Mutations in the C-terminal region of the bacteriophage exclusion protein PglX can selectively inactivate restriction in Salmonella.

Abstract

Salmonella enterica serovar Typhimurium strain LT2 is protected by two DNA restriction-modification systems (HsdRMS and Mod-Res) and a Type I bacteriophage exclusion (BREX) system (BrxA-L). The LB5000 strain was constructed to inactivate restriction but not methylation in all three systems and has been available for decades (L. R. Bullas and J. I. Ryu, J Bacteriol 156:471-474, 1983, https://doi.org/10.1128/jb.156.1.471-474.1983). However, this strain had been heavily mutagenized and contains hundreds of other mutations, including a few in DNA repair genes. Here, we describe the development of a strain that is only mutated for DNA restriction by the three systems and remains competent for DNA modification. We transferred mutations specific to DNA restriction from LB5000 to a wild-type LT2 background. The hsdR and res mutations affected only restriction in the wild-type background, but the brxC and pglZ mutations for the poorly understood BREX system also reduced modification. Amino acids in an unannotated conserved region of PglX in the BREX system were then randomized. Mutations were identified that specifically affected restriction at 37°C but were found to be temperature sensitive for restriction and methylation when tested at 30°C and 42°C. These mutations in PglX are consistent with a domain that communicates DNA methylation information to other BREX effector proteins. Finally, mutations generated in the specificity domain of PglX may have changed the DNA binding site recognized by the BREX system. IMPORTANCE The restriction system mutants constructed in this study will be useful for cloning DNA and transferring plasmids from other bacterial species into Salmonella. We verified which mutations in strain LB5000 resulted in loss of restriction for each restriction-modification system and the BREX system by moving these mutations to a wild-type Salmonella background. The methylase PglX was then mutagenized, which adds to our knowledge of the BREX system that is found in many bacteria but is not well understood. These PglX mutations affected restriction and methylation at different temperatures, which suggests that the C-terminal region of PglX may coordinate interactions between the methylase and other BREX system proteins.