TONB: FOCAL PROTEIN FOR E. COLI IRON TRANSPORT

Project: Research project

Project Details

Description

Iron is an essential nutrient for the growth of Escherichia coli K12, and
the tonB gene is an essential component of all high affinity iron transport
systems in E.coli. The TonB protein has an apparent molecular weight of
36,000 daltons in denaturing polyacrylamide gels; it is located in the cell
envelope fraction; the details of its role in iron transport remain an
enigma. Recombinant plasmids carrying the tonB gene have been constructed
and the DNA sequence of the tonB gene determined. The tonB promoter and
transcription terminator have been deduced from S1-nuclease mapping of tonB
RNA. Preliminary experiments suggest that transcription of tonB is
elevated under conditions of iron limitation. The specific aims of this
proposal are (i) to purify the TonB protein to homogeneity, (ii) to
determine the NH2-terminal amino acid sequence of TonB protein, (iii) to
determine the biochemical half-life of TonB protein in wild-type and
protease deficient strains, (iv) to localize TonB protein more precisely
within the cell envelope, and (v) to determine the molecular mechanism(s)
by which tonB gene expression is regulated in response to iron limitation.
Purified TonB protein will be used to prepare anti-TonB antisera in order
to develop a quantitative assay for TonB protein in cell extracts.
Recombinant DNA techniques will be used for the construction of strains
that overproduce TonB protein, and for the construction of tonB-lacZ and
tonB-tetA transcriptional fusion strains in order to study tonB regulation
and to obtain tonB regulatory mutants. In vitro mutagenesis with sodium
bisulfite and DNA sequence analysis will be used to isolate and
characterize mutations in cis-acting regulatory elements. Transposon Tn5
mutagenesis will be used to isolate mutations in trans-acting regulatory
elements. RNA-DNA hybridization experiments will be performed in order to
determine levels of tonB RNA synthesis under different growth conditions.
The proposed research will provide insight into the regulation and
synthesis of bacterial envelope proteins, in general, and it will lead to a
better understanding of the role of the tonB gene product in envelope
functions and iron transport in particular. The growing awareness that the
course of bacterial infection is determined, in part, by the availability
of iron in the blood and in part by the ability of pathogenic
microorganisms to sequester iron lends medical relevance to this proposal.
StatusFinished
Effective start/end date1/1/8512/31/85

Funding

  • National Institute of Allergy and Infectious Diseases

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