title: Molecular characterization of eutF mutants of Salmonella typhimurium LT2 identifies eutF lesions as partial-loss-of-function tonB alleles.
abstract: The eutF locus of Salmonella typhimurium LT2 was identified as a locus necessary for the utilization of ethanolamine as a sole carbon source. Initial models suggested that EutF was involved in either ethanolamine transport or was a transcriptional regulator of an ethanolamine transporter. Phenotypic characterization of eutF mutants suggested EutF was somehow involved in 1,2-propanediol, propionate, and succinate utilization. Here we provide evidence that two alleles defining the eutF locus, Delta903 and eutF1115, are partial-loss-of-function tonB alleles. Both mutations were complemented by plasmids containing a wild-type allele of the Escherichia coli tonB gene. Immunoblot analysis using TonB monoclonal antibodies detected a TonB fusion protein in strains carrying eutF alleles. Molecular analysis of the Delta903 allele identified a deletion that resulted in the fusion of the 3' end of tonB with the 3' end of trpA. In-frame translation of the tonB-trpA fusion resulted in the final 9 amino acids of TonB being replaced by a 45-amino-acid addition. We isolated a derivative of a strain carrying allele Delta903 that regained the ability to grow on ethanolamine as a carbon and energy source. The molecular characterization of the mutation that corrected the Eut- phenotype caused by allele Delta903 showed that the new mutation was a deletion of two nucleotides at the tonB-trpA fusion site. This deletion resulted in a frameshift that replaced the 45-amino-acid addition with a 5-amino-acid addition. This change resulted in a TonB protein with sufficient activity to restore growth on ethanolamine and eut operon expression to nearly wild-type levels. It was concluded that the observed EutF phenotypes were due to the partial loss of TonB function, which is proposed to result in reduced cobalamin and ferric siderophore transport in an aerobic environment; thus, the eutF locus does not exist.
authors.affiliation: Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin 53706-1567, USA.
authors.forename: M G
authors.initials: MG
authors.lastname: Thomas
authors.forename: G A
authors.initials: GA
authors.lastname: O'Toole
authors.forename: J C
authors.initials: JC
authors.lastname: Escalante-Semerena
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001692:Biological Transport; D002874:Chromosome Mapping; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D019856:Ethanolamine / Q000378:metabolism; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D016364:Reading Frames; D012486:Salmonella typhimurium / Q000235:genetics* / Q000378:metabolism; D017384:Sequence Deletion; D014161:Transduction, Genetic
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; C057616:tonB protein, Bacteria; C489181:tonB protein, E coli; D019856:Ethanolamine