PHAGE: Therapy,Applications,and Research

Gene transfer agents (GTAs) are phage-like particles that transfer cellular genomic DNA between cells. A hurdle faced in studying GTA function and interactions with cells is the difficulty in obtaining pure and functional GTAs from cultures.

We used a novel two-step method for purification of GTAs from R. capsulatus by monolithic chromatography.

Our efficient and simple process had advantages compared to previous approaches. The purified GTAs retained gene transfer activity and the packaged DNA could be used for further studies.

This method is applicable to GTAs produced by other species and small phages, and could be useful for therapeutic applications.

The experimental determination of a bacteriophage host is a laborious procedure. Thus, there is a pressing need for reliable computational predictions of bacteriophage hosts.

We developed the program vHULK for phage host prediction based on 9504 phage genome features, which consider alignment significance scores between predicted proteins and a curated database of viral protein families. The features were fed to a neural network, and two models were trained to predict 77 host genera and 118 host species.

In controlled random test sets with 90% redundancy reduction in terms of protein similarity, vHULK obtained on average 83% precision and 79% recall at the genus level, and 71% precision and 67% recall at the species level. The performance of vHULK was compared against three other tools on a test data set with 2153 phage genomes. On this data set, vHULK achieved better performance at both the genus and the species levels than the other tools.

Our results suggest that vHULK represents an advance on the state of art in phage host prediction.

Gibson assembly and assembly-in-yeast are strategies to create long synthetic DNAs from diverse fragments, for example, when engineering bacteriophage genomes. Design for these methods requires terminal sequence overlaps in the fragments, determining the order of assembly. Design to rebuild a genomic fragment that is too long for a single PCR presents a puzzle since some candidate joint regions cannot yield satisfactory primers for the overlap. No existing overlap assembly design software is open-source, and none explicitly supports rebuilding.

We describe here bigDNA software that solves the rebuilding puzzle by recursive backtracking, with options to remove or introduce genes; it also tests for mispriming on the template DNA. BigDNA was tested with 3082 prophages and other genomic islands (GIs), from 20 to 100 kb, and the synthetic Mycoplasma genitalium genome.

Rebuilding assembly design succeeded for all but ∼1% of GIs.

BigDNA will speed and standardize assembly design.

Non-O157 Shiga toxigenic Escherichia coli (STEC) are one of the most important food and waterborne pathogens worldwide. Although bacteriophages (phages) have been used for the biocontrol of these pathogens, a comprehensive understanding of the genetic characteristics and lifestyle of potentially effective candidate phages is lacking.

In this study, 10 non-O157-infecting phages previously isolated from feedlot cattle and dairy farms in the North-West province of South Africa were sequenced, and their genomes were analyzed.

Comparative genomics and proteomics revealed that the phages were closely related to other E. coli-infecting Tunaviruses, Seuratviruses, Carltongylesviruses, Tequatroviruses, and Mosigviruses from the National Center for Biotechnology Information GenBank database. Phages lacked integrases associated with a lysogenic cycle and genes associated with antibiotic resistance and Shiga toxins.

Comparative genomic analysis identified a diversity of unique non-O157-infecting phages, which could be used to mitigate the abundance of various non-O157 STEC serogroups without safety concerns.