Evidence for the involvement of the incC locus of broad host range plasmid RK2 in plasmid maintenance

Abstract

Plasmid pRK2501 is a deletion derivative of broad host range plasmid RK2 and encodes two trfB-regulated operons: the trfA operon which codes for both kilD, which interferes with plasmid maintenance if unregulated, and trfA whose protein product(s) is essential for replication from oriVRK2; and part of the trfB operon, containing both trfB/korA/korD, whose product negatively regulates transcription of both trfA and trfB operons, and incC, the product of which interferes in trans with inheritance of RK2 and certain of its derivatives. Plasmid pRK2501ts3 is a derivative with a point mutation in trfB, rendering plasmid maintenance temperature sensitive. Transcriptional fusions of the trfB operon and the galK gene demonstrate that this mutation derepresses trfB operon transcription at both 30 and 42 degrees C. The trfA operon is also derepressed by this mutation. Since the trfB gene product appears to be defective at both permissive and non-permissive temperatures the temperature sensitivity of pRK2501ts3 must be due to a secondary effect. In fact, it appears to arise from the inhibitory behavior of derepressed incC at the non-permissive temperature since a major class of "revertant" of pRK2501ts3 contains deletions inactivating incC and a reconstruction experiment demonstrates that such a deletion is sufficient for "reversion." Maxicell experiments show that at the non-permissive temperature the trfA operon polypeptide products are produced at much lower levels, an effect partly reversed by a deletion affecting incC. It is proposed that incC normally plays a role in maintenance of IncP plasmids by modulation of trfA operon expression.