Pseudomonas aeruginosareaches collective decisions via transient segregation of quorum sensing activities across cells

Abstract

Bacteria engage in a cell-to-cell communication process called quorum sensing (QS) to coordinate expression of cooperative exoproducts at the group level. While population-level QS-responses are well studied, we know little about commitments of single cells to QS. Here, we use flow cytometry to track the investment of Pseudomonas aeruginosa individuals into their intertwined Las and Rhl QS-systems. Using fluorescent reporters, we show that QS gene expression (signal synthase, receptor and exoproduct) was heterogenous and followed a gradual instead of a sharp temporal induction pattern. The simultaneous monitoring of two QS genes revealed that cells transiently segregate into low receptor (lasR) expressers that fully commit to QS, and high receptor expressers that delay QS commitment. Our mathematical model shows that such gene expression segregation could mechanistically be spurred by transcription factor limitation. In evolutionary terms, temporal segregation could serve as a QS-brake to allow for a bet-hedging strategy in unpredictable environments.

Bacterial quorum sensing allows graded and bimodal cellular responses to variations in population density

“…To further consider the functional context of the graded reaction norms, we turn to the single cell scale data, which reveals how the graded population response is built from the contributions of individual cells. In agreement with previous work in multiple quorum-sensing organisms [9,[30][31][32][33][34][35][36]48,49], we find cell-scale heterogeneity. In addition, our results illustrate how cellular heterogeneity changes with the environment, demonstrating the onset of ON/OFF bimodality at intermediate densities, with both the proportion of cells ON and the intensity of the cellular ON states increasing with increases in culture carrying capacity (Figures 3 & 4).…”

“…In the context of QS, positive feedback loops (signal autoregulation [52]) and the presence of cooperative transcription factor binding [53] provides recognized regulatory ingredients for bimodal expression [54]. Recently, the presence of heterogeneous QS response at the single-cell scale has been ascribed to a potential bet-hedge against mis-directed QS induction [48], suggesting that our OFF cells are poised to more quickly resume growth in the event of a rapid return to a growth-friendly environment.…”

Evolution of the quorum sensing systems in Pseudomonas aeruginosa can involve both loss of regulon function and network modulation

“…A single copy of the double reporter construct was chromosomally integrated in the experimentally evolved clones at the neutral attTn7 site using the mini-Tn7 system (Choi and Schweizer, 2006). Detailed step-by-step cloning protocol is described elsewhere (Jayakumar et al, 2021). We used Escherichia coli CC118 λpir for all intermediary steps in our cloning work (see Table S2 for a full list of non-experimentally evolved strains and plasmids used).…”

Single-cell imaging reveals that Staphylococcus aureus is highly competitive against Pseudomonas aeruginosa on surfaces

“…For time-lapse experiments, we used a constitutively expressed green fluorescent protein ( attTn7::ptac-gfp ) in the chromosome of PA as a marker to distinguish PA from SA. For experiments with PA gene reporters, we used PA strains carrying constructs with promoters of interest fused to mCherry together with the housekeeping gene promoter of rpsL fused to gfp ( attTn7::lasR-mCherry;rpsL- gfp and attTn7::rhlR-mCherry;rpsL-gfp ) (40). Prior to imaging, bacterial overnight cultures were grown in 10 ml tryptic soy broth (TSB, Becton Dickinson) in 50 ml falcon tubes for ± 16 hours at 37 °C and 220 rpm with aeration.…”