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 phage restriction. The analysis identified a previously uncharacterized gene at the 5-prime end of 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 pseudo-palindromic DNA target site upstream of its transcription start site. Precise deletion of the BrxR gene causes cell toxicity, reduces phage restriction, and significantly increases the expression of BrxC. In contrast, the introduction of a premature stop codon into the BrxR gene has little effect, implying that the BrxR coding sequence and BrxR protein have independent functional roles in BREX regulation. We speculate that the BrxR coding sequence is involved in cis regulation of BREX activity and that the BrxR protein may play an additional regulatory role, perhaps during horizontal transfer of the system.

Control of bacterial immune signaling by a WYL domain transcription factor

“…( A ) Structure of the Sm CapW dimer, with wHTH domain colored blue, WYL domain yellow, and WYL C-terminal extension (WCX) domain orange. ( B ) Model of the CapW wHTH domains binding DNA, generated by docking the structure of DNA-bound Acinetobacter BrxR (PDB ID 7T8K) ( 13 ) onto the structure of Sm CapW (overall Cα r.m.s.d. 2.9 Å; see Supplementary Figure S2d ).…”

Reassembling a cannon in the DNA defense arsenal: Genetics of StySA, a BREX phage exclusion system in Salmonella lab strains

“…This might be expected if BrxB were a toxin and BrxA an antitoxin. Two other systems yielded conflicting results for ΔbrxA : deletion abolished both R and M with the Acinetobacter system [ 59 ]; in the E . coli system neither R or M depended on it [ 30 ].…”

“…Both distant and close PglX homologs have been shown to be required for modification. Distant examples are in high-GC ( Streptomyces coelicolor [ 25 ]) and low-GC ( Bacillus cereus [ 28 ] and Lactobacillus casei [ 32 ]) taxa, while closer relatives include Acinetobacter [ 59 ], E . coli HS [ 30 ] and the plasmid borne pEFER of Escherichia fergusonii ATCC35469 [ 17 ].…”

“…In two other BREX systems, a clean deletion of brxB alone resulted in loss of modification [ 30 , 59 ]. In agreement with that result, replacement of brxB with cat in the WT context (M + strain J) results in loss of modification in resulting M - strain O.…”

“…The BrxL protein is critical to restriction action but not required for modification in other systems [ 30 , 59 ]. Our results agree that host modification does not require BrxL.…”