Protein fusions of beta-galactosidase to the ferrichrome-iron receptor of Escherichia coli K-12
Open Access
- 31 December 1985
- journal article
- research article
- Published by American Society for Microbiology in Journal of Bacteriology
- Vol. 165 (1), 181-192
- https://doi.org/10.1128/jb.165.1.181-192.1986
Abstract
The fusion-generating phage lambda plac Mu1 was used to produce fusions of lacZ to fhuA, the gene encoding the ferrichrome-iron receptor (FhuA protein) in the outer membrane of Escherichia coli K-12. Fusions to the fhuA gene in a delta (lac) strain were selected by their resistance to bacteriophage phi 80 vir. Ten independent (fhuA'-'lacZ) fusions were all Lac+ and were resistant to the lethal agents which require the FhuA protein as receptor, i.e., phi 80 vir, T5, T1, UC-1, and colicin M; none could utilize ferrichrome as the sole iron source. Specialized transducing phages were obtained by illegitimate excision from the chromosome of each of the fusion-bearing strains, and EcoRI fragments which encoded the fusions were subcloned into the high-copy plasmid pMLB524. Physical mapping of the fusion-containing plasmids confirmed the presence of three restriction sites which were also located on the chromosomal DNA of sequences near the fhuA gene. The direction of transcription of the fhuA gene was deduced from the direction of transcription of the (fhuA'-'lacZ) gene fusion. Identification of the chimeric proteins was made by both radiolabeling cells and immunoprecipitating the LacZ-containing proteins with antibody to beta-galactosidase and by preparing whole cell extracts from Lac+ cells containing the cloned gene fusions. Two sizes of (FhuA'-'LacZ) proteins were detected, 121 kDa and 124 kDa. The DNA sequences at the unique fusion joints were determined. The sequence information allowed us to identify three distinct fusion joints which were grouped as follows, type I fusions, 5'-ACT GCT CAG CCA A-3'; type IIa fusions, 5'-GCG GTT GAA CCG A-3'; and type IIb fusions: 5'-ACC GCT GCA CCT G-3'. To orient these fhuA fusion joints, the complete nucleotide sequence of the fhuA gene was determined from a 2,902-base-pair fragment of DNA. A single open reading frame was found which translated into a 747-amino acid polypeptide. The signal sequence of 33 amino acids was followed by a mature protein with a molecular weight of 78,992. Alignment of the amino acid sequence of the FhuA protein with the amino acid sequences presented for two other tonB-dependent receptor proteins in the outer membrane of E. coli showed an area of local homology at the amino terminus of all three proteins.This publication has 51 references indexed in Scilit:
- The nucleotide sequences of the ponA and ponB genes encoding penicillin‐binding proteins 1A and 1B of Escherichia coli K12European Journal of Biochemistry, 1985
- Characterization of the pColV-K30 encoded cloacin DF13/aerobactin outer membrane receptor protein ofEscherichia coli; isolation and purification of the protein and analysis of its nucleotide sequence and primary structureFEMS Microbiology Letters, 1985
- Molecular architecture and functioning of the outer membrane of Escherichia coli and other gram-negative bacteriaBiochimica et Biophysica Acta (BBA) - Reviews on Biomembranes, 1983
- The ferrichrome-iron receptor of Escherichia coli K-12 antigenicity of the fhuA proteinBiochimica et Biophysica Acta (BBA) - General Subjects, 1982
- A simple method for displaying the hydropathic character of a proteinJournal of Molecular Biology, 1982
- Sequence information within the lamB gene is required for proper routing of the bacteriophage λ receptor protein to the outer membrane of Escherichia coli K-12Journal of Molecular Biology, 1982
- The importance of theexbBgene for vitamin B12 and ferric iron transportFEMS Microbiology Letters, 1981
- The ferrichrome receptor protein (tonA) ofEscherichia coliis synthesised as a precursor in vitroFEBS Letters, 1981
- E. coli RNA polymerase interacts homologously with two different promotersCell, 1980
- Electrophoretic resolution of the ‘major outer membrane protein’ of Escherichia coli K12 into four bandsFEBS Letters, 1975