ABSTRACT
Phages are one of the key ecological drivers of microbial community dynamics, function, and evolution. Despite their importance in bacterial ecology and evolutionary processes, phage genes are poorly characterized, hampering their usage in a variety of biotechnological applications. Methods to characterize such genes, even those critical to the phage life cycle, are labor intensive and are generally phage specific. Here, we develop a systematic gene essentiality mapping method scalable to new phage-host combinations that facilitate the identification of nonessential genes. As a proof of concept, we use an arrayed genome-wide CRISPR interference (CRISPRi) assay to map gene essentiality landscape in the canonical coliphages λ and P1. Results from a single panel of CRISPRi probes largely recapitulate the essential gene roster determined from decades of genetic analysis for lambda and provide new insights into essential and nonessential loci in P1. We present evidence of how CRISPRi polarity can lead to false positive gene essentiality assignments and recommend caution towards interpreting CRISPRi data on gene essentiality when applied to less studied phages. Finally, we show that we can engineer phages by inserting DNA barcodes into newly identified inessential regions, which will empower processes of identification, quantification, and tracking of phages in diverse applications.
Subject(s)
Bacteriophages , Bacteriophages/genetics , DNA , Genes, Essential/geneticsABSTRACT
Lytic phages can be potent and selective inhibitors of microbial growth and can have profound impacts on microbiome composition and function. However, there is uncertainty about the biogeochemical conditions under which phage predation modulates microbial ecosystem function, particularly in terrestrial systems. Ionic strength is critical for infection of bacteria by many phages, but quantitative data is limited on the ion thresholds for phage infection that can be compared with environmental ion concentrations. Similarly, while carbon composition varies in the environment, we do not know how this variability influences the impact of phage predation on microbiome function. Here, we measured the half-maximal effective concentrations (EC50) of 80 different inorganic ions for the infection of E. coli with two canonical dsDNA and ssRNA phages, T4 and MS2, respectively. Many alkaline earth metals and alkali metals enabled lytic infection but the ionic strength thresholds varied for different ions between phages. Additionally, using a freshwater nitrate-reducing microbiome, we found that the ability of lytic phages to influence nitrate reduction end-products depended upon the carbon source as well as ionic strength. For all phage:host pairs, the ion EC50s for phage infection exceeded the ion concentrations found in many terrestrial freshwater systems. Thus, our findings support a model where phages most influence terrestrial microbial functional ecology in hot spots and hot moments such as metazoan guts, drought influenced soils, or biofilms where ion concentration is locally or transiently elevated and nutrients are available to support the growth of specific phage hosts.
ABSTRACT
Bacteriophage P1 is the premier transducing phage of E. coli. Despite its prominence in advancing E. coli genetics, modern molecular techniques have not been applied to thoroughly understand P1 structure. Here, we report the proteome of the P1 virion as determined by liquid chromatography tandem mass-spectrometry. Additionally, a library of single-gene knockouts identified the following five previously unknown essential genes: pmgA, pmgB, pmgC, pmgG, and pmgR. In addition, proteolytic processing of the major capsid protein is a known feature of P1 morphogenesis, and we identified the processing site by N-terminal sequencing to be between E120 and S121, producing a 448-residue, 49.3 kDa mature peptide. Furthermore, the P1 defense against restriction (Dar) system consists of six known proteins that are incorporated into the virion during morphogenesis. The largest of these, DarB, is a 250 kDa protein that is believed to translocate into the cell during infection. DarB deletions indicated the presence of an N-terminal packaging signal, and the N-terminal 30 residues of DarB are shown to be sufficient for directing a heterologous reporter protein to the capsid. Taken together, the data expand on essential structural P1 proteins as well as introduces P1 as a nanomachine for cellular delivery.
Subject(s)
Bacteriophage P1 , Escherichia coli , Bacteriophage P1/genetics , Bacteriophage P1/metabolism , Capsid/metabolism , Capsid Proteins/genetics , Capsid Proteins/metabolism , DNA, Viral/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
We report the genome sequence of bacteriophage U136B, which is reliant on the lipopolysaccharide and the antibiotic efflux protein TolC for infection of Escherichia coli and is a useful model for studying trade-offs and trade-ups that shape evolution. Phage U136B has a 49,233-bp genome with 87 predicted genes.
ABSTRACT
Bacteriophages (phages) are critical players in the dynamics and function of microbial communities and drive processes as diverse as global biogeochemical cycles and human health. Phages tend to be predators finely tuned to attack specific hosts, even down to the strain level, which in turn defend themselves using an array of mechanisms. However, to date, efforts to rapidly and comprehensively identify bacterial host factors important in phage infection and resistance have yet to be fully realized. Here, we globally map the host genetic determinants involved in resistance to 14 phylogenetically diverse double-stranded DNA phages using two model Escherichia coli strains (K-12 and BL21) with known sequence divergence to demonstrate strain-specific differences. Using genome-wide loss-of-function and gain-of-function genetic technologies, we are able to confirm previously described phage receptors as well as uncover a number of previously unknown host factors that confer resistance to one or more of these phages. We uncover differences in resistance factors that strongly align with the susceptibility of K-12 and BL21 to specific phage. We also identify both phage-specific mechanisms, such as the unexpected role of cyclic-di-GMP in host sensitivity to phage N4, and more generic defenses, such as the overproduction of colanic acid capsular polysaccharide that defends against a wide array of phages. Our results indicate that host responses to phages can occur via diverse cellular mechanisms. Our systematic and high-throughput genetic workflow to characterize phage-host interaction determinants can be extended to diverse bacteria to generate datasets that allow predictive models of how phage-mediated selection will shape bacterial phenotype and evolution. The results of this study and future efforts to map the phage resistance landscape will lead to new insights into the coevolution of hosts and their phage, which can ultimately be used to design better phage therapeutic treatments and tools for precision microbiome engineering.
Subject(s)
Bacteriophages/physiology , Escherichia coli/virology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacteriophages/drug effects , Biosynthetic Pathways/drug effects , CRISPR-Cas Systems/genetics , Cyclic GMP/analogs & derivatives , Cyclic GMP/pharmacology , DNA/genetics , Down-Regulation/drug effects , Down-Regulation/genetics , Escherichia coli/drug effects , Escherichia coli/genetics , Gene Expression Regulation, Bacterial/drug effects , Genes, Essential , Genome, Bacterial , Mutation/genetics , Phenotype , Reproducibility of Results , Suppression, GeneticABSTRACT
The host factor requirements of phages and mechanisms of mutational phage insensitivity must be characterized for rational design of phage cocktails. To characterize host dependencies of two novel Escherichia coli phages, the T1-like siphophage LL5 and the V5-like myophage LL12, forward genetic screens were conducted against the Keio collection, a library of single non-essential gene deletions in E. coli str. BW25113. These screens and subsequent experiments identified genes required by phages LL5 and LL12. E. coli mutants deficient in heptose II and the phosphoryl substituent of heptose I of the inner core lipopolysaccharide (LPS) were unable to propagate phage LL5, as were mutants deficient in the outer membrane protein TolC. Mutants lacking glucose I of the LPS outer core failed to propagate LL12. Two additional genes encoding cytoplasmic chaperones, PpiB and SecB, were found to be required for efficient propagation of phage LL5, but not LL12. This screening approach may be useful for identifying host factors dependencies of phages, which would provide valuable information for their potential use as therapeutics and for phage engineering.
Subject(s)
Coliphages/physiology , Escherichia coli/genetics , Escherichia coli/virology , Host-Pathogen Interactions , Coliphages/ultrastructure , Escherichia coli Proteins/genetics , Genome, Bacterial , Genome-Wide Association Study , Genomics/methods , Host-Pathogen Interactions/geneticsABSTRACT
This work describes the complete genome sequence of the virulent myophage LL12. The 136-kb LL12 genome is related to coliphage V5 and is a component in the prebiotic PreforPro. LL12 was isolated against enterotoxigenic Escherichia coli, which causes traveler's diarrhea.
ABSTRACT
Here, we describe the complete genome sequence of siphophage LL5. LL5 is a T1-like phage isolated against enterotoxigenic Escherichia coli, which causes traveler's diarrhea. LL5 is included as a component phage in the commercial prebiotic product PreforPro.
ABSTRACT
Bacterial Type I restriction-modification (R-M) systems present a major barrier to foreign DNA entering the bacterial cell. The temperate phage P1 packages several proteins into the virion that protect the phage DNA from host restriction. Isogenic P1 deletion mutants were used to reconstitute the previously described restriction phenotypes associated with darA and darB. While P1ΔdarA and P1ΔdarB produced the expected phenotypes, deletions of adjacent genes hdf and ddrA also produced darA-like phenotypes and deletion of ulx produced a darB-like phenotype, implicating several new proteins of previously unknown function in the P1 dar antirestriction system. Interestingly, disruption of ddrB decreased P1's sensitivity to EcoB and EcoK restriction. Proteomic analysis of purified virions suggests that packaging of antirestriction components into P1 virions follows a distinct pathway that begins with the incorporation of DarA and Hdf and concludes with DarB and Ulx. Electron microscopy analysis showed that hdf and darA mutants also produce abnormally high proportions of virions with aberrant small heads, which suggests Hdf and DarA play a role in capsid morphogenesis. The P1 antirestriction system is more complex than previously realized and is comprised of multiple proteins including DdrA, DdrB, Hdf, and Ulx in addition to DarA and DarB.
Subject(s)
Bacteriophage P1/metabolism , Capsid/physiology , Bacterial Proteins/metabolism , Bacteriophage P1/genetics , Bacteriophages/genetics , DNA Restriction-Modification Enzymes/genetics , DNA, Viral/metabolism , Escherichia coli/genetics , Morphogenesis , Proteomics , Viral Proteins/metabolism , Virion/geneticsABSTRACT
Here, we describe the complete genome sequence of siphophage Shivani, a T5-like constituent phage in the therapeutic phage cocktail IntestiPhage developed for bacterial gastroenteritis. Shivani was isolated against a foodborne pathogen, Salmonella enterica, which is one of the leading causes of gastroenteritis.