Adaptation and preadaptation of Salmonella enterica to Bile

Abstract

Bile possesses antibacterial activity because bile salts disrupt membranes, denature proteins, and damage DNA. This study describes mechanisms employed by the bacterium Salmonella enterica to survive bile. Sublethal concentrations of the bile salt sodium deoxycholate (DOC) adapt Salmonella to survive lethal concentrations of bile. Adaptation seems to be associated to multiple changes in gene expression, which include upregulation of the RpoS-dependent general stress response and other stress responses. The crucial role of the general stress response in adaptation to bile is supported by the observation that RpoS(-) mutants are bile-sensitive. While adaptation to bile involves a response by the bacterial population, individual cells can become bile-resistant without adaptation: plating of a non-adapted S. enterica culture on medium containing a lethal concentration of bile yields bile-resistant colonies at frequencies between 10(-6) and 10(-7) per cell and generation. Fluctuation analysis indicates that such colonies derive from bile-resistant cells present in the previous culture. A fraction of such isolates are stable, indicating that bile resistance can be acquired by mutation. Full genome sequencing of bile-resistant mutants shows that alteration of the lipopolysaccharide transport machinery is a frequent cause of mutational bile resistance. However, selection on lethal concentrations of bile also provides bile-resistant isolates that are not mutants. We propose that such isolates derive from rare cells whose physiological state permitted survival upon encountering bile. This view is supported by single cell analysis of gene expression using a microscope fluidic system: batch cultures of Salmonella contain cells that activate stress response genes in the absence of DOC. This phenomenon underscores the existence of phenotypic heterogeneity in clonal populations of bacteria and may illustrate the adaptive value of gene expression fluctuations.

Figure 1. Percentages of live and dead bacteria and relative numbers of colony-forming units.

Top panel: Percentages of live and dead bacteria (green and red histograms, respectively) found in 1 ml aliquots of an exponential culture of S. enterica SL1344 incubated in the presence of different concentrations of sodium deoxycholate (1%, 3%, 5%, 7% and 9%) during 30 minutes at 37°C. Bottom panel: Relative numbers of colony forming-units (CFU) after incubation of S. enterica SL1344 in the presence of different concentrations of sodium deoxycholate (1%, 3%, 5%, 7% and 9%) during 30 minutes at 37°C. The number of CFU in the absence of DOC is shown as 100%.

Figure 2. Minimal inhibitory concentrations (MICs) of sodium deoxycholate (DOC) for Salmonella cultures pre-exposed to various concentrations of DOC, and MICs for the same cultures after overnight growth in LB.

Figure 3. Validation of transcriptomic analysis: comparison of gene expression differences between LB and LB+5% deoxycholate as measured by RNA content (microarray analysis) and activity of lac fusions.

Figure 4. Minimal inhibitory concentration of sodium deoxycholate for bile resistant isolates after non-selective growth in LB.

The isolates had been originally obtained on plates containing 18% ox bile.

Figure 5. Lipopolysaccharide profiles of bile-resistant derivatives of S. enterica SL1344, as observed by electrophoresis and silver staining.

The lane marked “wt” shows the LPS profile of the wild type strain. Lanes 1–6 show the LPS profiles of bile-resistant mutants #1, #2, #3, #4, #5, and #6.

Figure 6. Levels of osmY gene expression in individual bacterial cells.

Panels A and B show the distribution of fluorescence intensity in individual cells (N>300) of S. enterica SV6562 (osmY::GFP) in two independent experiments. In both cases, strain SV6562 was grown during 5 h in LB with or without 5% sodium deoxycholate. Histograms represent the proportion of bacterial cells showing distinct fluorescence levels in LB (grey) and LB+DOC (red). Fluorescence intensities are shown in an arbitrary scale (0–1). Panel C shows the distribution of fluorescence intensity in individual cells (N>300) of S. enterica SV6780 (osmY::GFP RpoS⁻) under conditions identical to those of experiments A and B.

Figure 7. Time course of osmY::GFP expression in individual cells in the presence and in the absence of DOC.

Aliquots from an exponential culture (O.D.₆₀₀ = 0.5) of S. enterica SV6562 (osmY::GFP) grown in LB were transferred to agar pads containing or not 5% sodium deoxycholate. Bacterial cells were fixed in situ, and GFP fluorescence intensity was measured at 10 min intervals during 90 min.

Figure 8. Levels of cspD gene expression in individual bacterial cells.

The distribution of fluorescence intensity was measured in individual cells (N>300) of S. enterica SV6802 (cspD::GFP) after growth during 5 h in LB with or without 5% sodium deoxycholate. Histograms represent the proportion of bacterial cells showing distinct fluorescence levels in LB (grey) and LB+DOC (red). Fluorescence intensities are shown in an arbitrary scale (0–1).