Expression of the bacterioopsin gene in Halobacterium halobium using a multicopy plasmid.
Bacteriorhodopsin (bR) was expressed in Halobacterium halobium by using a multicopy plasmid containing the bop gene. The plasmid contains pGRB1, a 1.8-kilobase-pair plasmid; a 70-base-pair fragment from ISH11, a recently characterized insertion sequence; and a 1.6-kilobase-pair fragment carrying the bop gene from H. halobium S9. When transformed with this plasmid, a bop- insertion mutant of H. halobium yielded purple (Pum+) colonies. The insertion at the chromosomal bop locus remained intact in transformed cells, indicating that the plasmid bop gene was responsible for the Pum+ phenotype. bR was induced in early stationary phase in both wild-type and transformed cells. The final level of bR in transformed cells was 25-40% of that in wild type. The lower level of expression was presumably due to plasmid instability. Purple membrane purified from transformed strains had absorption and visible CD properties similar to wild type and contained bR in a hexagonal lattice with the same unit-cell dimension as wild type. The structure of bR from wild-type and transformed strains was identical at a resolution of 7.2 A. When reconstituted into vesicles, the purple membrane from wild-type and transformed strains showed similar light-dependent proton-pumping activity.
Krebs
M P
MP
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Hauss
T
T
Heyn
M P
MP
RajBhandary
U L
UL
Khorana
H G
HG
D001436:Bacteriorhodopsins / Q000235:genetics* / Q000378:metabolism / Q000528:radiation effects; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D003624:Darkness; D015870:Gene Expression; D005798:Genes, Bacterial; D006217:Halobacterium / Q000235:genetics*; D007700:Kinetics; D008027:Light; D008969:Molecular Sequence Data; D010957:Plasmids; D015183:Restriction Mapping; D014169:Transformation, Bacterial
D004269:DNA, Bacterial; D001436:Bacteriorhodopsins