Role of an autorepression system in the control of lambda dv plasmid copy number and incompatibility

Abstract

The lambda dv plasmid genome is composed of two regions: (1) the autorepressor region which consists of promoter-operator (pRoR) and autorepressor (tof) and (2) the origin region which consists of the origin of replication (ori) and two initiator genes (O and P) (Matsubara 1976). Replication of this plasmid is directly connected with transcription from pRoR. Using lambda dvs having various mutations in pRoR, the transcription ability was examined in detail in connection with the control mechanism of replication. The transcription ability of the autorepressor region is controlled by the binding affinity of the tof protein and pRoR. Thus, at steady-state, lambda dvs carrying a highly-constitutive ('strong') pRoR produced the autorepressor at high levels, whereas those carrying a low-constitutive ('weak') pRoR produced the autorepressor at low levels. This relationship did not change even when a fragment of lambda dv genome covering the autorepressor region was cloned into the plasmids pBR322 and pSC138, which could be maintained in a fixed amount within a cell. It was also shown that the autorepressor level at steady-state is a function of copy number of the DNA carrying the region for autorepression. These observations fit the autorepression model of Sompayrac and Maaløe (1973), which predicts that a decreased level of autorepressor activates pRoR and initiates transcription which leads to the production of tof protein until a new steady-state is established. By the same token, if the affinity of autorepressor and pRoR is decreased, pRoR remains active until a higher level of autorepressor is produced. Transcription of the autorepressor region directly affects the level of transcription of the origin region which is located distal to the promoter. Thus, the ability to replicate is connected with an ability to produce autorepressor. The lambda dv plasmids with 'strong' or 'weak' pRoR were maintained at a high or low copy number, respectively. The phenomenon of incompatibility of lambda dv was also examined using pBR322 and pSC138 plasmids carrying the cloned autorepressor region of lambda dv. The chimeric plasmids with 'strong' pRoR exhibited strong incompatibility with lambda dv, whereas those with 'weak' pRoR showed weak incompatibility. This indicates that interaction of the autorepressor and pRoR is related to the incompatibility of lambda dv plasmids.