A major limitation with traditional phage preparations is the variability in titer, salts, and bacterial contaminants between successive propagations. Here we introduce the Phage On Tap (PoT) protocol for the quick and efficient preparation of homogenous bacteriophage (phage) stocks. This method produces homogenous, laboratory-scale, high titer (up to 1010–11 PFU·ml−1), endotoxin reduced phage banks that can be used to eliminate the variability between phage propagations and improve the molecular characterizations of phage. The method consists of five major parts, including phage propagation, phage clean up by 0.22 μm filtering and chloroform treatment, phage concentration by ultrafiltration, endotoxin removal, and the preparation and storage of phage banks for continuous laboratory use. From a starting liquid lysate of > 100 mL, the PoT protocol generated a clean, homogenous, laboratory phage bank with a phage recovery efficiency of 85% within just two days. In contrast, the traditional method took upwards of five days to produce a high titer, but lower volume phage stock with a recovery efficiency of only 4%. Phage banks can be further purified for the removal of bacterial endotoxins, reducing endotoxin concentrations by over 3,000-fold while maintaining phage titer. The PoT protocol focused on T-like phages, but is broadly applicable to a variety of phages that can be propagated to sufficient titer, producing homogenous, high titer phage banks that are applicable for molecular and cellular assays.

BackgroundDiversity-generating retroelements (DGRs) are genetic cassettes that selectively mutate target genes to produce hypervariable proteins. First characterized in Bordetella bacteriophage BPP-1, the DGR creates a hypervariable phage tail fiber that enables host tropism switching. Subsequent surveys for DGRs conclude that the majority identified to date are bacterial or archaeal in origin. This work examines bacteriophage and bacterial genomes for novel phage-encoded DGRs.ResultsThis survey discovered 92 DGRs that were only found in phages exhibiting a temperate lifestyle. The majority of phage-encoded DGRs were identified as prophages in bacterial hosts from the phyla Bacteroidetes, Proteobacteria, and Firmicutes. Sequence reads from these previously unidentified prophages were present in viral metagenomes (viromes), indicating these prophages can produce functional viruses. Five phages possessed hypervariable proteins with structural similarity to the tail fiber of BPP-1, whereas the functions of the remaining DGR target proteins were unknown. A novel temperate phage that harbors a DGR cassette targeting a protein of unknown function was induced from Bacteroides dorei. This phage, here named Bacteroides dorei Hankyphage, lysogenizes 13 different Bacteroides species and was present in 34% and 21% of whole-community metagenomes and human-associated viromes, respectively.ConclusionsHere, the number of known DGR-containing phages is increased from four to 92. All of these phages exhibit a temperate lifestyle, including a cosmopolitan human-associated phage. Targeted hypervariation by temperate phages may be a ubiquitous mechanism underlying phage-bacteria interaction in the human microbiome.Electronic supplementary materialThe online version of this article (10.1186/s40168-018-0573-6) contains supplementary material, which is available to authorized users.

A major limitation with traditional phage preparations is the variability in titer, salts, and bacterial contaminants between successive propagations. Here we introduce the Phage On Tap (PoT) protocol for the quick and efficient preparation of homogenous bacteriophage (phage) stocks. This method produces homogenous, laboratory-scale, high titer (up to 1010-11 PFU∙ml-1), endotoxin reduced phage banks that can be used to eliminate the variability between phage propagations and improve the molecular characterizations of phage. The method consists of five major parts, including phage propagation, phage clean up by 0.22 µm filtering and chloroform treatment, phage concentration by ultrafiltration, endotoxin removal, and the preparation and storage of phage banks for continuous laboratory use. From a starting liquid lysate of >100 mL, the PoT protocol generated a clean, homogenous, laboratory phage bank with a phage recovery efficiency of 85% within just two days. In contrast, the traditional method took upwards of five days to produce a high titer, but lower volume phage stock with a recovery efficiency of only 4%. Phage banks can be further purified for the removal of bacterial endotoxins, reducing endotoxin concentrations by over 3,000-fold while maintaining phage titer. The PoT protocol focused on T-like phages, but is broadly applicable to a variety of phages that can be propagated to sufficient titer, producing homogenous, high titer phage banks that are applicable for molecular and cellular assays.

14 A major limitation with traditional phage preparations is the variability in titer, salts, and 15 bacterial contaminants between successive propagations. Here we introduce the Phage On Tap 16 (PoT) protocol for the quick and efficient preparation of homogenous bacteriophage (phage) 17 stocks. This method produces homogenous, laboratory-scale, high titer (up to 10 10-11 PFU•ml -1 ), 18 endotoxin reduced phage banks that can be used to eliminate the variability between phage 19 propagations and improve the molecular characterizations of phage. The method consists of five 20 major parts, including phage propagation, phage clean up by 0.22 µm filtering and chloroform 21 treatment, phage concentration by ultrafiltration, endotoxin removal, and the preparation and 22 storage of phage banks for continuous laboratory use. From a starting liquid lysate of >100 mL, 23 the PoT protocol generated a cleaned, homogenous, laboratory phage bank with a phage 24 recovery efficiency of 85% within just two days. In contrast, the traditional method took upwards 25 of five days to produce a high titer, but lower volume phage stock with a recovery efficiency of 26 only 4%. Phage banks can be further purified for the removal of bacterial endotoxins, reducing 27 endotoxin concentrations by over 3,000-fold while maintaining phage titer. The PoT protocol 28 focused on T-like phages, but is broadly applicable to a variety of phages that can be propagated 29 to sufficient titer, producing homogenous, high titer phage banks that are applicable for 30 molecular and cellular assays. 31 32

Eukaryotic life benefits from—and ofttimes critically relies upon—the de novo biosynthesis and supply of vitamins and micronutrients from bacteria. The micronutrient queuosine (Q), derived from diet and/or the gut microbiome, is used as a source of the nucleobase queuine, which once incorporated into the anticodon of tRNA contributes to translational efficiency and accuracy. Here, we report high-resolution, substrate-bound crystal structures of the Sphaerobacter thermophilus queuine salvage protein Qng1 (formerly DUF2419) and of its human ortholog QNG1 (C9orf64), which together with biochemical and genetic evidence demonstrate its function as the hydrolase releasing queuine from queuosine-5′-monophosphate as the biological substrate. We also show that QNG1 is highly expressed in the liver, with implications for Q salvage and recycling. The essential role of this family of hydrolases in supplying queuine in eukaryotes places it at the nexus of numerous (patho)physiological processes associated with queuine deficiency, including altered metabolism, proliferation, differentiation and cancer progression.