Heterologous expression of genes encoding bacterial light-harvesting complexes in Rhodobacter sphaeroides.
One of the major problems in structural work on membrane-spanning proteins is the identification of an expression system which will allow the production of enough pure protein for structural studies; an inadequate expression system can lead, for example, to the formation of unwanted protein inclusion bodies. In the present work we report the expression of genes encoding the light-harvesting 2 (LH2) membrane-spanning proteins from a number of species of purple bacteria in mutants of Rhodobacter sphaeroides that lack the native LH2 antenna. The LH2 structural genes (pucBA) from the photosynthetic bacteria Rhodopseudomonas acidophila and Rubrivivax gelatinosus were amplified and tailed by polymerase chain reaction, and cloned into an LH2 expression vector, which was then introduced into three LH2-minus Rb. sphaeroides mutants; DBC omega/G5 and DD13 (DD13/G1); the resulting transconjugant strains synthesized LH2 complexes that were examined using absorption and fluorescence spectroscopy, and Western blotting. Thus, we have created a heterologous expression system which supports the assembly of a functional "foreign" light-harvesting complex. This work opens up the possibility of creating site-directed LH2 mutants from bacteria for which no genetic system is available; this is particularly significant in the case of Rps. acidophila, since this bacterium has been the source of the LH2 complex that has recently been structurally resolved to atomic resolution.
Fowler
G J
GJ
Department of Molecular Biology and Biotechnology, University of Sheffield, United Kingdom.
Gardiner
A T
AT
Mackenzie
R C
RC
Barratt
S J
SJ
Simmons
A E
AE
Westerhuis
W H
WH
Cogdell
R J
RJ
Hunter
C N
CN
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004735:Energy Transfer; D015870:Gene Expression; D005798:Genes, Bacterial; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics* / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D012241:Rhodopseudomonas / Q000235:genetics / Q000378:metabolism; D012244:Rhodospirillaceae / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D045322:Photosynthetic Reaction Center Complex Proteins