Use of a two-color genetic screen to identify a domain of the global regulator Lrp that is specifically required for pap phase variation.

Abstract

The global regulator Lrp plays a central role as both a repressor and an activator in Pap phase variation. Unlike most other members of the Lrp regulon such as ilvIH, activation of papBA transcription requires the coregulator PapI and is methylation dependent. We developed a two-color genetic screen to identify Lrp mutations that inhibit Pap phase variation but still activate ilvIH transcription, reasoning that such mutations might identify PapI binding or methylation-responsive domains. Amino acid substitutions in Lrp at position 126, 133, or 134 greatly reduced the rate of Pap switching from phase off to phase on but had much smaller effects on ilvIH transcription. In vitro analyses indicated that the T134A and E133G Lrp variants maintained affinities for pap and ilvIH DNAs similar to those of wild-type Lrp. In addition, both mutant Lrp's were as responsive to PapI as wild-type Lrp, evidenced by an increase in affinity for pap Lrp binding sites 4, 5, and 6. Thus, in vitro analyses did not reveal the step(s) in Pap phase variation where these Lrp mutants were inhibited. In vivo analyses showed that both the T134A and E133G Lrp mutants activated transcription of a phase-on-locked pap derivative containing a mutation in Lrp binding site 3. Further studies indicated that the T134A Lrp mutant was blocked in a step in Pap phase variation that does not involve PapI. Our data suggest that these mutant Lrp's are defective in a previously unidentified interaction required for the switch from the phase-off to the phase-on pap transcription state.