Promoter mapping and transcriptional regulation of the iron assimilation system of plasmid ColV-K30 in Escherichia coli K-12

Abstract

The promoter of the high-affinity iron assimilation system coded in an approximately 8-kilobase-pair segment of the large Escherichia coli plasmid ColV-K30 was localized to a 0.7-kilobase HindIII-SalI fragment by in vitro runoff transcription. By an S1 nuclease protection assay, with in vitro-transcribed RNA and total in vivo-synthesized RNA, the major start site for transcription was mapped within this fragment and found to be identical in vitro and in vivo. A minor initiation site was located about 50 base pairs upstream from the major site. DNA sequencing of the HindIII-SalI fragment revealed the presence of two promoter-like structures within an extremely AT-rich region with transcriptional initiation sites at 30 and about 80 base pairs upstream from the initiation codon for the first structural gene. Numerous potential secondary structures were found in the DNA sequence around the major promoter. The major transcriptional start site was determined precisely by sequencing the 5' end of in vitro-transcribed RNA. The effect of iron on both the level of specific RNA, as determined by a quantitative S1 nuclease mapping assay, and on beta-galactosidase activity in a iucA'-'lacZ protein fusion, showed that the aerobactin operon is regulated at the transcriptional level. The iron-regulatory sequences are contained within a 152-base-pair Sau3A fragment of the promoter region.