1887

Abstract

A strain that allows the cloning of Fur-regulated loci was constructed. The strain, named FUR-SEL1, contains a chromosomal ′–′ transcriptional fusion that is expressed from the Fur-regulated promoter, . Therefore, Fur boxes introduced on a multicopy plasmid can cause derepression of the fusion by titrating the Fur repressor and thereby confer chloramphenicol resistance, which can be used as a selectable phenotype for cloning Fur-regulated loci. However, a number of additional mutations had to be introduced before FUR-SEL1 could be used for cloning Fur-regulated genes. The principal approach consisted of introducing a leaky mutation that ensures a more than 10-fold increase in chloramphenicol resistance for FUR-SEL1 transformants carrying FUR-box-containing plasmids. To verify that the cloning procedure selects Fur-regulated genes, 10 recombinant plasmids chosen at random among the ones selected with FUR-SEL1 were analysed by FURTA (Fur-titration assay), a method for identification of Fur-regulated genes. In addition, the nucleotide sequences of their chromosomal inserts were determined. Besides known Fur-regulated genes, seven loci which have not previously been shown to be Fur regulated were found, including the and genes, predicted ORF and four promoters identified first in this study. Three of the promoters preceded the gene, and ORFs and . The fourth was located upstream of predicted in this work. The regulation of the promoter activities by iron and the involvement of Fur in this regulation were shown. Employing FUR-SEL1 for cloning Fur-regulated loci from other enterobacteria is discussed.

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2000-12-01
2020-01-21
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