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

Abstract

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.

Figure 1.

Expression of loci harboring undermethylated GATC sites at putative promoters or regulatory regions, monitored by single cell analysis. (A) Flow cytometry analysis of strains carrying gfp transcriptional fusions downstream of the loci under study. The GFP fluorescence intensity distribution was examined in cultures grown in LB under aerobiosis, LB under miare OKcroaerophilia and intracellular salts medium (ISM). Dot plots represent the forward scatter (cell size) versus fluorescence intensity. (B) Visualization of Salmonella cells by fluorescence microscopy. For each locus, the image shown was obtained under the growth condition in which bistability was most conspicuous (LB under microaerophilia for carA, dgoR, gtr, holA, STM1290, STM3276 and STM5308, and LB under aerobiosis for opvAB and ssaN). White arrows signalize ON cells. Scale bars indicate 5 μm.

Figure 2.

Single cell analysis of Dam methylation-dependent control of carA, dgoR, gtr, holA, nanA, opvAB, ssaN, STM1290 and STM3726. Gene expression was measured by flow cytometry in the wild type (left column and red line in the histograms), in a dam mutant (middle column and blue line in the histograms) and under Dam methylase overproduction (right column and green line in the histograms). Dot plots represent the forward scatter (cell size) versus fluorescence intensity. Histograms represent the frequency of cells with different GFP intensities, and changes in the pattern of expression are indicated with arrows. The growth condition shown for dgoR, STM1290, holA and carA is LB under microaerophilia; for gtr, nanA, opvAB and STM3726 is LB under aerobiosis; and for ssaN is ISM minimal medium. More detailed information is provided in Supplementary Figure S2.

Figure 3.

Flow cytometry analysis of carA, dgoR, gtr, holA, nanA, opvAB, ssaN and STM1290 expression in the absence of individual transcriptional factors. Dot plots represent the forward scatter (cell size) versus fluorescence intensity. For dgoR, holA, nanA, STM1290 and STM3726, the growth condition shown is LB under microaerophilia; for gtr and opvAB is LB under aerobiosis; and for carA and ssaN is ISM. More detailed information is provided in Supplementary Figure S3.

Figure 4.

Tests of co-ordinated and/or independent switching of loci under transcriptional control by Dam methylation. Fluorescence images of S. enterica cells are shown, visualizing the expression of 10 pairwise combinations of gene fusions (ssaN versus gtr, ssaN versus carA, nanA versus gtr, nanA versus ssaN, opvAB versus gtr, opvAB versus ssaN, dgoR versus gtrA, dgoR versus ssaN, holA versus gtr and holA versus ssaN). Each row shows a strain containing a GFP and either an mCherry or an mOrange transcriptional fusion. The growth condition shown for ssaN versus gtr, nanA versus gtr, opvAB versus gt, ssaN versus carA, nanA versus ssaN, opvAB versus ssaN, dgoR versus ssaN and holA versus ssaN is LB under aerobiosis; for dgoR versus gtr, and holA versus gtr, the growth condition shown is LB under microaerophilia. Scale bars indicate 1 μm.