Single-cell regulation of Pseudomonas aeruginosa virulence reveals new insights into bet-hedging strategies in bacteria

Pseudomonas aeruginosa is an opportunistic human pathogen that utilizes a broad arsenal of virulence determinants to invade and kill the host cells. A major virulence factor of P. aeruginosa is the type 3 secretion system (T3SS), a mega-complex that functions as a molecular needle to translocate toxins into eukaryotic host cells. The system is composed of ~36 different genes and is assembled by hundreds of protein copies and can thus carry a high cost to its producers. In addition, to energetic costs, the T3SS is highly immunogenic and can attract unwanted attention. We observed that even in the absence of a host, or inducing conditions, a sub-population of ~30% of P. aeruginosa cells expresses the T3SS genes. While this behavior can be thought of as classic bet-hedging, it imposes a potential dilemma between fitness and virulence. How is this activity maintained when the bacteria pay the cost but do not reap any of the benefits? Here, we show, using imaging-based single-cell transcriptomics and immunofluorescence assays, that P. aeruginosa tightly regulates the T3SS by coupling its expression to rapid growth. We hypothesize that this reflects a strategy in which the relative cost of producing the T3SS is lower when the population’s metabolic capacity is high. However, we find that the proportion of this T3SS+ sub-population decreased linearly with the population growth, suggesting an effect of division-based dilution. We predict this conflict between metabolic regulation and population growth represents a general tradeoff in the optimization of bet-hedging behavior in bacteria.