A method for direct cloning of fur-regulated genes: identification of seven new fur-regulated loci in Escherichia coli.
A strain that allows the cloning of Fur-regulated loci was constructed. The strain, named FUR-SEL1, contains a chromosomal fhuA'-'cat transcriptional fusion that is expressed from the Fur-regulated promoter, fhuA(p). 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 fur mutation that ensures a more than 10(6)-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 Escherichia coli loci which have not previously been shown to be Fur regulated were found, including the pgmA and nrdHIEF genes, predicted ORF yhhY and four promoters identified first in this study. Three of the promoters preceded the nohA gene, and ORFs ygaC and yhhX. The fourth was located upstream of orf78 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.
Vassinova
Natalia
N
Department of Biophysics, St. Petersburg State Technical University, 29 Polytechnicheskaya Str, St. Petersburg 195251, Russia1.
Kozyrev
Dmitri
D
Department of Biophysics, St. Petersburg State Technical University, 29 Polytechnicheskaya Str, St. Petersburg 195251, Russia1.
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D007501:Iron / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C068477:ferric uptake regulating proteins, bacterial; D007501:Iron