Contribution of DNA adenine methylation to gene expression heterogeneity in Salmonella enterica

Expression of Salmonella enterica loci harboring undermethylated GATC sites at promoters or regulatory regions was monitored by single cell analysis. Cell-to-cell differences in expression were detected in ten such loci (carA, dgoR, holA, nanA, ssaN, STM1290, STM3276, STM5308, gtr and opvAB), with concomitant formation of ON and OFF subpopulations. The ON and OFF subpopulation sizes varied depending on the growth conditions, suggesting that the population structure can be modulated by environmental control. All the loci under study except STM5308 displayed altered patterns of expression in strains lacking or overproducing Dam methylase, thereby confirming control by Dam methylation. Bioinformatic analysis identified potential binding sites for transcription factors OxyR, CRP and Fur, and analysis of expression in mutant backgrounds confirmed transcriptional control by one or more of such factors. Surveys of gene expression in pairwise combinations of Dam methylation-dependent loci revealed independent switching, thus predicting the formation of a high number of cell variants. This study expands the list of S. enterica loci under transcriptional control by Dam methylation, and underscores the relevance of the DNA adenine methylome as a source of phenotypic heterogeneity.

Regulation of bistability in the std fimbrial operon of Salmonella enterica by DNA adenine methylation and transcription factors HdfR, StdE and StdF

Bistable expression of the Salmonella enterica std operon is controlled by an AND logic gate involving three transcriptional activators: the LysR-type factor HdfR and the StdE and StdF regulators encoded by the std operon itself. StdE activates transcription of the hdfR gene, and StdF activates std transcription together with HdfR. Binding of HdfR upstream of the std promoter is hindered by methylation of GATC sites located within the upstream activating sequence (UAS). Epigenetic control by Dam methylation thus antagonizes formation of the StdE-StdF-HdfR loop and tilts the std switch toward the StdOFF state. In turn, HdfR binding hinders methylation of the UAS, permitting activation of the StdE-StdF-HdfR loop and concomitant formation of StdON cells. Bistability is thus the outcome of competition between DNA adenine methylation and the StdE-StdF-HdfR activator loop.

Replication of Vibrio cholerae Chromosome I in Escherichia coli: Dependence on Dam Methylation

ABSTRACT We successfully substituted Escherichia coli's origin of replication oriC with the origin region of Vibrio cholerae chromosome I (oriCIVc ). Replication from oriCIVc initiated at a similar or slightly reduced cell mass compared to that of normal E. coli oriC. With respect to sequestration-dependent synchrony of initiation and stimulation of initiation by the loss of Hda activity, replication initiation from oriC and oriCIVc were similar. Since Hda is involved in the conversion of DnaAATP (DnaA bound to ATP) to DnaAADP (DnaA bound to ADP), this indicates that DnaA associated with ATP is limiting for V. cholerae chromosome I replication, which similar to what is observed for E. coli. No hda homologue has been identified in V. cholerae yet. In V. cholerae, dam is essential for viability, whereas in E. coli, dam mutants are viable. Replacement of E. coli oriC with oriCIVc allowed us to specifically address the role of the Dam methyltransferase and SeqA in replication initiation from oriCIVc . We show that when E. coli's origin of replication is substituted by oriCIVc , dam, but not seqA, becomes important for growth, arguing that Dam methylation exerts a critical function at the origin of replication itself. We propose that Dam methylation promotes DnaA-assisted successful duplex opening and replisome assembly at oriCIVc in E. coli. In this model, methylation at oriCIVc would ease DNA melting. This is supported by the fact that the requirement for dam can be alleviated by increasing negative supercoiling of the chromosome through oversupply of the DNA gyrase or loss of SeqA activity.