No abstract

The region of DNA coding for incompatibility (inc) and copy number control (cop) of the IncFII plasmid NRl is transcribed in both the rightward and leftward directions. The rightward transcripts serve as mRNA for the repAl protein, which is required for replication. A small, 91-base leftward transcript is synthesized from the opposite DNA strand and is complementary to a portion of the rightward mRNA near its 5' end. A 262-basepair Sau3A restriction fragment that encodes the small leftward transcript, but does not include the rightward transcription promoters, was cloned into the vector pBR322 or pUC8. The same fragment was cloned from an Incmutant of NRl that does not make the small leftward transcript. Transcription through the cloned fragments in these derivatives was under control of the tetracycline resistance gene in pBR322 or the lac promoter-operator in pUC8. In one orientation of the inserted DNA, a hybrid transcript containing rightward NRl RNA sequences was synthesized. In the other orientation, a hybrid transcript containing leftward NRl RNA sequences was synthesized. These plasmids were used to vary the intracellular levels of the rightward or leftward NRl RNA transcripts and to test their effects in trans on various coresident derivatives of NRl. An excess of rightward NR1 RNA in trans stimulated expression of the essential repAl gene and caused an increase in the copy number of a coresident NR1 plasmid. An excess of leftward NRl RNA in trans inhibited the expression of the repAl gene and lowered the coresident NR1 copy number, thereby causing incompatibility. A pBR322 derivative with no transcription through the cloned NRl DNA had no effect in trans. These results suggest that the small leftward transcript is the incompatibility inhibitor of NRl and that its target is the complementary portion of the rightward mRNA.

Replication of the IncFII plasmid NRl is controlled by regulating the amount of synthesis of the repAl initiator protein at both the transcriptional and translational levels. We have examined mutations which have altered each of these levels of regulation, resulting in different plasmid copy numbers. The genes which encode each of the individual wild-type or mutant regulatory components from the replication control region of NRl have been cloned independently into pBR322 vectors, and their effects in trans, either individually or in various combinations, on plasmid incompatibility, stability, copy number, and repAl gene expression have been defined.

DNA from the replication control region of plasmid NRl or of the Inccopy mutant pRR12 was cloned into a pBR322 vector plasmid. These pBR322 derivatives were mutagenized in vitro with hydroxylamine and transformed into Escherichia col cells that harbored either NR1 or pRR12. After selection for the newly introduced pBR322 derivatives only, those cells which retained the unselected resident NR1 or pRR12 plasmids were examined further. By this process, 134 plasmids with Incmutations in the cloned NRl or pRR12 DNA were obtained. These mutants fell into 11 classes. Two of the classes had plasmids with deletions or insertions in the NRl DNA and were not examined further. Plasmids with apparent point mutations were classified by examining (i) their ability to reconstitute a functional NRl-derived replicon (Rep' or Rep-), (ii) the copy numbers of the Rep' reconstituted replicons, (iii) the cross-reactivity of incompatability among the various mutant classes and parental plasmids, and (iv) the trans effects of the mutants on the copy number and stable inheritance of a coresident plasmid.