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. volcaniiH. 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.

Keywords: C50 carotenoid; UbiA prenyltransferase; carotenoid biosynthesis; cofactor biosynthesis; membrane protein biogenesis; microbial rhodopsin; proteorhodopsin.

FIG 1

Lycopene is the last shared intermediate in retinal and bacterioruberin biosynthesis. CrtY, the lycopene cyclase that catalyzes the conversion of lycopene to β-carotene that is subsequently cleaved to form retinal. Lye, lycopene elongase that catalyzes the prenylation of lycopene to the first bacterioruberin intermediate. Broken line, the regulatory mechanism of BO inhibition of Lye activity.

FIG 2

The H. volcanii lye gene is required for bacterioruberin synthesis. RP-UHPLC traces of carotenoid extracts from H. volcanii Δlye, H. volcanii H1209 (parental strain), and strains harboring plasmids expressing lye from H. volcanii, H. salinarum, and H. vallismortis as labeled. Positions of bacterioruberin and lycopene (lyc) standards are noted. Traces were normalized for total carotenoid concentration, corrected for slight differences in retention time using an internal standard, and offset along the vertical axis for clarity. Traces are representative of at least 3 replicates for each strain.

FIG 3

Expression of BO in H. volcanii specifically inhibits H. salinarum Lye. (A) Box plot (Tukey's [43, 44]) indicating bacterioruberin levels as determined by RP-UHPLC analysis of H. volcanii cultures with H. volcanii or H. salinarum lye. Strains harbored a BO expression vector or empty vector (control) as indicated. Where noted, retinal was added during the growth of the culture. Carotenoid levels were quantified by RP-UHPLC as described in Materials and Methods. Heavy horizontal bars indicate the median values, and boxes demarcate the upper and lower quartiles. Whiskers extend to the smaller value of 1.5 times the interquartile range or the most extreme value, n ≥ 3. (B) Photographs of representative colonies of H. volcanii strains with native lye replaced with H. salinarum lye. Empty vector control (left), strain with plasmid allowing BO expression (middle), strain with plasmid allowing BO expression with retinal added to the plate to convert BO to BR (right). (C) Box plot indicating bacterioruberin levels (ruberin metric) as determined by analysis of colony color. H. volcanii cultures were plated on Hv-YPC medium supplemented with retinal as noted. Four-day-old colonies were photographed and digital images were analyzed as described in Materials and Methods. Heavy horizontal bars indicate the median values, and boxes demarcate the upper and lower quartiles. Whiskers extend to the smaller value of 1.5 times the interquartile range or the most extreme value, n ≥ 9. Asterisks indicate Bonferroni adjusted P of <0.05. NS, not significantly different.

FIG 4

A specific region within H. salinarum Lye is necessary for BO-mediated regulation. (A) Protein alignment map of H. salinarum and H. volcanii Lye homologs and hybrids. The x axis represents the consensus alignment amino acid position, and the vertical lines within the protein sequences represent gaps in the alignment. Dashed lines delineate the H. salinarum Lye region required for inhibition by BO. (B) Box plot indicating bacterioruberin levels of strains expressing different versions of lye in the absence or presence of BO. Images were obtained and analyzed, and data were plotted as described for Fig. 3. n ≥ 9. Asterisks indicate Bonferroni adjusted P of <0.05. NS, not significantly different. (C) Template-based modeling of H. salinarum and H. volcanii Lye proteins. Structure predictions of H. salinarum (left) and H. volcanii (right) Lye. Structures are oriented so that the cytoplasm (in) is at the bottom and the extracellular environment (out) is at the top of the image. The regions highlighted in blue (H. salinarum) and cyan (H. volcanii) represent the identified 52-aa region required for inhibition by BO. The structural models for the two Lye homologs were generated using PHYRE2 (20) and superimposed using SuperPose (45). The structure files were highlighted and exported to image files using Geneious version 7 (46).

FIG 5

BO lacking covalent retinal binding inhibits Lye; H. salinarum sensory opsin II does not inhibit Lye. (A) Box plot indicating bacterioruberin levels of H. volcanii strains with H. salinarum lye expressing BO, BO K216A, or SOII in the absence or presence of retinal. Images were obtained and analyzed, and data were plotted as described for Fig. 3. n ≥ 9. Asterisk indicates Bonferroni adjusted P of <0.05. NS, not significantly different. (B) Quantification of H. salinarum BO and SOII and H. vallismortis CO expression. H. volcanii strains harboring expression plasmids for indicated opsins with C-terminal His₆ tags were grown in cultures with the same procedures used for carotenoid analysis. Cell lysates were normalized using total protein concentration and separated by polyacrylamide gel electrophoresis. Blots were probed with anti-His antibody and bands were quantified by densitometry. Median values compared to BO median set to 1. Error bars indicate 1 standard deviation, n = 3. Inset, representative immunoblot of His-tagged BO, SOII, and CO.

FIG 6

H. vallismortis Lye is specifically inhibited by H. vallismortis CO. Box plot indicating bacterioruberin levels of H. volcanii strains with H. volcanii, H. salinarum, or H. vallismortis lye and the indicated opsin in the absence or presence of retinal. Images were obtained and analyzed, and data were plotted as described for Fig. 3. n ≥ 9. Asterisks indicate Bonferroni adjusted P of <0.05. NS, not significantly different.