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

Abstract

Halophilic Archaea are a distinctive pink color due to a carotenoid pigment called bacterioruberin. To sense or utilize light, many halophilic Archaea also produce rhodopsins, complexes of opsin proteins with a retinal prosthetic group. Both bacterioruberin and retinal are synthesized from isoprenoid precursors, with lycopene as the last shared intermediate. We previously described a regulatory mechanism by which Halobacterium salinarum bacterioopsin and Haloarcula vallismortis cruxopsin inhibit bacterioruberin synthesis catalyzed by lycopene elongase. In this work, we found that opsins in all three major Halobacteria clades inhibit bacterioruberin synthesis, suggesting that this regulatory mechanism existed in the common Halobacteria ancestor. Halophilic Archaea, which are generally heterotrophic and aerobic, likely evolved from an autotrophic, anaerobic methanogenic ancestor by acquiring many genes from Bacteria via lateral gene transfer. These bacterial "imports" include genes encoding opsins and lycopene elongases. To determine if opsins from Bacteria inhibit bacterioruberin synthesis, we tested bacterial opsins and found that an opsin from Curtobacterium, in the Actinobacteria phylum, inhibits bacterioruberin synthesis catalyzed by its own lycopene elongase, as well as that catalyzed by several archaeal enzymes. We also determined that the lycopene elongase from Halococcus salifodinae, a species from a family of Halobacteria lacking opsin homologs, retained the capacity to be inhibited by opsins. Together, our results indicate that opsin-mediated inhibition of bacterioruberin biosynthesis is a widely distributed mechanism found in both Archaea and Bacteria, possibly predating the divergence of the two domains. Further analysis may provide insight into the acquisition and evolution of the genes and their host species.IMPORTANCE All organisms use a variety of mechanisms to allocate limited resources to match their needs in their current environment. Here, we explore how halophilic microbes use a novel mechanism to allow efficient production of rhodopsin, a complex of an opsin protein and a retinal prosthetic group. We previously demonstrated that Halobacterium salinarum bacterioopsin directs available resources toward retinal by inhibiting synthesis of bacterioruberin, a molecule that shares precursors with retinal. In this work, we show that this mechanism can be carried out by proteins from halophilic Archaea that are not closely related to H. salinarum and those in at least one species of Bacteria Therefore, opsin-mediated inhibition of bacterioruberin synthesis may be a highly conserved, ancient regulatory mechanism.

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

FIG 1

(A) Carotenoid biosynthesis in halophilic Archaea. Enzyme names are indicated next to reactions catalyzed. A broken line indicates the regulatory mechanism of an opsin inhibiting the committed step in bacterioruberin synthesis. GGPP, geranylgeranyl pyrophosphate. (B) Experimental approach for testing opsin-mediated inhibition of Lye in H. volcanii. The H. volcanii lye locus is shown approximately to scale on the left and the carotenoid produced is indicated on the right. The lye gene encoding lycopene elongase is directly downstream and in a putative operon with crtI, the gene that encodes phytoene dehydrogenase. No bacterioruberin is produced in the H. volcanii Δlye strain (Fig. 2). lye homologs are incorporated into the lye locus, maintaining the integrity of crtI, and bacterioruberin synthesis is restored (Fig. 2). Opsins are then expressed by introducing pTA963 (34), harboring the gene encoding the opsin, and bacterioruberin production is quantified to determine if the opsin inhibits Lye activity.

FIG 2

Expression of Lye homologs in H. volcanii restores bacterioruberin production. (A) Photographs of representative colonies of H. volcanii strains with the native lye deleted (Δlye) or replaced with lye homologs from the indicated species. Colonies were prepared from cultures, grown for 4 days, and photographed as described in Materials and Methods. (B) Reverse-phase ultra-high-performance liquid chromatography (RP-UHPLC) traces of carotenoid extracts from H. volcanii Δlye, H. volcanii H1209 (parental strain), and strains expressing Lye homologs from the indicated species. 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 2 replicates for each strain, except for Halorubrum sp. A07HR67, where only 1 trace was available. Values indicate bacterioruberin as a molar percentage of total lycopene and bacterioruberin.

FIG 3

Expression of opsins inhibits Lye-catalyzed bacterioruberin production in Halobacteria species. Box plot (Tukey’s [37]) indicating proportionate bacterioruberin levels compared to those of the empty vector control in H. volcanii strains expressing Lye homologs from the indicated species. Bacterioruberin levels were determined by colony pigment analysis, as described previously (11). Heavy horizontal bars indicate the median value, and boxes demarcate the upper and lower quartiles. Whiskers extend to the smaller value of 1.5 times the interquartile range or to the most extreme value. Asterisks indicate a Bonferroni adjusted P value of <0.05 (n ≥ 6).

FIG 4

Expression of opsins in H. volcanii. H. volcanii strains harboring expression plasmids for indicated opsins with C-terminal His₆ tags were grown in cultures using the same procedures as that for carotenoid analysis. Cell lysates were normalized by total protein concentration and separated by polyacrylamide gel electrophoresis. Blots were probed with anti-His₆ primary antibody. For standardization across multiple blots, cell lysates from H. volcanii expressing H. salinarum BO were included on all blots. Subsequently, each immunoblot image was adjusted (as an entire image) so that the BO bands from all blots had identical net (band subtracting background) mean gray values. The proportion indicated on the image was determined by dividing the net mean gray value of each opsin band into the net mean gray value of H. salinarum BO. Bands shown are from different blots, as indicated by black lines, and each is representative of at least 3 biological replicates. All full immunoblots are shown in Fig. S1 in the supplemental materials.

FIG 5

A bacterial opsin inhibits its own Lye and several archaeal Lyes. Box plot indicating proportionate bacterioruberin levels of H. volcanii strains expressing the indicated Lye and, if noted, the Curtobacterium opsin homolog. Relative bacterioruberin levels were determined by colony color analysis, and data were plotted as described for Fig. 3. Asterisks indicate a Bonferroni adjusted P value of <0.05 compared to empty vector controls (n ≥ 6).

FIG 6

Lye from a Halobacteria species that lacks opsins is inhibited by H. salinarum BO. Box plot indicating proportionate bacterioruberin levels of H. volcanii strains expressing H. salifodinae Lye and the indicated opsin homologs. Relative bacterioruberin levels were determined by colony color analysis, and data were plotted as described for Fig. 3. The asterisk indicates a Bonferroni adjusted P value of <0.05 compared to empty vector controls (n ≥ 6).