Expression of the bacterioopsin gene in Halobacterium halobium using a multicopy plasmid

Area of Science:

• Molecular Biology
• Biophysics
• Microbiology

Background:

• Bacteriorhodopsin (bR) is a light-driven proton pump found in Halobacterium halobium.
• Genetic manipulation of bR expression is crucial for understanding its function.
• Previous studies focused on endogenous bR production.

Purpose of the Study:

• To develop a method for expressing bacteriorhodopsin (bR) in Halobacterium halobium using a multicopy plasmid.
• To characterize the functional and structural properties of bR produced via this method.
• To compare bR from transformed cells with wild-type bR.

Main Methods:

• Construction of a multicopy plasmid (pGRB1) containing the bop gene from H. halobium S9.
• Transformation of a bop- insertion mutant of H. halobium with the constructed plasmid.
• Analysis of bR expression levels, purple membrane properties (absorption, CD), and proton-pumping activity.

Main Results:

• Transformed H. halobium yielded purple colonies (Pum+ phenotype) due to plasmid-borne bop gene expression.
• bR levels in transformed cells reached 25-40% of wild-type levels, attributed to plasmid instability.
• Purified purple membrane exhibited similar absorption, CD properties, and bR structure to wild-type.

Conclusions:

• The multicopy plasmid successfully enabled bacteriorhodopsin expression in H. halobium.
• The expressed bR retained native structural and functional characteristics, including proton-pumping activity.
• This approach provides a valuable tool for studying bacteriorhodopsin in a controlled genetic background.