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

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 pglX, 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 PglX 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.

Genome archaeology of two laboratory Salmonella enterica enterica sv Typhimurium.

The Salmonella research community has used strains and bacteriophages over decades, exchanging useful new isolates among laboratories for the study of cell surface antigens, metabolic pathways and restriction-modification (RM) studies. Here we present the sequences of two laboratory Salmonella strains (STK005, an isolate of LB5000; and its descendant ER3625). In the ancestry of LB5000, segments of ∼15 and ∼42 kb were introduced from Salmonella enterica sv Abony 803 into S. enterica sv Typhimurium LT2, forming strain SD14; this strain is thus a hybrid of S. enterica isolates. Strains in the SD14 lineage were used to define flagellar antigens from the 1950s to the 1970s, and to define three RM systems from the 1960s to the 1980s. LB5000 was also used as a host in phage typing systems used by epidemiologists. In the age of cheaper and easier sequencing, this resource will provide access to the sequence that underlies the extensive literature.

Complete Annotated Genome Sequence of the Salmonella enterica Serovar Typhimurium LT7 Strain STK003, Historically Used in Gene Transfer Studies.

The genome of Salmonella enterica serovar Typhimurium LT7 comprises a chromosome and two plasmids. One plasmid is very close to pSLT of Salmonella Typhimurium LT2; the second harbors a shufflon region. Prophage content is distinct: LT7 lacks Fels-1, while Gifsy-1 and Fels-2 show island-like divergence and likely programmed inversion, respectively.

Genome analysis of Salmonella enterica serovar Typhimurium bacteriophage L, indicator for StySA (StyLT2III) restriction-modification system action.

Bacteriophage L, a P22-like phage of Salmonella enterica sv Typhimurium LT2, was important for definition of mosaic organization of the lambdoid phage family and for characterization of restriction-modification systems of Salmonella. We report the complete genome sequences of bacteriophage L cI-40 13-am43 and L cII-101; the deduced sequence of wildtype L is 40,633 bp long with a 47.5% GC content. We compare this sequence with those of P22 and ST64T, and predict 72 Coding Sequences, 2 tRNA genes and 14 intergenic rho-independent transcription terminators. The overall genome organization of L agrees with earlier genetic and physical evidence; for example, no secondary immunity region (immI: ant, arc) or known genes for superinfection exclusion (sieA and sieB) are present. Proteomic analysis confirmed identification of virion proteins, along with low levels of assembly intermediates and host cell envelope proteins. The genome of L is 99.9% identical at the nucleotide level to that reported for phage ST64T, despite isolation on different continents ∼35 years apart. DNA modification by the epigenetic regulator Dam is generally incomplete. Dam modification is also selectively missing in one location, corresponding to the P22 phase-variation-sensitive promoter region of the serotype-converting gtrABC operon. The number of sites for SenLTIII (StySA) action may account for stronger restriction of L (13 sites) than of P22 (3 sites).