Whole genome sequencing identifies an allele responsible for clear vs. turbid plaque morphology in a Mycobacteriophage

Abstract

Background: Whole genome sequencing promises to revolutionize our ability to link genotypic and phenotypic variation in a wide range of model and non-model species.

Results: Here we describe the isolation and characterization of a novel mycobacteriophage named BGlluviae that grows on Mycobacterium smegmatis mc²155. BGlluviae normally produces turbid plaques but a spontaneous clear plaque was also recovered. The genomic DNA from pure populations of the BGlluviae phage and the clear plaque mutant were sequenced. A single substitution, at amino acid 54 (I to T), in the immunity repressor protein resulted in a clear plaque phenotype.

Conclusions: This substitution is predicted to be located at the subunit interaction interface of the repressor protein, and thus prevents the establishment of lysogeny.

Keywords: Immunity repressor protein; Mycobacteriophage; Mycobacterium smegmatis.

Fig. 1

Plaque and phage morphology of Mycobacteriophage BGlluviae. We isolated both (a) turbid/bulls-eye and (b) clear plaques from M. smegmatus mc²155 infected with BGlluviae. c Phage head and tail structure of BGlluviae isolated from turbid, bulls-eye plaques

Fig. 2

Maximum Likelihood phylogeny of BGlluviae Gene_43 Immunity Repressor with homologous Repressor proteins from other viral taxa. Maximum likelihood phylogeny built using the LG + G4 model of protein substitution with Gamma shape parameter α = 0.1. The Nearest Neighbor Interchange heuristic search option was used to search for the tree with the highest log likelihood, shown here (− 5638.001). Support for the tree was assessed using 1000 Ultrafast pseudoreplicates implemented in IQ-TREE (Trifinopoulos et al., 2016). Branch lengths are measured in the number of substitutions per site

Fig. 3

Predicted primary and secondary structure of BGlluviae Immunity Repressor protein. Highlighted residues indicate the location of potential helices as predicted by PSIPRED (Buchan et al., 2013). Rectangles indicate the location of different helix-turn-helix domains and motifs identified via the Conserved Domains Database (Lu et al., 2020) and Protein Data Bank (Berman et al., 2000). The Ile54Thr substitution (red triangle) identified between BGlluviae phages isolated from turbid and clear plaques interrupts helix 4, found within the protein dimerization motif. For reference, also shown are additional missense substitutions found within the Immunity Repressor at residues 23, 55, and 71 (blue triangles) of clear strains of the mycobacteriophage Adephagia (Petrova et al., 2015)

Fig. 4

Predicted Tertiary and Quaternary Structure of BGlluviae Immunity Repressor protein. a Comparison of BGlluviae turbid plaque (gold) and clear plaque (blue) repressor protein tertiary structures. Helices are sequentially numbered from the N-terminus. Amino acid 54 is Ile in the turbid and Tyr in the clear plaques (side chains shown in helix 4). Helices 2 and 3 are the DNA binding helix-turn-helix motifs as shown in b. b BGlluviae Gene 43 models superimposed on Lambda CI Repressor Protein (PDB ID 1LMB, gold model). BGlluviae wildtype with amino acid 54 = Ile (blue model) and BGlluviae mutant with amino acid 54 = Thr, (pink model) were each oriented to one of the Lambda CI repressor subunits (gold). The helix numbers correspond to the model shown in a

Fig. 5

Dimer Interface between subunits of Lambda CI Repressor and superimposed BGlluviae Gene 43 models. BGlluviae Gene 43 wildtype (amino acid 54 = Ile, blue model) and BGlluviae Gene 43 mutant (amino acid 54 = Thr, pink model) models were each superimposed onto each one of the Lambda CI repressor N-terminal dimer proteins (PDB ID 1LMB, gold model). The helix numbers correspond to those in Fig. 4