USE OF A 2-COLOR GENETIC SCREEN TO IDENTIFY A DOMAIN OF THE GLOBAL REGULATOR LRP THAT IS SPECIFICALLY REQUIRED FOR PAP PHASE VARIATION

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 ilvlH transcription, reasoning that such mutations might identify PapI binding or methylation-responsive domain s. amino acid substitutions in Lrp at position 126, 133, or 134 greatl y reduced the rate of Pap switching from abase odf to phase on hut had much smaller effects on ilvIH transcription, In vitro analyses indica ted that the T134A and E133G Lrp variants maintained affinities for pa p 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 wh ere these Lrp mutants were inhibited, In vivo analyses showed that bot h the T134A and E133G Lrp mutants activated transcription of a phase-o n-locked pap derivative containing a mutation in Lrp binding site 3, F urther studies indicated that the T134A Lrp mutant was blocked in a st ep in Pap phase variation that does not involve PapI. Our data suggest that these mutant Lrp's are defective in a previously unidentified in teraction required for the switch from the phase-off to the phase-an p np transcription state.