Mini-F E protein: the carboxy-terminal end is essential for E gene repression and mini-F copy number control

Mini-F is a segment of the conjugative plasmid F consisting of two origins of replication flanked by regulatory regions, which ensure a normal control of replication and partitioning. Adjacent to the ori-2 origin is a complex coding region that consists of the E gene overlapped by three open reading frames with the coding potential for 9000 M r polypeptides here designated 9kd-1, 9kd-2 and 9kd-3. In this paper, we show that open reading frame 9 kd-3 is preceded by active promoter and Shine-Dalgarno sequences. The E coding region specifies: (1) an initiator of replication, which acts at the ori-2 site; (2) a function that negatively regulates the expression of the E gene; and (3) a function involved in mini-F copy number control. To assign one of these functions to one of the overlapping coding sequences, we have isolated, characterized and sequenced mutations mapping in the E coding region. In this paper, we analyse two mutations ( cop5 and pla25) that abolish the repression of the E gene. As these mutations affect the primary structure of protein E itself but not the 9kd polypeptides, we conclude that protein E takes part in the negative regulation of its own synthesis. In addition, the localization of the cop5 and pla25 mutations indicates that the carboxy-terminal end of the E protein is involved in the autorepression function. The cop5 mutation causes an eightfold increase of the mini-F copy number. The pla25 mutation leads to the inability of the derived mini-F plasmid to give rise to plasmid-harbouring bacteria. The ways in which the cop5 and pla25 mutations may lead to such phenotypes are discussed in relation to the different functions mapping in the E coding sequence.