CS31A produced by septicemic and diarrheic Escherichia coli belongs to the Pap-regulatory family of adhesive factors, which are under methylation-dependent transcriptional regulation. Common features of operons encoding members of this family include two conserved GATC sites in the upstream regulatory region, and transcriptional regulators homologue to the PapB and PapI proteins. Methylation protection of GATC sites was previously shown to be dependent on the leucine-responsive regulatory protein (Lrp). Lrp and ClpB, the PapB equivalent, repressed clp basal transcription. A PapI homologue (AfaF) was required together with Lrp to establish the phase variation control, which gave rise to phase-ON cells that expressed CS31A and phase-OFF cells that did not express CS31A. In phase-OFF cells, the GATC(dist) site was methylated and the GATC(prox) site was protected from methylation, whereas in phase-ON cells, the inverse situation was found. Unlike Pap fimbriae, CS31A synthesis was dramatically reduced in media containing L-alanine or L-leucine. L-Alanine prevented the OFF-to-ON switch, locking clp expression in the OFF phase, whereas L-leucine repressed transcription without obvious effect on the switch frequency of phase variation. In phase-variable cells, leucine and alanine promoted methylation of GATC(dist) and methylation protection of GATC(prox), increasing the methylation pattern characteristic of repressed cells. Furthermore, alanine prevented the AfaF-dependent methylation protection of GATC(dist) and thus the appearance of phase-ON cells. In addition, analysis of clp expression in a Lrp-negative background indicated that alanine and leucine also repressed clp transcription by a methylation-independent mechanism.