Efficient traceless modification of the P1 bacteriophage genome through homologous recombination with enrichment in double recombinants: A new perspective on the functional annotation of uncharacterized phage genes

Abstract

The advent of high-throughput omic technologies has caused unprecedented progress in research on bacteriophages, the most abundant and still the least explored entities on earth. Despite the growing number of phage genomes sequenced and the rejuvenation of interest in phage therapy, the progress in the functional analysis of phage genes is slow. Simple and efficient techniques of phage genome targeted mutagenesis that would allow one to knock out particular genes precisely without polar effects in order to study the effect of these knock-outs on phage functions are lacking. Even in the case of model phages, the functions of approximately half of their genes are unknown. P1 is an enterobacterial temperate myophage of clinical significance, which lysogenizes cells as a plasmid. It has a long history of studies, serves as a model in basic research, is a gene transfer vector, and is a source of genetic tools. Its gene products have structural homologs in several other phages. In this perspective article, we describe a simple and efficient procedure of traceless P1 genome modification that could also serve to acquire targeted mutations in the genomes of certain other temperate phages and speed up functional annotations of phage genes.

Keywords: ampicillin resistance; bacteriophage P1; bacteriophage engineering; homologous recombination; multicopy plasmid; prophage; targeted mutagenesis; temperate bacteriophage.

Figure 1

Scheme of P1-targeted mutation acquisition by homologous recombination and enrichment of recombinant pool in double recombinants. The P1 designation in the figure indicates the P1 c1-100 mod749::IS5 IS1::Tn9 mutant. E. coli strain N99 (galK2, str^R) (Gottesman and Yarmolinsky, 1968) was used as a host for the P1 prophage in all experiments. Black arrows indicate the order of laboratory procedures and red arrows indicate the revevant changes in the content of cells at various stages of the experiment. Created with BioRender.com.