Regulation of phase variation in type I pili formation in Escherichia coli: Role of alkylresorcinols, microbial autoregulators

Formation of virulence-associated type I pili in Escherichia coli should be considered as one of the most efficient models for investigating the mechanisms of regulating the heterogeneity of populations of genetically identical microbial cells. The present work focused on the role of alkylhydroxybenzenes (AHBs), density-dependent intercellular regulators, in controlling phase variations in type I pili formation (fimbriogenesis). The tested AHB homologue was C12-AHB; a genetically constructed strain E. сoli dsp250 containing the fimA-lacZ hybrid operon was used. In this operon, the fimA gene encodes the main subunit of the pili protein, and its expression results in β-galactosidase synthesis; pili-forming cells, therefore, become blue on the medium with the Х-gal substrate. Expression of fimA depends on the inversion of the fimS region that is located upstream of it. If the inversion is on, pili formation takes place, if it is off, no pili are formed. An increase in C12-AHB concentration (within the 5 × 10^–5–2 × 10^–4 M range) in the exponential-phase culture of strain dsp250 causes a dose-dependent change in the dominant phenotype that is displayed by up to 98–99% of the cells. Cells with this phenotype form colonies with a blue center and white edges. Up to 60% of the cells with this phenotype assume a metastable state and up to 11% and 44% of them transition to the alternative phenotypes of pili-forming and pili-less cells, respectively. The influence of C12-AHB on off-switching, i.e. the formation of the avirulent phenotype, was observed irrespective of the growth conditions of strain dsp250. Addition of glucose to the LB medium (5 or 10 mg/mL) resulted in catabolic repression via regulation by the cAMP-CNR complex and predictably induced pili formation in 49 and 75% of the cells, respectively. Against this background, C12-AHB caused a dose-dependent decrease in the share of pili-forming cells to 33–61% and an increase in the share of pili-less cells to 32–61%. If glucose was added in excess (2.5, 5 or 10 mg/mL) to the diluted LB/2 medium, pili formation was completely repressed, while C12-AHB still induced the off inversion to the pili-less phenotype in up to 30% of the cells. The conclusion can be drawn that C12-AHB is not involved in the pathway of fimbriogenesis regulation via cAMP. Since C12-AHB functions as an extracellular alarmon (activating the rpoS regulon and the SOS response as shown earlier, see Golod et al., 2009), its mechanism of action apparently involves stress signal transduction. It induces the synthesis of global regulators RpoS and H-NS and of intracellular alarmon (p) ppGpp; these factors are responsible for the on → off inversion and the proliferation of pili-less cells.