Identification and characterization of the WYL BrxR protein and its gene as separable regulatory elements of a BREX phage restriction system

Abstract

Bacteriophage exclusion (‘BREX’) phage restriction systems are found in a wide range of bacteria. Various BREX systems encode unique combinations of proteins that usually include a site-specific methyltransferase; none appear to contain a nuclease. Here we describe the identification and characterization of a Type I BREX system from Acinetobacter and the effect of deleting each BREX ORF on growth, methylation, and restriction. We identified a previously uncharacterized gene in the BREX operon that is dispensable for methylation but involved in restriction. Biochemical and crystallographic analyses of this factor, which we term BrxR (‘BREX Regulator’), demonstrate that it forms a homodimer and specifically binds a DNA target site upstream of its transcription start site. Deletion of the BrxR gene causes cell toxicity, reduces restriction, and significantly increases the expression of BrxC. In contrast, the introduction of a premature stop codon into the BrxR gene, or a point mutation blocking its DNA binding ability, has little effect on restriction, implying that the BrxR coding sequence and BrxR protein play independent functional roles. We speculate that elements within the BrxR coding sequence are involved in cis regulation of anti-phage activity, while the BrxR protein itself plays an additional regulatory role, perhaps during horizontal transfer.

Control of bacterial immune signaling by a WYL domain transcription factor

“…Our inability to isolate these mutants in the context of a full CBASS system, when the same mutants are readily obtainable in our GFP reporter system, suggests that elimination of CapWmediated repression and the resulting high-level expression of CBASS genes is likely toxic to host cells. This model is consistent with a parallel study on the related BrxR transcription factor, in which deletion of BrxR leads to increased expression of BREX genes and toxicity to host cells (13,14).…”

“…We modelled a DNA-bound structure of CapW based on a structure of Acinetobacter BrxR bound to its cognate palindromic DNA sequence (13), revealing that the two wHTH domains in the CapW dimer are perfectly aligned to bind a palindromic DNA sequence ~20 base pairs in length (Fig. 3b), close to the length of palindromic sequences we identified as CapW binding sites (Fig.…”

“…CapW shares a similar overall architecture to another recently-identified transcription factor, BrxR, which controls the expression of BREX immune systems (13,14). Like CapW, BrxR binds BREX promoter sequences to repress expression in uninfected cells (13,14).…”

“…CapW shares a similar overall architecture to another recently-identified transcription factor, BrxR, which controls the expression of BREX immune systems (13,14). Like CapW, BrxR binds BREX promoter sequences to repress expression in uninfected cells (13,14). Curiously, introduction of an early stop codon into Acinetobacter BrxR to eliminate protein production does not compromise the antiphage activity of its cognate BREX system (13).…”

“…These data suggest that CapW may mediate increased CBASS expression, and concomitant host-cell toxicity, in response to a signal other than phage infection. Parallel discovery of CapW-like transcription factors in other bacterial immune systems including BREX (13,14) suggests that CapW and its relatives make up a family of ligand-responsive transcriptional switches, which directly sense stress signals and activate expression of diverse immune systems.…”