Molecular Microbiology (1990) 4(3), 427-437
Sequence of the fA?i/E outer-membrane receptor gene of Escherichia coii K12 and properties of mutants M. Sauer, K. Hantke and V. Braun* Mikrobioiogie Ii, Auf der Morgensteile 28, Universitat Tubingen, D-7400 Tubingen, FRG. Summary The fhuE gene of Escherichia coli codes for an outermembrane receptor protein required for the uptake of iron(tU) via coprogen, ferrioxamine B and rhodotorulic acid. The amino acid sequence, deduced from the nucleotide sequence, consisted of 729 residues. The mature form, composed of 693 residues, has a calculated molecular weight of 77453, which agrees with the molecular weight of 76000 determined by polyacrylamide gel electrophoresis. The FhuE protein contains four regions of homology with other TonBdependent receptors. A valine to proline exchange in the 'TonB box' absoMshed transport activity. Phenotypic revertants with substitutions of arginine, glutamine, or leucine at the valine position exhibited Increasing iron-coprogen transport rates. Point mutations resulting in the replacement of glycine (127) in the second homology region with either alanine, aspartate, valine, asparagine or histidine exhibited decreased transport rates (listed in descending order). A truncated FhuE protein lacking 24 amino acids at the C-terminal end was exported to the periptasm but failed to be inserted into the outer membrane.
the /V-terminus. This sequence is common to al! receptors involved in TonB-dependent uptake systems across the outer membrane (Sauer etai., 1987), and is also found in colicins whose uptake requires the TonB function (Roos et ai., 1989), We have also shown that the FhuE protein is synthesized with a rather long signal sequence of 36 amino acids, and is processed slowly, which is further delayed in truncated polypeptides derived from the fhuE gene carrying TntOOO insertions (Sauer e( at., 1987). In this paper we report the complete nucleotide sequence of fhuE and the derived amino acid sequence. Point mutations were introduced into the TonB box' region of the fhuE gene and into two additional regions which exhibit similar amino acid sequences among outermembrane receptor proteins {Lundrigan and Kadner, 1986; Braun e^ at,, 1987; Nau and Konisky, 1989). In addition, integration of a truncated FhuE protein into the outer membrane was studied. Strains containing TonB box' mutations affecting the flexibility of this region were transport-inactive. Mutations in the other two consensus sequences showed various transport defects. FhuE with a C-terminal 24-amino-acid deletion was exported across the cytoplasmic membrane but not properly integrated into the outer membrane.
Results Nucteotide sequence of the fhuE gene
Introduction Escherichia coii transports iron(lll) via the fungal siderophores coprogen, desferrioxamine B (Desferal) and rhodotorulic acid. All three ferric siderophores are bound to the outer-membrane FhuE receptor protein (Hantke, 1983), then transported across the outer membrane in a TonB, ExbBD and energy-dependent step, and subsequently taken up across the cytoplasmic membrane through the common transport system for ferric hydroxamates encoded by the fhuCDBgenes (Eick-Helmerich and Braun, 1989; Kdster and Braun, 1989; Schoffler and Braun, 1989). Previously, we published a partial sequence of the fhuE gene which contained the TonB box' sequence close to Received 21 August, 1989; revised 1 November. 1989. "For correspondence. Tel. (7071) 292096,
Previously, the fhuE-encoding region and the transcription polarity on plasmid pKH6 was determined by Tn 7000 transposon mutagenesis (Sauer ef al., 1987). Both strands of a 2.900 bp fragment were completely sequenced. It contained an open reading frame of 2187bp (Fig. 1). A possible promoter with a homology score (Mulligan ef ai., 1984) of 60.4 is indicated in Fig, 1. The ribosome binding site deviates in two out of six base pairs from the Shine-Dalgarno (SD) consensus sequence. The sequence from bp 294 to 312 overlapping the ' - 1 0 ' region, and the sequence from bp 316 to 334 conform in 13 and 12, respectively, out of 19 positions with the consensus sequence of the Fur iron repressor binding site (Fig. 2) (Calderwood and Mekalanos, 1988; deLorenzo ef al., 1987; Pressler et ai., 1988). A potential stem and loopforming sequence which could serve as a transcription termination site extends from bp 2611 to 2632 (Fig. 1).
428
M. Sauer, K. Hanfke and V. Braun
o « Oo * O -(^ O
Sequence of the fA?i/E outer-membrane receptor gene of Escherichia coii K12 and properties of mutants M. Sauer, K. Hantke and V. Braun* Mikrobioiogie Ii, Auf der Morgensteile 28, Universitat Tubingen, D-7400 Tubingen, FRG. Summary The fhuE gene of Escherichia coli codes for an outermembrane receptor protein required for the uptake of iron(tU) via coprogen, ferrioxamine B and rhodotorulic acid. The amino acid sequence, deduced from the nucleotide sequence, consisted of 729 residues. The mature form, composed of 693 residues, has a calculated molecular weight of 77453, which agrees with the molecular weight of 76000 determined by polyacrylamide gel electrophoresis. The FhuE protein contains four regions of homology with other TonBdependent receptors. A valine to proline exchange in the 'TonB box' absoMshed transport activity. Phenotypic revertants with substitutions of arginine, glutamine, or leucine at the valine position exhibited Increasing iron-coprogen transport rates. Point mutations resulting in the replacement of glycine (127) in the second homology region with either alanine, aspartate, valine, asparagine or histidine exhibited decreased transport rates (listed in descending order). A truncated FhuE protein lacking 24 amino acids at the C-terminal end was exported to the periptasm but failed to be inserted into the outer membrane.
the /V-terminus. This sequence is common to al! receptors involved in TonB-dependent uptake systems across the outer membrane (Sauer etai., 1987), and is also found in colicins whose uptake requires the TonB function (Roos et ai., 1989), We have also shown that the FhuE protein is synthesized with a rather long signal sequence of 36 amino acids, and is processed slowly, which is further delayed in truncated polypeptides derived from the fhuE gene carrying TntOOO insertions (Sauer e( at., 1987). In this paper we report the complete nucleotide sequence of fhuE and the derived amino acid sequence. Point mutations were introduced into the TonB box' region of the fhuE gene and into two additional regions which exhibit similar amino acid sequences among outermembrane receptor proteins {Lundrigan and Kadner, 1986; Braun e^ at,, 1987; Nau and Konisky, 1989). In addition, integration of a truncated FhuE protein into the outer membrane was studied. Strains containing TonB box' mutations affecting the flexibility of this region were transport-inactive. Mutations in the other two consensus sequences showed various transport defects. FhuE with a C-terminal 24-amino-acid deletion was exported across the cytoplasmic membrane but not properly integrated into the outer membrane.
Results Nucteotide sequence of the fhuE gene
Introduction Escherichia coii transports iron(lll) via the fungal siderophores coprogen, desferrioxamine B (Desferal) and rhodotorulic acid. All three ferric siderophores are bound to the outer-membrane FhuE receptor protein (Hantke, 1983), then transported across the outer membrane in a TonB, ExbBD and energy-dependent step, and subsequently taken up across the cytoplasmic membrane through the common transport system for ferric hydroxamates encoded by the fhuCDBgenes (Eick-Helmerich and Braun, 1989; Kdster and Braun, 1989; Schoffler and Braun, 1989). Previously, we published a partial sequence of the fhuE gene which contained the TonB box' sequence close to Received 21 August, 1989; revised 1 November. 1989. "For correspondence. Tel. (7071) 292096,
Previously, the fhuE-encoding region and the transcription polarity on plasmid pKH6 was determined by Tn 7000 transposon mutagenesis (Sauer ef al., 1987). Both strands of a 2.900 bp fragment were completely sequenced. It contained an open reading frame of 2187bp (Fig. 1). A possible promoter with a homology score (Mulligan ef ai., 1984) of 60.4 is indicated in Fig, 1. The ribosome binding site deviates in two out of six base pairs from the Shine-Dalgarno (SD) consensus sequence. The sequence from bp 294 to 312 overlapping the ' - 1 0 ' region, and the sequence from bp 316 to 334 conform in 13 and 12, respectively, out of 19 positions with the consensus sequence of the Fur iron repressor binding site (Fig. 2) (Calderwood and Mekalanos, 1988; deLorenzo ef al., 1987; Pressler et ai., 1988). A potential stem and loopforming sequence which could serve as a transcription termination site extends from bp 2611 to 2632 (Fig. 1).
428
M. Sauer, K. Hanfke and V. Braun
o « Oo * O -(^ O
