Construction of Enterococcus faecalis pAD1 miniplasmids: Identification of a minimal pheromone response regulatory region and evaluation of a novel pheromone-dependent growth inhibition

Abstract

Several pAD1 miniplasmids were constructed that consisted of all or a portion of the pAD1 EcoRI B fragment with pheromone-inducible "E region" lacZ transcriptional fusions. Miniplasmids containing the entire EcoRI B fragment (the "E miniplasmids") were found to regulate LacZ expression normally, indicating that sufficient information is present on this fragment to regulate a pheromone response. These plasmids also encoded normal replication functions. The E miniplasmids were further reduced by deleting a KpnI fragment. These "K miniplasmids" were able to perform most of the functions central to a pheromone response but failed to induce the fused transcripts to levels observed in the parental plasmids. This defect was found to be due to a pheromone-dependent growth inhibition of cells containing the K miniplasmids. Evidence indicated that this inhibition was due to transcriptional readthrough beyond the lacZ gene and into the putative replication region of the plasmid. Possible mechanisms of this inhibition as well as its potential usefulness in further examining the characteristics of the pheromone response are discussed.