Reverse Genetic Systems for Pseudomonas aeruginosa Leviphages

Abstract

Reverse genetic systems for RNA viruses are the platforms to introduce mutations into the RNA genomes and thus have helped understand their life cycle and harness them for human purposes to develop vaccines and delivery systems. These systems are based on the complementary DNA (cDNA) of the RNA viruses, whose transcripts derived from bacterial RNA polymerases act not only as the primary mRNA for phage protein synthesis, but also as the template for phage RNA replicases (aka. RNA-dependent RNA polymerases). Here, we present a protocol optimized for the small RNA phages of Leviviridae (i.e., leviphages) infecting Pseudomonas aeruginosa. This protocol includes three fundamental steps: (i) Creation of a promoter-fused cDNA, (ii) generation of a clone into mini-Tn7-based vector, and (iii) introduction of the clone into non-susceptible hosts. As the representative example, we describe the reverse genetic system for PP7, which infects a set of P. aeruginosa strains such as PAO1. The cDNA was fused to the T7 promoter, which was cloned in mini-Tn7-Gm. This construct was introduced into P. aeruginosa PAK and E. coli HB101. Functional assembly of PP7 phages from the culture supernatants were assessed by plaque formation on PAO1 and the phage particles were observed under transmission microscope. We found that the host cells should be cultured at 30 °C for the maximal phage production (~1012 pfu/mL). The reverse genetic systems will provide a new insight into the life cycle of the RNA phages and help develop engineered variants with new traits for phage applications regarding selective diagnosis and efficient therapy.

An outer membrane determinant for RNA phage genome entry in Pseudomonas aeruginosa

“…Phage lysates of MPK7, MP29, and PRR1 were prepared by plate lysate method, using P. aeruginosa strain PAO1, while PP7 lysates were prepared using PAK cells containing the PP7 cDNA that had been cultured in 200 ml LB broth for 24 h at 30°C as described elsewhere. 41 Phage particles were precipitated with 1M NaCl and 10% polyethylene glycol (average molecular weight, 8,000 Da) (Sigma-Aldrich) at 4°C overnight, pelleted by centrifugation, and dissolved in phage buffer (100 mM NaCl, 50 mM Tris [pH7.5], 10 mM MgSO 4 , 1 mM EDTA) (5 ml). Phage particles were further concentrated by ultracentrifugation at 100,000 × g for 3 h and then resuspended in phage buffer (1 ml).…”

A phage protein-derived antipathogenic peptide that targets type IV pilus assembly

“…The RNA samples were subjected to RNeasy Cleanup kit (Qiagen, USA) to completely remove residual DNA. For quantitative measurement of the P1 RNA levels in the transgenic flies, quantitative PCR (qPCR) followed by cDNA synthesis was done by using 1 μg of the RNA samples, ReverTra Ace™ qPCR RT Kit (Toyobo, Japan) and TUNDERBIRD™ SYBR® qPCR Mix (Toyobo, Japan) as described previously [ 46 ]. The primers used for RNA assay are listed in Table S2, with the rp49 gene as the control.…”

RNA and Single-Stranded DNA Phages: Unveiling the Promise from the Underexplored World of Viruses

“…Thanks to the small genome size, engineering of ssDNA and RNA phage genomes can be performed through standard molecular cloning and plasmid transformation techniques into host cells. The reverse genetic system for RNA viruses uses complementary DNA (cDNA) to introduce desired mutations into the RNA genomes [118]. Modifications of cDNA, serving as templates for phage protein synthesis and RNA replication, in turn allow for studies of the viral life cycle and engineering of these viruses for practical applications [119,120].…”