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

Abstract

Understanding mechanisms that shape horizontal exchange in prokaryotes is a key problem in biology. A major limit on DNA entry is imposed by restriction-modification (RM) processes that depend on the pattern of DNA modification at host-specified sites. In classical RM, endonucleolytic DNA cleavage follows detection of unprotected sites on entering DNA. Recent investigation has uncovered BREX (BacteRiophage EXclusion) systems. These RM-like activities employ host protection by DNA modification, but immediate replication arrest occurs without evident of nuclease action on unmodified phage DNA. Here we show that the historical stySA RM locus of Salmonella enterica sv Typhimurium is a variant BREX system. A laboratory strain disabled for both the restriction and methylation activity of StySA nevertheless has wild type sequence in brxX, the modification gene homolog. Instead, flanking genes pglZ and brxC each carry multiple mutations (μ) in their C-terminal domains. We further investigate this system in situ, replacing the mutated pglZμ and brxCμ genes with the WT counterpart. PglZ-WT supports methylation in the presence of either BrxCμ or BrxC-WT but not in the presence of a deletion/insertion allele, ΔbrxC::cat. Restriction requires both BrxC-WT and PglZ-WT, implicating the BrxC C-terminus specifically in restriction activity. These results suggests that while BrxC, PglZ and BrxX are principal components of the BREX modification activity, BrxL is required for restriction only. Furthermore, we show that a partial disruption of brxL disrupts transcription globally.

Structure, substrate binding and activity of a unique AAA+ protein: the BrxL phage restriction factor

“…The mechanism by which type 1 BREX systems restrict phage is not yet understood but occurs at a step prior to phage replication and does not involve cleavage of the phage genome ( 12 ). BREX systems presumably employ a DNA scanning mechanism that differentiates methylated (host) from unmethylated (foreign) sites; consistent with this, the Salmonella enterica BREX system (StySA) is observed to much more effectively restrict phage containing a higher number of unmodified PglX recognition sequences ( 21 ). Furthermore, given that all six or seven conserved genes in various type I BREX systems so far characterized, including BrxL, are required for restriction, it seems likely that a multi-protein BREX complex carries out this scanning function.…”

“…Furthermore, given that all six or seven conserved genes in various type I BREX systems so far characterized, including BrxL, are required for restriction, it seems likely that a multi-protein BREX complex carries out this scanning function. In the StySA BREX system, the authors identified a BrxC mutant containing a handful of relatively conserved mutations in its C-terminal domain that is competent for methylation but deficient for restriction ( 21 ). They proposed that BrxC’s C-terminal domain is critical for scanning and identifying unmethylated target sites and perhaps for licensing BrxL for phage restriction.…”

“…The sequence similarities between BrxL and either Lon or RadA are comparable (both less than 20% identity versus BrxL) and is highest across recognizable sequence motifs required for nucleotide binding and ATPase activity. Although BrxL is often annotated in various sequence databases as a homologue of Lon protease (a designation that led to its name), it does not contain an obviously conserved Lon protease active site motif ( 21 ). The BrxL gene is limited to only Type 1 and 4 BREX systems; in alternative BREX systems (four total) it appears to have been replaced by a DNA helicase ( 9 ).…”

“…The BrxL gene is limited to only Type 1 and 4 BREX systems; in alternative BREX systems (four total) it appears to have been replaced by a DNA helicase ( 9 ). Furthermore, a recent study of a variant BREX system in Salmonella , termed the stySA RM system, showed that BrxL is required for restriction only, and concludes that it is unlikely to be a AAA+ protease ( 21 ).…”

Crystal structure of the BREX phage defence protein BrxA

“…2 C). Specifically, BrxA from E. fergusonii ATCC 35469 pEFER ( Picton et al., 2021 , 2022 ), E. coli HS2 ( Gordeeva et al., 2019 ; Isaev et al., 2020 ), Salmonella LT2 ( Zaworski et al., 2022 ), M. magneticum AMB-1, Acinetobacter NEB394 ( Luyten et al., 2022 ), Bacillus cereus H3081.97 ( Goldfarb et al., 2015 ), and Lactobacillus casei Zhang ( Hui et al., 2019 , 2022 ). All are annotated as domain of unknown function (DUF) 1819 proteins.…”