ppGppmediates the growth phase-dependent regulation ofagn43, a phase variable gene, by stimulating its promoter activity

Antigen 43 (Ag43) is a self-recognizing outer membrane protein ofEscherichia coliexpressed during intracellular growth and biofilm formation, suggesting a role in infection. The expression ofagn43is under phase variation control, meaning that there are regulatory mechanisms adjusting the percentage ofagn43-expressing cells in the population, in addition to mechanisms modulating the transcriptional expression level in each expressing cell. Phenotypic and transcriptional studies indicate that Ag43 expression is induced upon entry into the stationary phase in a ppGpp-dependent and RpoS-independent manner. The use of single-cell approaches and phase variation deficient strains let to conclude that ppGpp stimulatesagn43promoter activity, rather than affecting the percentage ofagn43-expressing cells. The data highlight the relevance that promoter activity regulation may have, without any involvement of the phase variation state, in the final Ag43 expression output. Theagn43promoter of the MG1655 strain carries an AT-rich discriminator between positions -10 and +1, which is highly conserved among theagn43genes present in the different pathotypes ofE. coli. Remarkably, the AT-rich discriminator is required for the positive transcriptional control mediated by ppGpp.