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We describe a portable epigenetic switch based on opvAB, a Salmonella enterica operon that undergoes bistable expression under DNA methylation control. A DNA fragment containing the opvAB promoter and the opvAB upstream regulatory region confers bistability to heterologous genes, yielding OFF and ON subpopulations. Bistable expression under opvAB control is reproducible in Escherichia coli, showing that the opvAB switch can be functional in a heterologous host. Subpopulations of different sizes can be produced at will using engineered opvAB variants. Controlled formation of antibiotic-resistant and antibiotic-susceptible subpopulations may allow use of the opvAB switch in the study of bacterial heteroresistance to antibiotics.

We describe a portable epigenetic switch based on opvAB, a Salmonella enterica operon that undergoes bistable expression under DNA methylation control. A DNA fragment containing the opvAB promoter and the opvAB upstream regulatory region confers bistability to heterologous genes, yielding OFF and ON subpopulations. Bistable expression under opvAB control is reproducible in Escherichia coli, showing that the opvAB switch can be functional in a heterologous host. Subpopulations of different sizes can be produced at will using engineered opvAB variants. Controlled formation of antibiotic-resistant and antibiotic-susceptible subpopulations may allow use of the opvAB switch in the study of bacterial heteroresistance to antibiotics., This study was supported by grant BIO2016-75235-P from the Ministerio de Ciencia, Innovación y Universidades of Spain (to JC) and the Swedish Research Council grant 2017-01527 (to DIA).

Formation of C(5)-methyl-cytosine, N(4)-methyl-cytosine, and N(6)-methyl-adenine in bacterial genomes is postreplicative, and occurs at specific targets. Base methylation can modulate the interaction of DNA-binding proteins with their cognate sites, and controls chromosome replication, correction of DNA mismatches, cell cycle-coupled transcription, and formation of epigenetic lineages by phase variation. During four decades, the roles of DNA methylation in bacterial physiology have been investigated by analyzing the contribution of individual methyl groups or small methyl group clusters to the control of DNA-protein interactions. Nowadays, single-molecule real-time sequencing can analyze the DNA methylation of the entire genome (the 'methylome'). Bacterial methylomes provide a wealth of information on the methylation marks present in bacterial genomes, and may open a new era in bacterial epigenomics.

Formation of bacterial lineages can have adaptive value either as a division of labor or as a bet-hedging strategy, and may facilitate bacterial adaptation to hostile and/or unpredictable environments. Lineage formation is often under epigenetic control, thus avoiding the burden of mutation. Epigenetic lineages can be formed whenever a cell-to-cell difference (e.g., generated by intrinsic noise) is propagated by a heritable feedback loop. In other cases, the basis of bacterial lineage formation is more complex and involves the formation of heritable DNA methylation patterns. In Salmonella enterica, a pathogen of humans and livestock, lineage formation plays roles in host colonization, bacteriophage resistance, motility, biofilm formation, and adaptive resistance to antibiotics.

The Salmonella enterica opvAB operon is a horizontally-acquired locus that undergoes phase variation under Dam methylation control. The OpvA and OpvB proteins form intertwining ribbons in the inner membrane. Synthesis of OpvA and OpvB alters lipopolysaccharide O-antigen chain length and confers resistance to bacteriophages 9NA (Siphoviridae), Det7 (Myoviridae), and P22 (Podoviridae). These phages use the O-antigen as receptor. Because opvAB undergoes phase variation, S. enterica cultures contain subpopulations of opvAB OFF and opvAB ON cells. In the presence of a bacteriophage that uses the O-antigen as receptor, the opvAB OFF subpopulation is killed and the opvAB ON subpopulation is selected. Acquisition of phage resistance by phase variation of O-antigen chain length requires a payoff: opvAB expression reduces Salmonella virulence. However, phase variation permits resuscitation of the opvAB OFF subpopulation as soon as phage challenge ceases. Phenotypic heterogeneity generated by opvAB phase variation thus preadapts Salmonella to survive phage challenge with a fitness cost that is transient only., Author Summary A tradeoff can increase the adaptive capacity of an organism at the expense of lowering the fitness conferred by specific traits. This study describes a tradeoff that confers bacteriophage resistance in Salmonella enterica at the expense of reducing its pathogenic capacity. Phase variation of the opvAB operon creates two subpopulations of bacterial cells, each with a distinct lipopolysaccharide structure. One subpopulation is large and virulent but sensitive to phages that use the lipopolysaccharide O-antigen as receptor, while the other is small and avirulent but phage resistant. In the presence of a phage that targets the O-antigen, only the avirulent subpopulation survives. However, phase variation permits resuscitation of the virulent opvAB OFF subpopulation as soon as phage challenge ceases. This transient tradeoff may illustrate the adaptive value of epigenetic mechanisms that generate bacterial subpopulations in a reversible manner.

Phenotypic heterogeneity is common in bacteria. Cases of epigenetic formation of bacterial lineages have been known for decades, and more examples have been unveiled by the advent of single-cell analysis. Epigenetic mechanisms establish cell fate in bacterial genera which undergo developmental programs. Lineage formation also occurs during biofilm formation and the colonization of animals by bacterial pathogens, and may be a frequent phenomenon when bacterial populations adapt to harsh environments. Lineage formation can be observed even in the laboratory, which suggests that phenotypic heterogeneity in clonal populations may be intrinsic to the bacterial lifestyle. The underlying mechanisms are diverse, ranging from relatively simple, inheritable feedback loops to complex self-perpetuating DNA methylation patterns. Keywords: DNA adenine methylation; Dam methylation pattern; Phase variation; Bistability; Noise