemented by high-throughput sequencing metagenomics technology, have greatly increased our understanding of the diversity of bacteriophages. Oceanospirillum, within the order Oceanospirillales, are important symbiotic marine bacteria associated with hydrocarbon degradation and algal blooms, especially in polar regions. However, until now there has been no isolate of an Oceanospirillum bacteriophage, and so details of their metagenome has remained unknown. Results Here, we reported the first Oceanospirillum phage, vB_OliS_GJ44, which was assembled into a 33,786 bp linear dsDNA genome, which includes abundant tail-related and recombinant proteins. The recombinant module was highly adapted to the host, according to the tetranucleotides correlations. Genomic and morphological analyses identified vB_OliS_GJ44 as a siphovirus, however, due to the distant evolutionary relationship with any other known siphovirus, it is proposed that this virus could be classified as the type phage of a new Oceanospirivirus genus within the Siphoviridae family. vB_OliS_GJ44 showed synteny with six uncultured phages, which supports its representation in uncultured environmental viral contigs from metagenomics. Homologs of several vB_OliS_GJ44 genes have mostly been found in marine metagenomes, suggesting the prevalence of this phage genus in the oceans. Conclusions These results describe the first Oceanospirillum phage, vB_OliS_GJ44, that represents a novel viral cluster and exhibits interesting genetic features related to phage–host interactions and evolution. Thus, we propose a new viral genus Oceanospirivirus within the Siphoviridae family to reconcile this cluster, with vB_OliS_GJ44 as a representative member.

A Cut above the Rest: Characterization of the Assembly of a Large Viral Icosahedral Capsid

The head of Salmonella virus SPN3US is composed of ~50 different proteins and is unusual because within its packaged genome there is a mass (>40 MDa) of ejection or E proteins that enter the Salmonella cell. The assembly mechanisms of this complex structure are poorly understood. Previous studies showed that eight proteins in the mature SPN3US head had been cleaved by the prohead protease. In this study, we present the characterization of SPN3US prohead protease mutants using transmission electron microscopy and mass spectrometry. In the absence of the prohead protease, SPN3US head formation was severely impeded and proheads accumulated on the Salmonella inner membrane. This impediment is indicative of proteolysis being necessary for the release and subsequent DNA packaging of proheads in the wild-type phage. Proteomic analyses of gp245- proheads that the normal proteolytic processing of head proteins had not occurred. Assays of a recombinant, truncated form of the protease found it was active, leading us to hypothesize that the C-terminal propeptide has a role in targeting the protease into the prohead core. Our findings provide new evidence regarding the essential role of proteolysis for correct head assembly in this remarkable parasite.

Phage T7 DNA mimic protein Ocr is a potent inhibitor of BREX defence

BREX (for BacteRiophage EXclusion) is a superfamily of common bacterial and archaeal defence systems active against diverse bacteriophages. While the mechanism of BREX defence is currently unknown, self versus non-self differentiation requires methylation of specific asymmetric sites in host DNA by BrxX (PglX) methyltransferase. Here, we report that T7 bacteriophage Ocr, a DNA mimic protein that protects the phage from the defensive action of type I restriction–modification systems, is also active against BREX. In contrast to the wild–type phage, which is resistant to BREX defence, T7 lacking Ocr is strongly inhibited by BREX, and its ability to overcome the defence could be complemented by Ocr provided in trans. We further show that Ocr physically associates with BrxX methyltransferase. Although BREX+ cells overproducing Ocr have partially methylated BREX sites, their viability is unaffected. The result suggests that, similar to its action against type I R–M systems, Ocr associates with as yet unidentified BREX system complexes containing BrxX and neutralizes their ability to both methylate and exclude incoming phage DNA.

Comparison of CRISPR and Marker-Based Methods for the Engineering of Phage T7

With the recent rise in interest in using lytic bacteriophages as therapeutic agents, there is an urgent requirement to understand their fundamental biology to enable the engineering of their genomes. Current methods of phage engineering rely on homologous recombination, followed by a system of selection to identify recombinant phages. For bacteriophage T7, the host genes cmk or trxA have been used as a selection mechanism along with both type I and II CRISPR systems to select against wild-type phage and enrich for the desired mutant. Here, we systematically compare all three systems; we show that the use of marker-based selection is the most efficient method and we use this to generate multiple T7 tail fibre mutants. Furthermore, we found the type II CRISPR-Cas system is easier to use and generally more efficient than a type I system in the engineering of phage T7. These results provide a foundation for the future, more efficient engineering of bacteriophage T7.

Comparison of CRISPR and marker based methods for the engineering of phage T7

With the recent rise in interest in using lytic bacteriophages as therapeutic agents, there is an urgent requirement to understand their fundamental biology to enable the engineering of their genomes. Current methods of phage engineering rely on homologous recombination, followed by a system of selection to identify recombinant phages. For bacteriophage T7, the host genes cmk or trx have been used as a selection mechanism along with both type I and II CRISPR systems to select against wild-type phage and enrich for the desired mutant. Here we systematically compare all three systems; we show that the use of marker-based selection is the most efficient method and we use this to generate multiple T7 tail fiber mutants. Furthermore, we found the type II CRISPR-Cas system is easier to use and generally more efficient than a type I system in the engineering of phage T7. These results provide a foundation for the future, more efficient engineering of bacteriophage T7.

Diverse, abundant and novel viruses infecting “unculturable” but abundant marine bacteria

Many major marine bacterial lineages such as SAR11, Prochlorococcus, SAR116, and several Roseobacter lineages have members that are abundant, relatively slow-growing, and genome-streamlined. The isolation of phages that infect SAR11 and SAR116 have demonstrated the dominance of these phages in the marine virosphere. However, no phages have been isolated from bacteria in the Roseobacter RCA lineage, another abundant group of bacteria in the ocean. In this study, seven RCA phages that infect three different RCA strains were isolated and characterized. All seven RCA phages belong to the Podoviridae family and have genome sizes ranging from 39.6 to 58.1 kb. Interestingly, three RCA phages (CRP-1, CRP-2 and CRP-3) show a similar genomic content and architecture with SAR116 phage HMO-2011, which represents one of the most abundant known viral groups in the ocean. The high degree of homology between CRP-1, CRP-2, CRP-3 and HMO-2011 resulted in contribution of the RCA phages to the dominance of HMO-2011-type phage in the ocean. CRP-4 and CRP-5 are similar to the Siovirus roseophages in terms of gene content and organization. The remaining two RCA phages, CRP-6 and CRP-7, show limited genomic similarity with known phages and appear to form two new phage genera. Metagenomic fragment recruitment analyses reveal that these RCA phage groups are much more abundant in the ocean compared to most existing marine roseophage groups. The characterization of these RCA phages has greatly expanded our understanding of the genomic diversity and evolution of marine roseophages. Metagenomic fragment recruitment analyses suggest the critical need for isolating phages from the abundant but “unculturable” bacteria in the marine ecosystem.

Phage Reduce Stability for Regaining Infectivity during Antagonistic Coevolution with Host Bacterium

The coevolution between phage and host bacterium is an important force that drives the evolution of the microbial community, yet the coevolution mechanisms have still not been well analyzed. Here, by analyzing the interaction between a Bacillus phage vB_BthS_BMBphi and its host bacterium, the coevolution mechanisms of the first-generation phage-resistant bacterial mutants and regained-infectivity phage mutants were studied. The phage-resistant bacterial mutants showed several conserved mutations as a potential reason for acquiring phage resistance, including the mutation in flagellum synthesis protein FlhA and cell wall polysaccharide synthesis protein DltC. All the phage-resistant bacterial mutants showed a deleted first transmembrane domain of the flagellum synthesis protein FlhA. Meanwhile, the regain-infectivity phage mutants all contained mutations in three baseplate-associated phage tail proteins by one nucleotide, respectively. A polymorphism analysis of the three mutant nucleotides in the wild-type phage revealed that the mutations existed before the interaction of the phage and the bacterium, while the wild-type phage could not infect the phage-resistant bacterial mutants, which might be because the synchronized mutations of the three nucleotides were essential for regaining infectivity. This study for the first time revealed that the synergism mutation of three phage baseplate-associated proteins were essential for the phages’ regained infectivity. Although the phage mutants regained infectivity, their storage stability was decreased and the infectivity against the phage-resistant bacterial mutants was reduced, suggesting the phage realized the continuation of the species by way of “dying to survive”.

A mutant bacteriophage evolved to infect resistant bacteria gained a broader host range

SummaryBacteriophages (phages) are the most abundant entities in nature, yet little is known about their capacity to acquire new hosts and invade new niches. By exploiting the Gram positive soil bacteriumBacillus subtilis(B. subtilis) and its lytic phage SPO1 as a model, we followed the co-evolution of bacteria and phages. After infection, phage resistant bacteria were readily isolated. These bacteria were defective in production of glycosylated wall teichoic acid (TA) polymers, served as SPO1 receptor. Subsequently, a SPO1 mutant phage that could infect the resistant bacteria evolved. The emerging phage contained mutations in two genes, encoding the baseplate and fibers required for host attachment. Remarkably, the mutant phage gained the capacity to infect non-hostBacillusspecies that are not infected by the wild type phage. We provide evidence that the evolved phage lost its dependency on the species specific glycosylation pattern of TA polymers. Instead, the mutant phage gained the capacity to directly adhere to the TA backbone, conserved among different species, thereby crossing the species barrier.

Cell Wall Glycans Mediate Recognition of the Dairy BacteriumStreptococcus thermophilusby Bacteriophages

ABSTRACTReceptors on the cell surfaces of bacterial hosts are essential during the infection cycle of bacteriophages. To date, the phage receptors of the industrial relevant dairy starter bacteriumStreptococcus thermophilusremain elusive. Thus, we set out to identify cell surface structures that are involved in host recognition by dairy streptococcal phages. Five industrialS. thermophilusstrains sensitive to different phages (pactype,costype, and the new type 987), were selected to generate spontaneous bacteriophage-insensitive mutants (BIMs). Of these, approximately 50% were deselected as clustered regularly interspaced short palindromic repeat (CRISPR) mutants, while the other pool was further characterized to identify receptor mutants. On the basis of genome sequencing data, phage resistance in putative receptor mutants was attributed to nucleotide changes in genes encoding glycan biosynthetic pathways. Superresolution structured illumination microscopy was used to visualize the interactions betweenS. thermophilusand its phages. The phages were either regularly distributed along the cells or located at division sites of the cells. The cell wall structures mediating the latter type of phage adherence were further analyzed via phenotypic and biochemical assays. Altogether, our data suggested that phage adsorption toS. thermophilusis mediated by glycans associated with the bacterial cell surface. Specifically, thepac-type phage CHPC951 adsorbed to polysaccharides anchored to peptidoglycan, while the 987-type phage CHPC926 recognized exocellular polysaccharides associated with the cell surface.IMPORTANCEStreptococcus thermophilusis widely used in starter cultures for cheese and yoghurt production. During dairy fermentations, infections of bacteria with bacteriophages result in acidification failures and a lower quality of the final products. An understanding of the molecular factors involved in phage-host interactions, in particular, the phage receptors in dairy bacteria, is a crucial step for developing better strategies to prevent phage infections in dairy plants.