Deletions or duplications in the BtuB protein affect its level in the outer membrane of Escherichia coli.
The Escherichia coli btuB product is an outer membrane protein that mediates the TonB-coupled active transport of cobalamins and the uptake of the E colicins and bacteriophage BF23. The roles of various segments of the BtuB protein in its function or cellular localization were investigated by analysis of several genetic constructs. Hybrid proteins in which various lengths from the amino terminus of BtuB were linked to alkaline phosphatase (btuB::phoA genes) were all secreted across the cytoplasmic membrane. The BtuB-PhoA proteins that carried up to 327 amino acids of BtuB appeared to reside in the periplasmic space, whereas hybrid proteins containing at least 399 amino acids of BtuB were associated with the outer membrane. Eleven in-frame internal deletion mutations that spanned more than half of the mature sequence were prepared by combining appropriate restriction fragments from btuB variants with 6-bp linker insertions. None of the deleted proteins was able to complement any BtuB functions, and only three of them were detectable in the outer membrane, suggesting that most of the deletions affected sequences needed for stable association with the outer membrane. Duplications covering the same portions of BtuB were prepared in the same manner. All of these partial duplication variants complemented all BtuB functions, although some gave substantially reduced levels of activity. These proteins were found in the outer membrane, although some were subject to proteolytic cleavage within or near the duplicated segment. These results indicate that the insertion of BtuB into the outer membrane requires the presence of several regions of teh BtuB protein and that the presence of extra or redundant segments of the protein can be tolerated during its insertion and function.
Köster
W
W
Department of Microbiology, School of Medicine, University of Virginia, Charlottesville 22908.
Gudmundsdottir
A
A
Lundrigan
M D
MD
Seiffert
A
A
Kadner
R J
RJ
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D001693:Biological Transport, Active; D002462:Cell Membrane / Q000378:metabolism; D002872:Chromosome Deletion; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins; D015183:Restriction Mapping; D013329:Structure-Activity Relationship; D014805:Vitamin B 12 / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D011993:Recombinant Fusion Proteins; D014805:Vitamin B 12