Carotenoid pigment of Halophilic archaeon Haloarcula sp. A15 induces apoptosis of breast cancer cells.

The halophilic microorganisms living in extreme environments contain high concentrations of carotenoids with notable medical abilities. The purpose of this study was to evaluate the anticancer effect of carotenoids extracted from native Iranian halophilic microorganisms with the ability to inhibit breast cancer cell line. To begin the study, 40 halophilic strains were cultured, and 8 strains capable of producing pigmented colonies were chosen from those cultured strains. In the next step, from among 8 strains using MTT assay, 1 capable of reducing cell viability of the breast cancer MCF-7 cell line was chosen as a selective strain. The principal carotenoid was characterized using UV-visible, FT-IR spectroscopic, and LC-MASS analyses. Using real time PCR technique, the expression of genes specific for apoptosis, in the presence or absence of carotenoid, was examined. Among all strains, carotenoid extracted from strain A15 had the most potent cytotoxic effect on breast cancer cell line (IC50 = 0.0645 mg/mL). 16S rRNA gene analysis showed that strain A15 had similarity with Haloarcula hispanica for about 99.5%. According to the analysis results, it could be estimated that the principal carotenoid extracted form Haloarcula sp. A15 was similar to bacterioruberin. Both early and late apoptosis were increased significantly about 10% and 39%, respectively, due to upregulation of CASP3, CASP8, BAX genes expression in MCF-7 cell line. In contrast, the expression of genes MKI67, SOX2 were significantly downregulated in treated MCF-7 cell line. The results of this study showed that Halophilic archaeon strain could be a good candidate for the production of high added-value bacterioruberin due to its possible anticancer properties.

Divergent degeneration of creA antitoxin genes from minimal CRISPRs and the convergent strategy of tRNA-sequestering CreT toxins.

Aside from providing adaptive immunity, type I CRISPR-Cas was recently unearthed to employ a noncanonical RNA guide (CreA) to transcriptionally repress an RNA toxin (CreT). Here, we report that, for most archaeal and bacterial CreTA modules, the creA gene actually carries two flanking 'CRISPR repeats', which are, however, highly divergent and degenerated. By deep sequencing, we show that the two repeats give rise to an 8-nt 5' handle and a 22-nt 3' handle, respectively, i.e., the conserved elements of a canonical CRISPR RNA, indicating they both retained critical nucleotides for Cas6 processing during divergent degeneration. We also uncovered a minimal CreT toxin that sequesters the rare transfer RNA for isoleucine, tRNAIleCAU, with a six-codon open reading frame containing two consecutive AUA codons. To fully relieve its toxicity, both tRNAIleCAU overexpression and supply of extra agmatine (modifies the wobble base of tRNAIleCAU to decipher AUA codons) are required. By replacing AUA to AGA/AGG codons, we reprogrammed this toxin to sequester rare arginine tRNAs. These data provide essential information on CreTA origin and for future CreTA prediction, and enrich the knowledge of tRNA-sequestering small RNAs that are employed by CRISPR-Cas to get addictive to the host.

Toxin-antitoxin RNA pairs safeguard CRISPR-Cas systems.

CRISPR-Cas systems provide RNA-guided adaptive immunity in prokaryotes. We report that the multisubunit CRISPR effector Cascade transcriptionally regulates a toxin-antitoxin RNA pair, CreTA. CreT (Cascade-repressed toxin) is a bacteriostatic RNA that sequesters the rare arginine tRNAUCU (transfer RNA with anticodon UCU). CreA is a CRISPR RNA-resembling antitoxin RNA, which requires Cas6 for maturation. The partial complementarity between CreA and the creT promoter directs Cascade to repress toxin transcription. Thus, CreA becomes antitoxic only in the presence of Cascade. In CreTA-deleted cells, cascade genes become susceptible to disruption by transposable elements. We uncover several CreTA analogs associated with diverse archaeal and bacterial CRISPR-cas loci. Thus, toxin-antitoxin RNA pairs can safeguard CRISPR immunity by making cells addicted to CRISPR-Cas, which highlights the multifunctionality of Cas proteins and the intricate mechanisms of CRISPR-Cas regulation.

Characterization of a GlgC homolog from extremely halophilic archaeon Haloarcula japonica.

Glycogen synthesis in bacteria is mainly organized by the products of glgB, glgC, and glgA genes comprising the widely known glg operon. On the genome of extremely halophilic archaeon Haloarcula japonica, there was a gene cluster analogous to the bacterial glg operon. In this study, we focused on a GlgC homolog of Ha. japonica, and its recombinant enzyme was prepared and characterized. The enzyme showed highest activity toward GTP and glucose-1-phosphate as substrates in the presence of 2.6 m KCl and predicted to be work as "GDP-glucose pyrophosphorylase" in Ha. japonica.

Agl22 and Agl23 are involved in the synthesis and utilization of the lipid-linked intermediates in the glycosylation pathways of the halophilic archaeaon Haloarcula hispanica.

Like both eukaryotes and bacteria, archaea can decorate proteins with N- and O-linked glycans. Whereas pathways and roles of N-glycosylation have been studied in several model archaeal organisms, little is known of O-glycosylation. To explore commonalities and variations of these two versions of glycosylation, we used Haloarcula hispanica as a model. Our previous work showed that H. hispanica S-layer glycoproteins are modified by an N-linked glucose-α-(1, 2)-[sulfoquinovosamine-ß-(1, 6)-]galactose trisaccharide and an O-linked glucose-α-(1, 4)-galactose disaccharide. Here, we found that H. hispanica membrane contains C60 dolichol phosphate (DolP) as a lipid carrier for glycosylation. As revealed by bioinformatics, gene deletion and phenotype analysis, gene HAH_1571, renamed agl22, encodes a predicted glucosyltransferase that transfers glucose from glucose-DolP onto galactose-DolP to form the glucose-α-(1, 4)-galactose-DolP precursor of the N-glycosylation. Gene HAH_2016, renamed agl23, encodes a putative flippase-associated protein responsible for flipping of hexose-DolPs across the membrane to face the exterior. Our results also suggested that the synthesis of the N- and O-linked glycans onto target protein occurs on the outer surface of the cell using hexose-DolPs as sugar donors. Deletion mutant showed that N- and O-glycosylation are required for growth in the defined medium mimicking the natural habitat of H. hispanica.

CTP synthase forms cytoophidia in archaea.

CTP synthase (CTPS) is an important metabolic enzyme that catalyzes the rate-limiting reaction of nucleotide CTP de novo synthesis. Since 2010, a series of studies have demonstrated that CTPS can form filamentous structures in bacteria and eukaryotes, which are termed cytoophidia. However, it is unknown whether cytoophidia exist in the third domain of life, archaea. Using Haloarcula hispanica as a model system, here we demonstrate that CTPS forms distinct intracellular compartments in archaea. Under stimulated emission depletion microscopy, we find that the structures of H. hispanica CTPS are elongated, similar to cytoophidia in bacteria and eukaryotes. When Haloarcula cells are cultured in low-salt medium, the occurrence of cytoophidia increases dramatically. In addition, treatment of H. hispanica with a glutamine analog or overexpression of CTPS can promote cytoophidium assembly. Our study reveals that CTPS can form cytoophidia in all three domains of life, suggesting that forming cytoophidia is an ancient property of CTPS.

Insights into gene regulation of the halovirus His2 infecting Haloarcula hispanica.

Gene expression in Haloarcula hispanica cells infected with the gammapleolipovirus His2 was studied using a custom DNA microarray. Total RNA from cells sampled at 0, 1, 2, 3, and 4.5 hr postinfection was reverse-transcribed into labeled cDNA and hybridized to microarrays, revealing temporal and differential expression in both host and viral genes. His2 gene expression occurred in three main phases (early, middle, and late), and by 4.5 hr p.i. the majority of genes were actively transcribed, including those encoding the major structural proteins. Eighty host genes were differentially regulated ≥twofold postinfection, with most of them predicted to be involved in transport, translation, and metabolism. Differentially expressed host genes could also be grouped into early-, middle-, and late-expressed genes based on the timing of their up- and downregulation postinfection. The altered host transcriptional pattern suggests regulation by His2 infection, which may reprogram host metabolism to facilitate its own DNA replication and propagation. This study enhances the characterization of many hypothetical viral genes and provides insights into the interaction between His2 and its host.

Primed adaptation tolerates extensive structural and size variations of the CRISPR RNA guide in Haloarcula hispanica.

Recent studies on CRISPR adaptation revealed that priming is a major pathway of spacer acquisition, at least for the most prevalent type I systems. Priming is guided by a CRISPR RNA which fully/partially matches the invader DNA, but the plasticity of this RNA guide has not yet been characterized. In this study, we extensively modified the two conserved handles of a priming crRNA in Haloarcula hispanica, and altered the size of its central spacer part. Interestingly, priming is insusceptible to the full deletion of 3' handle, which seriously impaired crRNA stability and interference effects. With 3' handle deletion, further truncation of 5' handle revealed that its spacer-proximal 6 nucleotides could provide the least conserved sequence required for priming. Subsequent scanning mutation further identified critical nucleotides within 5' handle. Besides, priming was also shown to tolerate a wider size variation of the spacer part, compared to interference. These data collectively illustrate the high tolerance of priming to extensive structural/size variations of the crRNA guide, which highlights the structural flexibility of the crRNA-effector ribonucleoprotein complex. The observed high priming effectiveness suggests that primed adaptation promotes clearance of the fast-replicating and ever-evolving viral DNA, by rapidly and persistently multiplexing the interference pathway.

Discovery of bacteriorhodopsins in Haloarchaeal species isolated from Indian solar salterns: deciphering the role of the N-terminal residues in protein folding and functional expression.

Interesting optical and photochemical properties make microbial rhodopsin a promising biological material suitable for various applications, but the cost-prohibitive nature of production has limited its commercialization. The aim of this study was to explore the natural biodiversity of Indian solar salterns to isolate natural bacteriorhodopsin (BR) variants that can be functionally expressed in Escherichia coli. In this study, we report the isolation, functional expression and purification of BRs from three pigmented haloarchaea, wsp3 (water sample Pondicherry), wsp5 and K1T isolated from two Indian solar salterns. The results of the 16S rRNA data analysis suggest that wsp3, wsp5 and K1T are novel strains belonging to the genera Halogeometricum, Haloferax and Haloarcula respectively. Overall, the results of our study suggest that 17 N-terminal residues, that were not included in the gene annotation of the close sequence homologues, are essential for functional expression of BRs. The primary sequence, secondary structural content, thermal stability and absorbance spectral properties of these recombinant BRs are similar to those of the previously reported Haloarcula marismortui HmBRI. This study demonstrates the cost-effective, functional expression of BRs isolated from haloarchaeal species using E. coli as an expression host and paves the way for feasibility studies for future applications.

Temperature-dependent expression of different guanine-plus-cytosine content 16S rRNA genes in Haloarcula strains of the class Halobacteria.

Haloarcula strains, which are halophilic archaea, harbour two to three copies of 16S rRNA genes (rrsA, rrsB and rrsC) in their genomes. While rrsB and rrsC (rrsBC) show almost identical sequences, rrsA shows 4-6% sequence difference and 1-3% guanine-plus-cytosine content (PGC) difference compared to rrsBC. Based on the strong correlation between the PGC of 16S rRNA genes and the growth temperatures of the prokaryotes, we hypothesised that high-PGCrrsA and low-PGCrrsBC are expressed at high and low temperatures, respectively. To verify the hypothesis, we performed sequence analyses and expression surveys of each 16S rRNA gene in eight Haloarcula strains. The secondary structure prediction of the 16S rRNA via computer simulation showed that the structural stability of 16S rRNAs transcribed from rrsA was higher than that of 16S rRNAs transcribed from rrsBC. We measured expression levels of rrsA and rrsBC under various temperature conditions by reverse-transcriptase quantitative PCR. The expression ratio of high-PGCrrsA to low-PGCrrsBC increased with cultivation temperatures in seven of eight Haloarcula strains. Our results suggest that the transcription of high-PGCrrsA and low-PGCrrsBC may be regulated in response to environmental temperature, and that 16S rRNAs transcribed from high-PGCrrsA function under high temperature conditions close to the maximum growth temperature.

Harnessing the native type I-B CRISPR-Cas for genome editing in a polyploid archaeon.

Research on CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated protein) systems has led to the revolutionary CRISPR/Cas9 genome editing technique. However, for most archaea and half of bacteria, exploitation of their native CRISPR-Cas machineries may be more straightforward and convenient. In this study, we harnessed the native type I-B CRISPR-Cas system for precise genome editing in the polyploid haloarchaeon Haloarcula hispanica. After testing different designs, the editing tool was optimized to be a single plasmid that carries both the self-targeting mini-CRISPR and a 600-800 bp donor. Significantly, chromosomal modifications, such as gene deletion, gene tagging or single nucleotide substitution, were precisely introduced into the vast majority of the transformants. Moreover, we showed that simultaneous editing of two genomic loci could also be readily achieved by one step. In summary, our data demonstrate that the haloarchaeal CRISPR-Cas system can be harnessed for genome editing in this polyploid archaeon, and highlight the convenience and efficiency of the native CRISPR-based genome editing strategy.

Opsin-Mediated Inhibition of Bacterioruberin Synthesis in Halophilic Archaea.

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.

An Acidic Exopolysaccharide from Haloarcula hispanica ATCC33960 and Two Genes Responsible for Its Synthesis.

A 1.1 × 106 Da acidic exopolysaccharide (EPS) was purified from an extremely halophilic archaeon Haloarcula hispanica ATCC33960 with a production of 30 mg L-1 when grown in AS-168 medium, which mainly composed of mannose and galactose with a small amount of glucose in a molar ratio of 55.9 : 43.2 : 0.9. Two glycosyltransferase genes (HAH_1662 and HAH_1667) were identified to be responsible for synthesis of the acidic EPS. Deletion of either HAH_1662 or HAH_1667 led to loss of the acidic EPS. The mutants displayed a different cell surface morphology, retarded growth in low salty environment, an increased adhesion, and swimming ability. Our results suggest that biosynthesis of the acidic EPS might act as an adaptable mechanism to protect the cells against harsh environments.

The spacer size of I-B CRISPR is modulated by the terminal sequence of the protospacer.

Prokaryotes memorize invader information by incorporating alien DNA as spacers into CRISPR arrays. Although the spacer size has been suggested to be predefined by the architecture of the acquisition complex, there is usually an unexpected heterogeneity. Here, we explored the causes of this heterogeneity in Haloarcula hispanica I-B CRISPR. High-throughput sequencing following adaptation assays demonstrated significant size variation among 37 957 new spacers, which appeared to be sequence-dependent. Consistently, the third nucleotide at the spacer 3΄-end (PAM-distal end) showed an evident bias for cytosine and mutating this cytosine in the protospacer sequence could change the final spacer size. In addition, slippage of the 5΄-end (PAM-end), which contributed to most of the observed PAM (protospacer adjacent motif) inaccuracy, also tended to change the spacer size. We propose that both ends of the PAM-protospacer sequence should exhibit nucleotide selectivity (with different stringencies), which fine-tunes the structural ruler, to a certain extent, to specify the spacer size.

Malate Synthase and ß-Methylmalyl Coenzyme A Lyase Reactions in the Methylaspartate Cycle in Haloarcula hispanica.

Haloarchaea are extremely halophilic heterotrophic microorganisms belonging to the class Halobacteria (Euryarchaeota). Almost half of the haloarchaea possesses the genes coding for enzymes of the methylaspartate cycle, a recently discovered anaplerotic acetate assimilation pathway. In this cycle, the enzymes of the tricarboxylic acid cycle together with the dedicated enzymes of the methylaspartate cycle convert two acetyl coenzyme A (acetyl-CoA) molecules to malate. The methylaspartate cycle involves two reactions catalyzed by homologous enzymes belonging to the CitE-like enzyme superfamily, malyl-CoA lyase/thioesterase (haloarchaeal malate synthase [hMS]; Hah_2476 in Haloarcula hispanica) and ß-methylmalyl-CoA lyase (haloarchaeal ß-methylmalyl-CoA lyase [hMCL]; Hah_1341). Although both enzymes catalyze the same reactions, hMS was previously proposed to preferentially catalyze the formation of malate from acetyl-CoA and glyoxylate (malate synthase activity) and hMCL was proposed to primarily cleave ß-methylmalyl-CoA to propionyl-CoA and glyoxylate. Here we studied the physiological functions of these enzymes during acetate assimilation in H. hispanica by using biochemical assays of the wild type and deletion mutants. Our results reveal that the main physiological function of hMS is malyl-CoA (not malate) formation and that hMCL catalyzes a ß-methylmalyl-CoA lyase reaction in vivo The malyl-CoA thioesterase activities of both enzymes appear to be not essential for growth on acetate. Interestingly, despite the different physiological functions of hMS and hMCL, structural comparisons predict that these two proteins have virtually identical active sites, thus highlighting the need for experimental validation of their catalytic functions. Our results provide further proof of the operation of the methylaspartate cycle and indicate the existence of a distinct, yet-to-be-discovered malyl-CoA thioesterase in haloarchaea. IMPORTANCE: Acetate is one of the most important substances in natural environments. The activated form of acetate, acetyl coenzyme A (acetyl-CoA), is the high-energy intermediate at the crossroads of central metabolism: its oxidation generates energy for the cell, and about a third of all biosynthetic fluxes start directly from acetyl-CoA. Many organic compounds enter the central carbon metabolism via this key molecule. To sustain growth on acetyl-CoA-generating compounds, a dedicated assimilation (anaplerotic) pathway is required. The presence of an anaplerotic pathway is a prerequisite for growth in many environments, being important for environmentally, industrially, and clinically important microorganisms. Here we studied specific reactions of a recently discovered acetate assimilation pathway, the methylaspartate cycle, functioning in extremely halophilic archaea.

First characterization of extremely halophilic 2-deoxy-D-ribose-5-phosphate aldolase.

2-Deoxy-d-ribose-5-phosphate aldolase (DERA) catalyzes the aldol reaction between two aldehydes and is thought to be a potential biocatalyst for the production of a variety of stereo-specific materials. A gene encoding DERA from the extreme halophilic archaeon, Haloarcula japonica, was overexpressed in Escherichia coli. The gene product was successfully purified, using procedures based on the protein's halophilicity, and characterized. The expressed enzyme was stable in a buffer containing 2 M NaCl and exhibited high thermostability, retaining more than 90% of its activity after heating at 70 °C for 10 min. The enzyme was also tolerant to high concentrations of organic solvents, such as acetonitrile and dimethylsulfoxide. Moreover, H. japonica DERA was highly resistant to a high concentration of acetaldehyde and retained about 35% of its initial activity after 5-h' exposure to 300 mM acetaldehyde at 25 °C, the conditions under which E. coli DERA is completely inactivated. The enzyme exhibited much higher activity at 25 °C than the previously characterized hyperthermophilic DERAs (Sakuraba et al., 2007). Our results suggest that the extremely halophilic DERA has high potential to serve as a biocatalyst in organic syntheses. This is the first description of the biochemical characterization of a halophilic DERA.

DNA motifs determining the accuracy of repeat duplication during CRISPR adaptation in Haloarcula hispanica.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) acquire new spacers to generate adaptive immunity in prokaryotes. During spacer integration, the leader-preceded repeat is always accurately duplicated, leading to speculations of a repeat-length ruler. Here in Haloarcula hispanica, we demonstrate that the accurate duplication of its 30-bp repeat requires two conserved mid-repeat motifs, AACCC and GTGGG. The AACCC motif was essential and needed to be ∼10 bp downstream from the leader-repeat junction site, where duplication consistently started. Interestingly, repeat duplication terminated sequence-independently and usually with a specific distance from the GTGGG motif, which seemingly served as an anchor site for a molecular ruler. Accordingly, altering the spacing between the two motifs led to an aberrant duplication size (29, 31, 32 or 33 bp). We propose the adaptation complex may recognize these mid-repeat elements to enable measuring the repeat DNA for spacer integration.

Exploring the multiple biotechnological potential of halophilic microorganisms isolated from two Argentinean salterns.

The biodiversity and biotechnological potential of microbes from central Argentinean halophilic environments have been poorly explored. Salitral Negro and Colorada Grande salterns are neutral hypersaline basins exploded for NaCl extraction. As part of an ecological analysis of these environments, two bacterial and seven archaeal representatives were isolated, identified and examined for their biotechnological potential. The presence of hydrolases (proteases, amylases, lipases, cellulases and nucleases) and bioactive molecules (surfactants and antimicrobial compounds) was screened. While all the isolates exhibited at least one of the tested activities or biocompounds, the species belonging to Haloarcula genus were the most active, also producing antimicrobial compounds against their counterparts. In general, the biosurfactants were more effective against olive oil and aromatic compounds than detergents (SDS or Triton X-100). Our results demonstrate the broad spectrum of activities with biotechnological potential exhibited by the microorganisms inhabiting the Argentinean salterns and reinforce the importance of screening pristine extreme environments to discover interesting/novel bioactive molecules.

Complete genome sequence of Haloarcula sp. CBA1115 isolated from non-purified solar salts.

Haloarcula sp. CBA1115, isolated from non-purified solar salts from South Korea, is a halophilic archaeon belonging to the family Halobacteriaceae. Here, we present the complete genome sequence of the strain Haloarcula sp. CBA1115 (4,225,046bp, with a G+C content of 61.98%), which is distributed over one chromosome and five plasmids. A comparison of the genome sequence of Haloarcula sp. CBA1115 with those of members of its closely related taxa showed that the closest neighbor is Haloarcula hispanica Y27, a popular model organism for archaeal studies. The strain was found to possess a number of genes predicted to be involved in osmo-regulatory strategies and metal regulation, suggesting that it might be useful for bioremediation in extreme environments.

Complete biosynthetic pathway of the C50 carotenoid bacterioruberin from lycopene in the extremely halophilic archaeon Haloarcula japonica.

UNLABELLED: Haloarcula japonica, an extremely halophilic archaeon that requires high concentrations of NaCl for growth, accumulates the C50 carotenoid bacterioruberin (BR). By homology analysis, a gene cluster, including c0507, c0506, and c0505, was found and predicted to be involved in the synthesis of bacterioruberin. To elucidate the function of the encoded enzymes, we constructed Ha. japonica mutants of these genes and analyzed carotenoids produced by the mutants. Our research showed that c0507, c0506, and c0505 encoded a carotenoid 3,4-desaturase (CrtD), a bifunctional lycopene elongase and 1,2-hydratase (LyeJ), and a C50 carotenoid 2",3"-hydratase (CruF), respectively. The above three carotenoid biosynthetic enzymes catalyze the reactions that convert lycopene to bacterioruberin in Ha. japonica. This is the first identification of functional CrtD and CruF in archaea and elucidation of the complete biosynthetic pathway of bacterioruberin from lycopene. IMPORTANCE: Haloarcula japonica, an extremely halophilic archaeon, accumulates the C50 carotenoid bacterioruberin (BR). In this study, we have identified three BR biosynthetic enzymes and have elucidated their functions. Among them, two enzymes were found in an archaeon for the first time. Our results revealed the biosynthetic pathway responsible for production of BR in Ha. japonica and provide a basis for investigating carotenoid biosynthetic pathways in other extremely halophilic archaea. Elucidation of the carotenoid biosynthetic pathway in Ha. japonica may also prove useful for producing the C50 carotenoid BR efficiently by employing genetically modified haloarchaeal strains.