Sequences of the Escherichia coli BtuB protein essential for its insertion and function in the outer membrane.
The Escherichia coli btuB gene encodes the outer membrane transporter for vitamin B12, the E colicins, colicin A, and bacteriophage BF23. Several series of mutant forms of BtuB resulting from the insertion of dipeptide sequences and from overlapping in-frame deletions and duplications were constructed. Strains expressing the variant genes in single and multiple copy numbers were analyzed for BtuB function, for the level of BtuB polypeptide in the outer membrane, and for changes in the outer membrane permeability barrier. Most dipeptide insertions had normal transport function and assembly in the membrane. Only 2 of the 27 deletions spanning residues 5 and 514 possessed transport function, and most of the remainder were not stably inserted in the membrane. Most duplications (19 of 21) retained transport function and were inserted in the outer membrane, although some were subject to proteolysis. Even long duplications containing as many as 340 repeated amino-terminal residues retained function, suggesting considerable plasticity in the sequence requirements for membrane insertion of BtuB. Expression of many deletion and duplication proteins conferred increased susceptibility to structurally unrelated inhibitors that are normally excluded by the outer membrane. These results could be consistent with the mutational disruption of extracellular loops or transmembrane segments of BtuB that constitute a gated channel, but the finding that alterations throughout the length of BtuB affect membrane permeability properties suggests that the altered proteins might perturb the outer membrane structure itself.
Lathrop
J T
JT
Department of Microbiology, School of Medicine, University of Virginia, Charlottesville 22908, USA.
Wei
B Y
BY
Touchie
G A
GA
Kadner
R J
RJ
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000235:genetics / Q000502:physiology*; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics / Q000502:physiology*; D029968:Escherichia coli Proteins; D008565:Membrane Proteins / Q000378:metabolism; D026901:Membrane Transport Proteins; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016364:Reading Frames; D018000:Receptors, Peptide / Q000235:genetics / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D014805:Vitamin B 12 / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; C044592:BtuB protein, E coli; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D018000:Receptors, Peptide; C057616:tonB protein, Bacteria; C489181:tonB protein, E coli; D014805:Vitamin B 12