Leucine-responsive regulatory protein-mediated repression of clp (encoding CS31A) expression by L-leucine and L-alanine in Escherichia coli

Abstract

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.

FIG. 1.

The clp regulatory region, sequences surrounding the clp operon, and operon fusion constructs. The nucleotide sequence of the clp regulatory region is shown at the top, with the GATC sites in bold. The GenBank accession number for this sequence is L48184. The organization of DNA surrounding the clp operon is shown below the regulatory region. Boxes with arrowheads indicate the positions of sequences with high homologies to sequences present in data banks as found using the BLASTn program (National Center for Biotechnology Information). The operon fusion constructs are shown in expanded scale at the bottom. The clpB gene is truncated in λ15 (black box) and is entire in λ6 (hatched box). P indicates the putative clp promoter.

FIG. 2.

Analysis of the in vivo GATC methylation states of the clp regulatory region. A Southern blot of chromosomal DNA obtained from MC4100.λ6 (A) and MC4100.λ6(ptrf1) (B) grown in M9-glycerol (lanes 2, 3, and 17 to 20), in M9-glycerol plus alanine (lanes 4, 5, and 13 to 16), or in M9-glycerol plus leucine, isoleucine, valine (lanes 6, 7, and 9 to 12) is shown. For MC4100.λ6, a colony grown on M9-glycerol was chosen at random among uniform colonies as the starting material. For MC4100.λ6(ptrf1), a Lac⁺ and a Lac⁻ colony were used as the starting material. The DNA was digested with BamHI plus EcoRI and either DpnI (D) or MboI (M). DNA hybridization was carried out with the digoxigenin-labeled probe indicated in panel C by a horizontal dotted line. The digoxigenin-labeled DNA molecular weight marker VI (Roche) was loaded in lanes 1 and 8. Its fragment sizes are indicated on the left. Size of DNA fragments that hybridized with the probe are shown on the right. (C) Schematic representation of DNA fragments that may hybridize with the probe.

FIG. 3.

Model for control of clp expression by Lrp, AfaF, ClpB, and alanine. D and P in open squares represent GATC_dist and GATC_prox sites, respectively. B in a triangle represents ClpB. The minus sign in an oval indicates a repressive effect on clp basal transcription. (A) Low level of basal transcription in the absence of AfaF and alanine; GATC_dist is methylated, whereas Lrp is bound to GATC_prox; ClpB is bound near to the promoter region. (B) Alanine represses the basal transcription independently of Lrp by an unknown mechanism and decreases Lrp binding to GATC_prox. (C and D) AfaF bound to Lrp allows Lrp binding to GATC_dist in phase-ON cells (C) and increases Lrp binding to GATC_prox in phase-OFF cells (D). (E) Alanine prevents the Lrp-AfaF complex from binding to GATC_dist and increases its binding to GATC_prox, locking cells in the OFF phase.