Characterization of novel recombinant mycobacteriophages derived from homologous recombination between two temperate phages

Abstract

Comparative analyses of mycobacteriophage genomes reveals extensive genetic diversity in genome organization and gene content, contributing to widespread mosaicism. We previously reported that the prophage of mycobacteriophage Butters (cluster N) provides defense against infection by Island3 (subcluster I1). To explore the anti-Island3 defense mechanism, we attempted to isolate Island3 defense escape mutants on a Butters lysogen, but only uncovered phages with recombinant genomes comprised of regions of Butters and Island3 arranged from left arm to right arm as Butters-Island3-Butters (BIBs). Recombination occurs within two distinct homologous regions that encompass lysin A, lysin B, and holin genes in one segment, and RecE and RecT genes in the other. Structural genes of mosaic BIB genomes are contributed by Butters while the immunity cassette is derived from Island3. Consequently, BIBs are morphologically identical to Butters (as shown by transmission electron microscopy) but are homoimmune with Island3. Recombinant phages overcome antiphage defense and silencing of the lytic cycle. We leverage this observation to propose a stratagem to generate novel phages for potential therapeutic use.

Keywords: bacteriophage evolution; homologous recombination; mycobacteriophage genomics; phage genetic diversity; phage genome mosaicism; phage therapy.

Graphical Abstract

Fig. 1.

Phage sensitivity assay (spot tests) showing mc²155 (butters) defense against Island3. Ten-fold serial dilutions (3 µl) of Island3 and Eponine (as control) were spotted onto lawns of Mycobacterium smegmatis mc²155 (mc²155) and Butters lysogen [mc²155(Butters)].

Fig. 2.

Genome characterization of Island3, butters, and recombinant phage, BIB. a) Phamerator maps (Cresawn et al. 2011) for Butters, and Island3 and a derived map for BIB. Genes are depicted by rectangular boxes and are aligned to their genomic ruler. Gene numbers are within the rectangular boxes and putative gene functions, if known, are indicated above the gene. Sections defined between genomes show nucleotide similarity between the genome segments with variable E value scores. Selected segments with over 300 bp aligned sequences and E value ≤ 1e−20 are shown within connecting sections. Within each square bracket is the fraction of number of nucleotide identities over total aligned sequences, percentage of nucleotide identity in parentheses and E value (e val). The bottom row is a representative map of a BIB genome. Blocks or modules derived from Butters are shaded darker and the module derived from Island3 is shaded lighter. b) Alignment of SNPs across the first homology region. The entire nucleotide sequences that defines the first homology region (genome coordinates: 21,825–23,917 [Butters] and 25,302–27,409 [Island3]) were aligned in Jalview (Waterhouse et al. 2009). Conserved sequences were redacted to yield only SNPs. From left to right, sequences of BIBs first map to Butters before transitioning into Island3. SNPs in some BIBs “flipflop” from one parental genome to another and back. c) Alignment of SNPs across the second homology region (genome coordinates: 33,050–35,516 [Butters] and 33,774–35,854 [Island3]. From left to right, BIB genomes align to Island3 before transitioning into Butters. d) Lengths of genetic segments exchanged during recombination. Length of Island3 module was estimated from the first occurrence of SNP originating from the Island3 genome in the first homology region to the first occurrence of a SNP originating from the Butters genome in the second homology region. The same coordinates were used to estimate the length of the Butters module that was swapped out from the Butters genome.

Fig. 3.

Physical and immunity properties of BIBs and their parental phages. a) Comparison of plaque morphologies of Butters, Island3, and BIBs. Plaque assay was performed by plating serial dilutions of the phage lysates onto mc²155 and plaque morphologies assessed. BIBs present an emergent phenotype distinguishable from that of both Butters and Island3. b) Electron micrograph of Butters, Island3 and BIB phages. BIBs and Island3 share identical capsid and tail lengths. c) Immunity relationships for Butters, Island3, and BIBs. Three microliters of serially diluted phages were spotted on lawns of mc²155, mc²155(Butters), and mc²155(Island3). BIBs are homoimmune with Island3.

Fig. 4.

PCR screening of plaques obtained from plating experiments. Primers specific for each possible phage type (BIB, Butters, Island3, and IBI) for first homology region were designed and used to screen plaques via PCR. PCR products were run on 0.8% agarose gel at 60 V. GeneRuler 1 kb DNA ladder, ThermoFisher Scientific (extreme right panel) was used as ladder (#). Expected product size is 1,367 bp for BIB (arrow). Nonspecific bands appear in some instances due to nonspecific primer annealing for PCR conditions used. Also, some of the nonspecific bands can be mapped to the mc²155 genome which is inevitably present when crude phage lysates are filtered through 0.2 µm filters. Samples from left to right are ladder, primary plaques (1p1, 1p2, and 1p3), no template control, M. smegmatis mc²155 (Ms), Butters lysate, Island3 lysate, BIB6 lysate, and ladder (#). Note that primary plaques show PCR products with BIB-specific primers but not with Butters- or Island3- or IBI-specific primers. This is representative of at least 3 independent biological replicates.

Fig. 5.

Model showing coinfection as a strategy to overcome antiphage systems. Bacteria (or lysogen) have antiphage defense systems that block lytic infection by both rectangular shaped head (left)- and hexagonal shaped head (right) phages. Antiphage defense system 1 (rectangular block) targets portions of rectangular shaped head phage and antiphage system 2 (rectangular block) targets portions of hexagonal shaped head phage. During coinfection, homologous regions (shown as small blocks flanking antiphage defense systems) may recombine, yielding recombinant phage that escape defense systems present in the cell.