Constraints on horizontal gene acquisition in bacteria: genetic analysis of a novel restriction-modification system

Bacterial lineages respond to changes in their physical and biotic environment by acquisition of niche-adaptive functions via Horizontal Gene Transfer (HGT). They are specified in lineage-variable segments called genome islands. For about half of these islands, the RecA-independent mechanism of assembly and dissemination is obscure, while site-specific recombinases and transposases play a role in circulation of others. We study an island region bearing highly variable restriction-modification (RM) systems. The island (dubbed the Immigration Control Region, ICR) varies within and between enteric species to protect against exogenous DNA entrance. In addition, unidentified site-specific HGT mechanisms may act here.
To initiate study of the mechanism of intergeneric transfer between E. coli and Salmonella enterica sv Typhimurium LT2, we chose a restriction-disabled derivative (LB5000) often used for molecular genetic constructions. Sequencing of this strain allowed identification of the mutations that potentially result in restriction-deficiency in three RM systems: two well-studied (SenLT2I (LT, StyLT in the early literature) and SenLT2II (SB, StySB)) and one poorly characterized, SenLT2III (SA, StySA). Surprisingly, in the genetic region expected for the StySA system, multiple mutations were found in domains of two separate genes. These identified homologs of a BREX-like architecture for the StySA system. Mutational states of the 8 gene cluster were tested for site-specific methylation level (PacBio RSII) and bacteriophage restriction. We compared wild type alleles with engineered deletions of individual genes and with the multiply mutated conserved domains of two genes. In addition, we performed transcriptomic analysis (Cappable-Seq and RACE) in order to unravel the operon structure of this gene cluster. This work should contribute to understanding the role played by this recently-discovered but widespread family of prokaryotic genome defense activities.