Bacterio-opsin is the sole protein found in the purple membrane of the halophilic archaebacterium, Halobacterium halobium. The complex of this protein with the chromophore, retinal, constitutes bacteriorhodopsin (bR) which functions as a light-driven proton pump to generate energy for the cell. Purple membrane synthesis is regulated by environmental factors such as light intensity and oxygen tension. Our discovery of two genes (brp and bac) associated with the expression of the bacterio-opsin (bop) gene has provided the first insight into a possible regulatory mechanism. The long term objectives of this proposal are: (i) to understand the regulation of bop gene expression in H. halobium, and (ii) to elucidate the function of bR at the structural level.
One aim i s to determine the functions of a cluster of genes flanking the bop gene and to define the role of light and oxygen in the regulation of these genes. Gene expression will be assayed under a variety of culture conditions which suppress or enhance purple membrane synthesis. Determination of mRNA levels expressed from these genes along with characterization of their protein products will assist in defining gene functions and regulatory mechanisms. Insights into the regulation of gene expression in the halophilic archaebacteria and the evolution of gene structure and function in all three cellular kingdoms will likely be revealed.
A second aim i s to establish a DNA transformation system for H. halobium. Halocin resistance and the bop gene will be developed for use as genetic markers. Shuttle vectors will be constructed and used as carriers for these markers and/or for genomic libraries from antibiotic or halocin resistant mutants. Once available, such a system can be used: (i) to augment investigations of the regulation of bop gene expression by complementation analysis and gene fusion, and (ii) to express site-directed mutations of the bop gene in the native organism.
A third aim i s to determine the specific amino acids of bacteriorhodopsin which are involved in transmembrane proton translocation and cation binding. Many individual site-directed mutations of the bop gene will be generated and expressed in E. coli until a DNA transformation system is available for H. halobium. Our collaborators, Prof. R. Stroud (UCSF) and Prof. D. Kliger (UCSC), will assess the function and structure of mutant bop proteins. Results obtained will contribute to an understanding of the basic processes of light/energy transduction and membrane biology.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM031785-11
Application #
3280106
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1983-06-01
Project End
1994-05-31
Budget Start
1993-06-01
Budget End
1994-05-31
Support Year
11
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of California San Francisco
Department
Type
Schools of Medicine
DUNS #
073133571
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Turner, G J; Reusch, R; Winter-Vann, A M et al. (1999) Heterologous gene expression in a membrane-protein-specific system. Protein Expr Purif 17:312-23
Turner, G J; Miercke, L J; Mitra, A K et al. (1999) Expression, purification, and structural characterization of the bacteriorhodopsin-aspartyl transcarbamylase fusion protein. Protein Expr Purif 17:324-38
Gropp, F; Gropp, R; Betlach, M C (1995) Effects of upstream deletions on light- and oxygen-regulated bacterio-opsin gene expression in Halobacterium halobium. Mol Microbiol 16:357-64
Shand, R F; Betlach, M C (1994) bop gene cluster expression in bacteriorhodopsin-overproducing mutants of Halobacterium halobium. J Bacteriol 176:1655-60
Turner, G J; Miercke, L J; Thorgeirsson, T E et al. (1993) Bacteriorhodopsin D85N: three spectroscopic species in equilibrium. Biochemistry 32:1332-7
Mitra, A K; Miercke, L J; Turner, G J et al. (1993) Two-dimensional crystallization of Escherichia coli-expressed bacteriorhodopsin and its D96N variant: high resolution structural studies in projection. Biophys J 65:1295-306
Gropp, R; Gropp, F; Betlach, M C (1992) Association of the halobacterial 7S RNA to the polysome correlates with expression of the membrane protein bacterioopsin. Proc Natl Acad Sci U S A 89:1204-8
Shand, R F; Betlach, M C (1991) Expression of the bop gene cluster of Halobacterium halobium is induced by low oxygen tension and by light. J Bacteriol 173:4692-9
Thorgeirsson, T E; Milder, S J; Miercke, L J et al. (1991) Effects of Asp-96----Asn, Asp-85----Asn, and Arg-82----Gln single-site substitutions on the photocycle of bacteriorhodopsin. Biochemistry 30:9133-42
Shand, R F; Miercke, L J; Mitra, A K et al. (1991) Wild-type and mutant bacterioopsins D85N, D96N, and R82Q: high-level expression in Escherichia coli. Biochemistry 30:3082-8

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