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

Bacteria undergo cycles of growth and starvation, to which they must adapt swiftly. One important strategy for adjusting growth rates relies on ribosomal levels. While high ribosomal levels are required for fast growth, their dynamics during starvation remain unclear. Here, we analyzed ribosomal RNA (rRNA) content of individual Salmonella cells using Fluorescence In-Situ Hybridization (rRNA-FISH), and measured during nutrient limitation a dramatic decrease in rRNA numbers only in a subpopulation, resulting in a bimodal distribution of cells with high and low rRNA content. During nutritional upshifts the two subpopulations were associated with distinct phenotypes. Using a transposon screen coupled with rRNA-FISH, we identified two mutants, DksA and RNase I, acting on rRNA transcription shutdown and degradation, that abolished the formation of the subpopulation with low rRNA content. Our work identifies a bacterial mechanism for regulation of ribosomal bimodality that may be beneficial for population survival during starvation. To identify the genetic factors driving ribosomal bimodality and the formation of 16Shigh and 16Slow subpopulations, we generated a saturated genome-wide library of S.Tm mutants via random Tn5 transposition. We grew the transposon mutant library in LB medium to the transition phase, stained for 16S rRNA-FISH and analyzed by flow cytometry. 16Shigh and 16Slow subpopulations were separately sorted and analyzed by Transposon insertion site sequencing (TIS).