Opsin-Mediated Inhibition of Bacterioruberin Synthesis in Halophilic Archaea

Abstract

Halophilic archaea often inhabit environments with limited oxygen, and many produce ion-pumping rhodopsin complexes that allow them to maintain electrochemical gradients when aerobic respiration is inhibited. Rhodopsins require a protein, an opsin, and an organic cofactor, retinal. We previously demonstrated that in Halobacterium salinarum, bacterioopsin (BO), when not bound by retinal, inhibits the production of bacterioruberin, a biochemical pathway that shares intermediates with retinal biosynthesis. In this work, we used heterologous expression in a related halophilic archaeon, Haloferax volcanii, to demonstrate that BO is sufficient to inhibit bacterioruberin synthesis catalyzed by the H. salinarum lycopene elongase (Lye) enzyme. This inhibition was observed both in liquid culture and in a novel colorimetric assay to quantify bacterioruberin abundance based on the colony color. Addition of retinal to convert BO to the bacteriorhodopsin complex resulted in a partial rescue of bacterioruberin production. To explore if this regulatory mechanism occurs in other organisms, we expressed a Lye homolog and an opsin from Haloarcula vallismortis in H. volcanii. H. vallismortis cruxopsin-3 expression inhibited bacterioruberin synthesis catalyzed by H. vallismortis Lye but had no effect when bacterioruberin synthesis was catalyzed by H. salinarum or H. volcanii Lye. Conversely, H. salinarum BO did not inhibit H. vallismortis Lye activity. Together, our data suggest that opsin-mediated inhibition of Lye is potentially widespread and represents an elegant regulatory mechanism that allows organisms to efficiently utilize ion-pumping rhodopsins obtained through lateral gene transfer. IMPORTANCE Many enzymes are complexes of proteins and nonprotein organic molecules called cofactors. To ensure efficient formation of functional complexes, organisms must regulate the production of proteins and cofactors. To study this regulation, we used bacteriorhodopsin from the archaeon Halobacterium salinarum. Bacteriorhodopsin consists of the bacterioopsin protein and a retinal cofactor. In this article, we further characterize a novel regulatory mechanism in which bacterioopsin promotes retinal production by inhibiting a reaction that consumes lycopene, a retinal precursor. By expressing H. salinarum genes in a different organism, Haloferax volcanii, we demonstrated that bacterioopsin alone is sufficient for this inhibition. We also found that an opsin from Haloarcula vallismortis has inhibitory activity, suggesting that this regulatory mechanism might be found in other organisms.

Species Widely Distributed in Halophilic Archaea Exhibit Opsin-Mediated Inhibition of Bacterioruberin Biosynthesis

“…Expression of opsins often inhibits the Lyes of their respective species. We had previously determined that expression of H. salinarum BO in H. volcanii cells allowed effective inhibition of bacterioruberin synthesis catalyzed by H. salinarum Lye (11). To assess potential opsin-mediated inhibition of bacterioruberin synthesis across the Halobacteria class, we expressed opsins in the H. volcanii strains harboring the lye genes from their respective organisms.…”

“…To assess if inhibition was correlated to opsin abundance, we constructed opsin genes with C-terminal hexahistidine (His 6 ) tags and analyzed expression via immunoblotting. We had previously used a His 6 -tagged H. salinarum BO to assess expression (11), and this BO also inhibited bacterioruberin synthesis catalyzed by H. salinarum Lye (Fig. 4).…”

“…Plasmids all harbored an ampicillin resistance marker and were propagated in Escherichia coli DH5␣ cells (New England Biolabs, Ipswich, MA) in LB media with 100 g/ml ampicillin (Gold Biotechnology, St. Louis, MO). For integration into the H. volcanii lye locus, synthetic genes encoding Lye were amplified with appropriate primers (Table S5 in the supplemental material) and ligated into pRFP128 (11), an integrative plasmid derived from pTA131 (33), using NdeI and BglII sites.…”

“…For carotenoid analysis from liquid cultures, H. volcanii cultures were grown in Hv-YPC medium (33) supplemented with 40 g/ml thymidine. The carotenoids were extracted and analyzed by reverse-phase ultra-high-performance liquid chromatography, as previously described (11).…”

“…We previously described a mechanism of regulation in which Halobacterium salinarum bacterioopsin (BO), when not bound by retinal, inhibits bacterioruberin synthesis catalyzed by Lye (6). We subsequently identified a similar inhibition by the opsin of Haloarcula vallismortis (11). This inhibition likely ensures that lycopene is conserved for retinal biosynthesis when opsins are available.…”