The rate of protein secretion dictates the temporal dynamics of flagellar gene expression.

Flagellar gene expression is temporally regulated in response to the assembly state of the growing flagellum. The key mechanism for enforcing this temporal hierarchy in Salmonella enterica serovar Typhimurium is the sigma(28)-FlgM checkpoint, which couples the expression of the late flagellar (P(class3)) genes to the completion of the hook-basal body. This checkpoint is triggered when FlgM is secreted from the cell. In addition to the sigma(28)-FlgM checkpoint, a number of other regulatory mechanisms respond to the secretion of late proteins. In this work, we examined how middle (P(class2)) and late (P(class3)) gene expression is affected by late protein secretion. Dynamic analysis of flagellar gene expression identified a novel mechanism where induction of P(class2) activity is delayed either when late protein secretion is abolished or when late protein secretion is increased. Using a number of different approaches, we were able to show that this mechanism did not involve any known flagellar regulator. Furthermore, the changes in P(class2) activity were not correlated with the associated changes in P(class3) activity, which was found to be proportional to late protein secretion rates. Our data indicate that both P(class2) and P(class3) promoters are continuously regulated in response to assembly and late protein secretion rates. These results suggest that flagellar regulation is more complex than previously thought.