Physiological stress drives the emergence of a Salmonella subpopulation through ribosomal RNA regulation

Protocol for comparing ribosomal levels in single bacterial cells at different growth stages using rRNA-FISH

“…This data is presented in Figure 1 in Ciolli Mattioli et al., 2023. 1 CRITICAL: Exponentially growing bacteria grown in LB are used for positive and negative controls, include six samples from this phase (OD 600 = 0.4–0.6), additionally to the other samples of interest. Once the growth phase of interest is reached, calculate the volume of bacteria to harvest using the formula (f1).…”

“…We used an inverted epifluorescence microscope (Eclipse Ti2-E, Nikon) equipped with a x100 NA 1.45 oil-immersion objective, filter sets (DAPI, GFP, Cy3, Cy5/Cy5.5), and a CMOS camera (Prime 95C, Photometrics), as previously described. 1 Data can be collected using any fluorescent microscope with RGB channels and filters to visualize red, green, and blue signals. Alternatively, a confocal microscope also can be used to observe signal localization.…”

“…Given the dependence of growth rate on protein synthesis and ribosome abundance, 11 exponentially growing bacteria are expected to show a uniform ribosome abundance per cell (as shown in Figure 8 , in yellow). On the other hand, when S .Tm bacteria exit the exponential phase and enter a nutrient-limited state, a bimodal distribution of cells characterized by high and low levels of ribosomes is expected to emerge 1 (as shown in Figure 8 , in gray).

Figure 8 In exponential phase S .Tm cells have uniform levels of ribosomes, while during transition to stationary phase, ribosomal levels display a bimodal distribution Density histograms show 16S rRNA-FISH total fluorescence intensity of individual S.Tm cells measured by flow cytometry during exponential growth (in yellow) and the transition from exponential growth to stationary phase (in dark gray) ( n = 9,000).

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“…For complete details on the use and execution of this protocol, please refer to Ciolli Mattioli et al. 1 …”

Time resolved multi-omics reveals diverse metabolic strategies ofSalmonelladuring diet-induced inflammation

“…Given their presumed role in initiation of infection and the inflammatory response, it was somewhat surprising that these genes were not discriminant of early expression but instead, seemed active over the course of infection. We consider it possible that subpopulations of Salmonella may be infecting host cells continually or inflammationinduced envelope stress might alters the expression of these genes, explaining their chronic expression (48,(68)(69)(70).…”