An Unusual Phage Repressor Encoded by Mycobacteriophage BPs.

Valerie M Villanueva,Lauren M Oldfield,Graham F Hatfull

doi:10.1371/journal.pone.0137187

Abstract

Temperate bacteriophages express transcription repressors that maintain lysogeny by down-regulating lytic promoters and confer superinfection immunity. Repressor regulation is critical to the outcome of infection—lysogenic or lytic growth—as well as prophage induction into lytic replication. Mycobacteriophage BPs and its relatives use an unusual integration-dependent immunity system in which the phage attachment site (attP) is located within the repressor gene (33) such that site-specific integration leads to synthesis of a prophage-encoded product (gp33103) that is 33 residues shorter at its C-terminus than the virally-encoded protein (gp33136). However, the shorter form of the repressor (gp33103) is stable and active in repression of the early lytic promoter PR, whereas the longer virally-encoded form (gp33136) is inactive due to targeted degradation via a C-terminal ssrA-like tag. We show here that both forms of the repressor bind similarly to the 33–34 intergenic regulatory region, and that BPs gp33103 is a tetramer in solution. The BPs gp33103 repressor binds to five regulatory regions spanning the BPs genome, and regulates four promoters including the early lytic promoter, PR. BPs gp33103 has a complex pattern of DNA recognition in which a full operator binding site contains two half sites separated by a variable spacer, and BPs gp33103 induces a DNA bend at the full operator site but not a half site. The operator site structure is unusual in that one half site corresponds to a 12 bp palindrome identified previously, but the other half site is a highly variable variant of the palindrome.

Highlights

Following adsorption and DNA injection, temperate phages must choose between two alternative outcomes: lytic growth in which the phage replicates and the cell lyses to release progeny phage particles, or lysogeny in which the lytic genes are switched off and a prophage genome is maintained either by site-specific chromosomal integration, or stable extrachromosomal replication [1]
In the well-studied example of phage lambda, lysogenic maintenance is achieved by expression of a repressor that binds to tripartite operators (OL and OR) at the early lytic promoters PL and PR [1]
Establishment of lambda lysogeny occurs by expression of cI from the promoter for lysogenic establishment (PRE), which is independent of cI, but requires the activator, cII [2]

Summary

Introduction

Following adsorption and DNA injection, temperate phages must choose between two alternative outcomes: lytic growth in which the phage replicates and the cell lyses to release progeny phage particles, or lysogeny in which the lytic genes are switched off and a prophage genome is maintained either by site-specific chromosomal integration, or stable extrachromosomal replication [1]. Occupancy of the ORep site is evident at the highest protein concentration in DNase I footprinting (Fig 3) and may depend on concomitant binding of gp33103 elsewhere in the DNA fragment, such as at OR To further explore these interactions we synthesized a series of small (40 bp) dsDNA substrates, containing segments of the 33–34 intergenic region (Fig 1C) and asked whether gp33103. A mutant (127b) containing a large deletion and missing much of the intergenic region including ORep and OR-L but retaining OR-R forms complexes with much faster relative mobility that those seen with other substrates, indicating that perhaps either a monomer or dimer of gp33103 is binding, the basis for such an unusual property is unclear

Discussion

Materials and Methods