Regulation of leading region genes on IncI1 plasmid ColIb-P9.

The psiB and ssb genes of IncI1 plasmid ColIb-P9 reside in the leading region, which is the first portion of the plasmid to be transferred during conjugation. These genes are expressed in a transient burst in newly-formed transconjugants via a process known as zygotic induction, and are thought to promote establishment of the immigrant plasmid. One hypothesis for the regulation of zygotic induction is that the genes are activated as part of a conjugation-induced heat-shock response. Both ssb and psiB are damage-inducible and have putative sigma 32-dependent promoter sequences. Data are presented showing that zygotic induction occurs when sigma 32 levels are limited and that conjugation fails to induce the heat- shock response, as measured using a sigma 32-dependent reporter construct. A second hypothesis is that zygotic induction results from a transient loss of negative supercoiling during plasmid transfer. The finding that neither psiB nor ssb expression is strongly induced in vegetative cells treated with coumermycin is inconsistent with this hypothesis. A third possibility is that psiB and ssb are regulated by a plasmid-encoded trans-acting repressor which is absent from the recipient cell. This hypothesis was tested with an entry-exclusion mutant of ColIb, created by insertional inactivation of the eex locus. When ColIb was transferred to a recipient harbouring this mutant, zygotic induction was observed. This finding also demonstrates that zygotic induction is independent of vegetative replication, since the incoming and resident plasmids are incompatible. Previous work suggested that psiB and ssb are closely linked to promoters. However, data obtained using RP4- and ColIb-mobilisable constructs carrying different portions of the leading region suggest that at least 5 kb of upstream DNA is necessary for zygotic induction of both genes, thus indicating that they are induced as part of a leading region operon.