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 may be considered as one of the best models for investigation of the mechanisms regulating population heterogeneity of genetically identical microbial cells. In the present work, the role of intracellular regulators alkylresorcinols (alkylhydroxyben-zenes, AHB) (a C12-AHB homolog) in control of phase variations in type I pili formation (fimbriogenesis) was studied using a genetically constructed strain E. coli dsp250 containing the fimA-lacZ hybrid operon. In this operon the fimA gene encodes the main subunit of the pili protein, and its expression results in 0-galac-tosidase synthesis; pili-forming cells are therefore stained blue on the medium with the х-gal substrate. Expression offimA depends on inversion of the fimS region (inversion: on-pili formation, off-no pili). Increase in C12-AHB concentration to 5 х 10<sup>-5</sup>-2 х 10<sup>-4</sup> M in the exponential-phase culture of strain dsp250 was shown to cause dose-dependent transition of the dominant phenotype (up to 98-99%) forming colonies with blue center and white edges to a metastable state (up to 60%), with transition to alternative phenotypes of pili-forming (up to 11%) and pili-less cells (up to 44%). Effect of C12-AHB on off-switching to the avirulent phenotype of strain dsp250 was observed under various growth conditions. Addition of glucose to LB medium (5 or 10 mg/mL) resulted in catabolite repression via regulation by the cAMP-CNR complex and induce pili formation in 49 and 75% of the cells, respectively. Under these conditions, addition of C12-AHB resulted in dose-dependent decrease in the share of pili-forming cells to 32-61% and an increase in the share of pili-less cells to 33-61%. Addition of glucose to diluted LB/2 medium (2.5, 5 or 10 mg/mL) repressed pili formation completely, while C12-AHB still induced the off inversion to the pili-less phenotype (up to 30%). Thus, C12-AHB is not involved in the pathway of fimbriogenesis regulation via cAMP. The probable mechanism of action of C12-AHB, which has the functions of an extracellular alarmon (we have previously reported activation of the rpoS regulon and of SOS response), is transduction of the signal of a stress situation in the cell, which induces the synthesis of RpoS and H-NS global regulators and of the (p) ppGpp intracellular alarmon, responsible for switching the on ^ off inversion and growth of pili-less cells.