Extending the Host Range of Bacteriophage Particles for DNA Transduction

Abstract

A major limitation in using bacteriophage-based applications is their narrow host range. Approaches for extending the host range have focused primarily on lytic phages in hosts supporting their propagation rather than approaches for extending the ability of DNA transduction into phage-restrictive hosts. To extend the host range of T7 phage for DNA transduction, we have designed hybrid particles displaying various phage tail/tail fiber proteins. These modular particles were programmed to package and transduce DNA into hosts that restrict T7 phage propagation. We have also developed an innovative generalizable platform that considerably enhances DNA transfer into new hosts by artificially selecting tails that efficiently transduce DNA. In addition, we have demonstrated that the hybrid particles transduce desired DNA into desired hosts. This study thus critically extends and improves the ability of the particles to transduce DNA into novel phage-restrictive hosts, providing a platform for myriad applications that require this ability.

High-throughput mapping of the phage resistance landscape inE. coli

“…In addition, these methods have the capability to identify fitness costs associated with broadly seen phage resistance phenotypes in a competitive natural environment, and thus improve our understanding of microbial ecology in general [13,114,206,207,[210][211][212]. Such systems-level insights will be valuable both in uncovering new mechanisms in host-phage interaction and perhaps in developing different design strategies for targeted microbial community interventions, engineering highly virulent or extended host-range phages and rationally formulated phage-cocktails for therapeutic applications [89,204,208,[213][214][215][216][217][218][219][220][221][222][223][224][225][226]. Alternatively, identifying phage resistance determinants may also enable engineering of bacterial strains with phage defense systems crucial in a number of bioprocesses such as in the dairy industry [227,228], biocontainment strategies for bioproduction industry [229,230] or to facilitate bacterial vaccine discovery and development [231][232][233].…”

Engineering Phage Host-Range and Suppressing Bacterial Resistance Through Phage Tail Fiber Mutagenesis

“…In particular, they engineered phages that lack a tail fiber gene and provided the missing function from a library of plasmids encoding various tail fiber homologs from phages with known host-range. The tail-encoding plasmids were then evolved through chemical mutagenesis and selected based on the capacity for phages to transduce the corresponding plasmid to a host (Yosef et al, 2017).…”

“…Various approaches have been undertaken to rationally expand the host-range of phages to combat resistance (Ando et al, 2015; Chen et al, 2017; Gebhart et al, 2017; Hawkins et al, 2008; Heilpern and Waldor, 2003; Lin et al, 2012; Nguyen et al, 2012; Scholl et al, 2009; Yoichi et al, 2005; Yosef et al, 2017). However, these approaches depend on hybridization between already characterized bacteriophages with known and desired host-ranges.…”

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

“…Within this study we were able to rapidly generate a number of tail fiber mutants (the majority of which ultimately proved to be nonfunctional in their ability to infect E. coli) using trxA-based selection. The functional T7-Yep-phi tail fiber hybrid, adds to the growing evidence that functional synthetic phages can be engineered [16,24,25].…”

“…Therefore, these genes have previously been used as markers to positively select for phage mutants in an E. coli background that lack trxA or cmk [14,16]. This marker-based approach has been used to knockout a number of T7 genes including genes 0.4, 11, 12, 17 [16][17][18]. These knockouts were achieved by replacing the target gene with trxA by homologous recombination, and selection on E. coli BW25113 DtrxA cells [16][17][18].…”