Halorubrum Sp Research Articles

Optimization of extraction procedure and antioxidant activity of C50 carotenoids from Halorubrum sp. HRM-150

C50 carotenoids are promising bioactive compounds produced by halophilic archaea. Relevant studies have focused on their outstanding antioxidant activity, whereas the extraction process has received less attention. In this study, the effect of factors on carotenoid extraction efficiency was explored through single-factor and orthogonal experiments. The results showed that the C50 carotenoids were effectively extracted by suspending and homogenizing cell pellets in methanol (1:30, w-v) for 10 min, subsequently incubating at 30 °C for 30 min in the dark, finally centrifuging and drying over vacuum rotary evaporation at 30 °C in the dark. The carotenoid content increased 1.84 times by the above procedure than that before optimization. Moreover, the UVVis spectra, TLC and HPLCMS analysis revealed that the main component of carotenoids extracted from Halorubrum sp. HRM-150 was bacterioruberin (84.12 %), followed by monoanhydrobacterioruberin (15.13 %). Both carotenoid extract and bacterioruberin exhibited antioxidant capacity, which were significantly higher than that of astaxanthin (p < 0.05) by scavenging assays of 2,2-azinobis-3-ethyl-benzothiazole-6-sulfonic acid, 2,2-diphenyl-1-picrylhydrazyl and hydroxyl radicals. In addition, the hydroxyl radical scavenging rate of bacterioruberin was significantly higher than that of carotenoid extract (p < 0.01), indicating its potential activity against or delaying diseases caused by reactive oxygen species. C50 carotenoids (especially BR) have great application prospects in human health due to their excellent antioxidant capacity.

Optimization of C50 Carotenoids Production by Open Fermentation of Halorubrum sp. HRM-150.

C50 carotenoids, as unique bioactive molecules, have many biological properties, including antioxidant, anticancer, and antibacterial activity, and have a wide range of potential uses in the food, cosmetic, and biomedical industries. The majority of C50 carotenoids are produced by the sterile fermentation of halophilic archaea. This study aims to look at more cost-effective and manageable ways of producing C50 carotenoids. The basic medium, carbon source supplementation, and optimal culture conditions for Halorubrum sp. HRM-150 C50 carotenoids production by open fermentation were examined in this work. The results indicated that Halorubrum sp. HRM-150 grown in natural brine medium grew faster than artificial brine medium. The addition of glucose, sucrose, and lactose (10g/L) enhanced both biomass and carotenoids productivity, with the highest level reaching 4.53 ± 0.32μg/mL when glucose was added. According to the findings of orthogonal studies based on the OD600 and carotenoids productivity, the best conditions for open fermentation were salinity 20-25%, rotation speed 150-200rpm, and pH 7.0-8.2. The up-scaled open fermentation was carried out in a 7 L medium under optimum culture conditions. At 96h, the OD600 and carotenoids productivity were 9.86 ± 0.51 (dry weight 10.40 ± 1.27g/L) and 7.31 ± 0.65μg/mL (701.40 ± 21.51μg/g dry weight, respectively). When amplified with both universal bacterial primer and archaeal primer in the open fermentation, Halorubrum remained the dominating species, indicating that contamination was kept within an acceptable level. To summarize, open fermentation of Halorubrum is a promising method for producing C50 carotenoids.

Genomic features of a new head–tail halovirus VOLN27B infecting a Halorubrum strain

Relatively few viruses infecting haloarchaea (haloviruses) have been reported. In this study, the genome sequence of VOLN27B, a recently described archaeal tailed virus (arTV) with a myovirus morphotype was described, along with the sequence of its host, Halorubrum spp. LN27. Halovirus VOLN27B contains a linear, dsDNA genome of 76,891 bp which is predicted to encode 109 proteins and four tRNAs (tRNAThr, tRNAArg, tRNAGly and tRNAAsn). The DNA G + C content of VOLN27B genome is 56.1 mol%, nearly 10% lower than that of its host strain. A 315 bp LTR (long terminal repeat) was detected in the genome. The genome of its host strain LN27 was 3,301,211 bp (chromosome and 1 plasmid) with a DNA G + C content of 68.3 mol% and 3142 annotated protein coding genes. At least two hypothetical proviruses were detected in the genome. It lacked a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) locus. Sequence similarity and phylogenetic tree reconstructions placed it within the genus Halorubrum as a potential new species. VOLN27B exhibits a distinct difference in the frequency of codon usage against its host strain Halorubrum sp. LN27. The organization of VOLN27B genome shows remarkable synteny and amino acid sequence similarity to the genomes and predicted proteins of HF1-like haloviruses (genus Haloferacalesvirus) and a provirus in the genome of Halorubrum depositum Y78. VOLN27B and its host Halorubrum sp. LN27 comprise a new virus-host system from a hypersaline ecosystem and can be used to further understand the novel biology at extreme salt concentration.

Halorubrum pleomorphic virus-6 Membrane Fusion Is Triggered by an S-Layer Component of Its Haloarchaeal Host.

(1) Background: Haloarchaea comprise extremely halophilic organisms of the Archaea domain. They are single-cell organisms with distinctive membrane lipids and a protein-based cell wall or surface layer (S-layer) formed by a glycoprotein array. Pleolipoviruses, which infect haloarchaeal cells, have an envelope analogous to eukaryotic enveloped viruses. One such member, Halorubrum pleomorphic virus 6 (HRPV-6), has been shown to enter host cells through virus-cell membrane fusion. The HRPV-6 fusion activity was attributed to its VP4-like spike protein, but the physiological trigger required to induce membrane fusion remains yet unknown. (2) Methods: We used SDS-PAGE mass spectroscopy to characterize the S-layer extract, established a proteoliposome system, and used R18-fluorescence dequenching to measure membrane fusion. (3) Results: We show that the S-layer extraction by Mg2+ chelating from the HRPV-6 host, Halorubrum sp. SS7-4, abrogates HRPV-6 membrane fusion. When we in turn reconstituted the S-layer extract from Hrr. sp. SS7-4 onto liposomes in the presence of Mg2+, HRPV-6 membrane fusion with the proteoliposomes could be readily observed. This was not the case with liposomes alone or with proteoliposomes carrying the S-layer extract from other haloarchaea, such as Haloferax volcanii. (4) Conclusions: The S-layer extract from the host, Hrr. sp. SS7-4, corresponds to the physiological fusion trigger of HRPV-6.

VOLN27B: A New Head-Tailed Halovirus Isolated from an Underground Salt Crystal and Infecting Halorubrum.

A novel halovirus, VOLN27B, was isolated from a drill core sample taken at a depth of approximately 430 m, from a layer formed during the Cretaceous period (Anhui, China). VOLN27B infects the halophilic archaeon Halorubrum sp. LN27 and has a head-tailed morphotype with a contractile tail, typical of myoviruses. The average head diameter is 64 ± 2.0 nm, and uncontracted tails are 15 ± 1.0 × 65 ± 2.0 nm. The latent period is about 10 h. The maturing time of VOLN27B in cells of Halorubrum sp. LN27 was nearly 8 h. The adsorption time of VOLN27B on cells of Halorubrum sp. LN27 was less than 1 min. Virus particles are unstable at pH values less than 5 or when the NaCl concentration is below 12% (w/v). VOLN27B and Halorubrum sp. LN27 were recovered from the same hypersaline environment and provide a new virus-host system in haloarchaea.

Effect of Carbon Sources in Carotenoid Production from Haloarcula sp. M1, Halolamina sp. M3 and Halorubrum sp. M5, Halophilic Archaea Isolated from Sonora Saltern, Mexico.

The isolation and molecular and chemo-taxonomic identification of seventeen halophilic archaea from the Santa Bárbara saltern, Sonora, México, were performed. Eight strains were selected based on pigmentation. Molecular identification revealed that the strains belonged to the Haloarcula, Halolamina and Halorubrum genera. Neutral lipids (quinones) were identified in all strains. Glycolipid S-DGD was found only in Halolamina sp. strain M3; polar phospholipids 2,3-O-phytanyl-sn-glycerol-1-phosphoryl-3-sn-glycerol (PG), 2,3-di-O-phytanyl-sn-glycero-1-phospho-3′-sn-glycerol-1′-methyl phosphate (PGP-Me) and sodium salt 1-(3-sn-phosphatidyl)-rac-glycerol were found in all the strains; and one unidentified glyco-phospholipid in strains M1, M3 and M4. Strains M1, M3 and M5 were selected for further studies based on carotenoid production. The effect of glucose and succinic and glutamic acid on carotenoid production was assessed. In particular, carotenoid production and growth significantly improved in the presence of glucose in strains Haloarcula sp. M1 and Halorubrum sp. M5 but not in Halolamina sp. M3. Glutamic and succinic acid had no effect on carotenoid production, and even was negative for Halorubrum sp. M5. Growth was increased by glutamic and succinic acid on Haloarcula sp. M1 but not in the other strains. This work describes for first time the presence of halophilic archaea in the Santa Bárbara saltern and highlights the differences in the effect of carbon sources on the growth and carotenoid production of haloarchaea.

Genomic analysis of facultatively oligotrophic haloarchaea of the genera Halarchaeum, Halorubrum, and Halolamina, isolated from solar salt.

Extremely halophilic archaea (haloarchaea) belonging to the phylum Euryarchaeota have been found in high-salinity environments. In this study, Halarchaeum sp. CBA1220, Halorubrum sp. CBA1229, and Halolamina sp. CBA1230, which are facultatively oligotrophic haloarchaea, were isolated from solar salt by culture under oligotrophic culture conditions. The complete genomes of strains CBA1220, CBA1229, and CBA1230 were sequenced and were found to contain 3,175,875, 3,582,278, and 3,465,332bp, with a G + C content of 68.25, 67.66, and 66.75mol%, respectively. In total, 60, 36, and 33 carbohydrate-active enzyme genes were determined in the respective strains. The strains harbored various genes encoding stress-tolerance proteins, including universal stress proteins, cold-shock proteins, and rubrerythrin and rubrerythrin-related proteins. The genome data produced in this study will facilitate further research to improve our understanding of other halophilic strains and promote their industrial application.

Exploration of haloarchaea for their potential applications in food industry

Extremophiles are a major source for many industrially important biomolecules. Haloarchaea have been extensively studied for their ability to grow in extreme environments and the array of metabolites especially osmolytes they produce with resistance to extreme physical and chemical conditions. In the present study, we have studied the haloarchaeal isolates for their ability to produce industrially important biomolecules like—proteases, amylases, surfactants, antifreeze and antidesiccation proteins. The haloarchaea used in this study were isolated from fish sauce fermentation broth and belonged to Halorubrum sp. SS1 (KY053875.1), Halobacterium sp. SS2 (KY053876.1), Halococcus sp. AMS1 (KU995303.1), Halorhabdus sp. AMS6 (KU995310.1) and Halobacterium sp. SFF3 (KY053871.1). All the archaea isolated in this report had negligible amylase and surfactant activities but showed marginal protease activity. The isolates were also found to be producing significant antifreeze protein stable till 192 h and desiccation tolerant till 240 min of studies. This is the first report of microbial extracellular proteins exhibiting antidesiccation and antifreeze properties, thus introducing a new family of industrially important microbial proteins. These applications have immense potential in food preservation and processing.

Bioprospecting and characterization of pigmented halophilic archaeal strains from Algerian hypersaline environments with analysis of carotenoids produced by Halorubrum sp. BS2.

A set of 110 extremely halophilic archaeal strains were isolated from seven distinct saline habitats located in different regions of Algeria. The physicochemical characterization of the samples showed that these habitats were thalassohaline. The carotenoid production from isolated strains varied from 0.1 to 3.68 µg/ml. Based on their physiological characteristics and pigment production, 43 strains were selected and identified by means of phenotypic tests and 16S ribosomal RNA gene sequencing. Phylogenetic analysis indicated that the isolates corresponded to the class Halobacteria and were closely related to genera Halorubrum, Haloarcula, Haloferax, Natrinema, Halogeometricum, Haloterrigena, and Halopiger. Carotenoids of the highest producer, strain Halorubrum sp. BS2 were identified using high-performance liquid chromatography-diode array detector and liquid chromatography-mass spectrometry. Bacterioruberin and bisanhydrobacterioruberin were the predominant carotenoids. The scavenging activity of these carotenoids reached 99% at a concentration of 18 μg/ml, which was much higher than that of ascorbic acid used as a reference compound. These carotenoids also exhibited significant antibacterial activities against four human-pathogenic strains and four fish-pathogenic strains. Variations in salinity, agitation rate, temperature, and light intensity were found to influence growth and carotenoid production of Halorubrum sp. BS2. Our results suggest that halophilic archaea represent a potential source for carotenoids, which are characterized by high antioxidant and antibacterial activities.

Characterization of a new exopolysaccharide produced by Halorubrum sp. TBZ112 and evaluation of its anti-proliferative effect on gastric cancer cells.

In the present study, we aimed to extract, purify, analyze monosaccharide composition of exopolysaccharide (EPS) produced by Halorubrum sp. TBZ112 (KCTC 4203 and IBRC-M 10773) and also to evaluate its possible antiproliferative activity against human gastric cancer (MKN-45) cell line and its biocompatibility effect on normal cells using human dermal fibroblast (HDF) cell line. Average molecular weight and monosaccharide composition were determined by high-pressure size exclusion chromatography (HPSEC) with multi-angle laser light scattering (MALLS) and high-pressure anion exchange chromatography (HPAEC), respectively. Fourier transform infrared (FTIR) spectroscopy was used for the partial characterization of the EPS. The EPS effect on the cell proliferation and viability of MKN-45 and HDF cells was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and trypan blue dye exclusion, respectively. Strain TBZ112 excreted 480mg.l-1 of the EPS under optimal growth conditions. The EPS had a molecular weight of 5.052kDa and was a heteropolysaccharide containing ten moieties mainly composed of mannose (19.95%), glucosamine (15.55%), galacturonic acid (15.43%), arabinose (12.24%), and glucuronic acid (12.05%). No significant difference of the EPS treatments on the proliferation activity of MKN-45 and HDF cells were observed (P > 0.05). For the first time, the EPS from Halorubrum sp. TBZ112, an extremely halophilic archaeon related to Halorubrum genus, was isolated and chemically characterized. The EPS from Halorubrum sp. TBZ112 possesses a relatively low molecular weight and might be applied as a biocompatible compound. More investigations are needed to determine other biological activities of the EPS along with further details of its chemical structure.

Sialic Acid-Like Sugars in Archaea: Legionaminic Acid Biosynthesis in the Halophile Halorubrum sp. PV6

N-glycosylation is a post-translational modification that occurs in all three domains. In Archaea, however, N-linked glycans present a degree of compositional diversity not observed in either Eukarya or Bacteria. As such, it is surprising that nonulosonic acids (NulOs), nine-carbon sugars that include sialic acids, pseudaminic acids, and legionaminic acids, are routinely detected as components of protein-linked glycans in Eukarya and Bacteria but not in Archaea. In the following, we report that the N-linked glycan attached to the S-layer glycoprotein of the haloarchaea Halorubrum sp. PV6 includes an N-formylated legionaminic acid. Analysis of the Halorubrum sp. PV6 genome led to the identification of sequences predicted to comprise the legionaminic acid biosynthesis pathway. The transcription of pathway genes was confirmed, as was the co-transcription of several of these genes. In addition, the activities of LegI, which catalyzes the condensation of 2,4-di-N-acetyl-6-deoxymannose and phosphoenolpyruvate to generate legionaminic acid, and LegF, which catalyzes the addition of cytidine monophosphate (CMP) to legionaminic acid, both heterologously expressed in Haloferax volcanii, were demonstrated. Further genome analysis predicts that the genes encoding enzymes of the legionaminic acid biosynthetic pathway are clustered together with sequences seemingly encoding components of the N-glycosylation pathway in this organism. In defining the first example of a legionaminic acid biosynthesis pathway in Archaea, the findings reported here expand our insight into archaeal N-glycosylation, an almost universal post-translational modification in this domain of life.

Halorubrum sp. TBZ112, an Extremely Halophilic Carotenoid- Producing Archaeon Isolated from Urmia Lake

Background: Carotenoids are organic pigments with substantial applications in neutraceuticals, pharmaceuticals, cosmetics and food industries. Considering the importance of carotenoids, we aimed to isolate and identify a carotenoid-producing microorganism in the present study. Methods: Gram-negative, aerobic and rod-shaped archaeon, Halorubrum sp. TBZ112 (KCTC 4203 and IBRC-M 10773) producing carotenoids with circle and red colonies were isolated from Urmia Lake in Northwest Iran. Phenotypic characterization and molecular identification of isolate were also conducted. In order to study the ability of TBZ112 to produce carotenoids as well as the production profile, carotenoids were extracted using acetone-methanol solution (7:3 v/v). Then, the carotenoid content of the extract was evaluated using UV spectroscopy and thin-layer chromatography (TLC). The carotenoid profile was analyzed using liquid chromatography-mass spectrometry (LC-MS) techniques. Results: The 16S rRNA analysis showed that TBZ112 had the highest similarity with Halorubrum chaoviator Halo-G*T (99.78%). The total carotenoid content of strain TBZ112 was found to be 11.7 mg/l. The LC-MS analytical results indicated that carotenoids that were produced included bacterioruberin, lycopene and β-carotene. Among them, bacterioruberin was predominant. Conclusion: Consequently, we can suggest that future studies should investigate this new and natural source for producing carotenoids.

Draft genome sequence of the extremely halophilic Halorubrum sp. SAH-A6 isolated from rock salts of the Danakil depression, Ethiopia

The draft genome sequence of Halorubrum sp. SAH-A6, isolated from commercial rock salts of the Danakil depression, Ethiopia. The genome comprised 3,325,770bp, with the G+C content of 68.0%. The strain has many genes which are responsible for secondary metabolites biosynthesis, transport and catabolism as compared to other Halorubrum archaea members. Abundant genes responsible for numerous transport systems, solute accumulation, and aromatic/sulfur decomposition were detected. The first genomic analysis encourages further research on comparative genomics, and biotechnological applications. The NCBI accession number for this genome is SAMN04278861 and ID: 4278861 and strain deposited with accession number KCTC 43215.

Characterization of a bacterioruberin-producing Haloarchaea isolated from the marshlands of the Odiel river in the southwest of Spain.

In this work, we describe the isolation, identification, pigment characterization, and optimization of the culture conditions for a haloarchaea strain isolated from salt evaporation ponds in the Odiel river, at Southwest of Spain. The haloarchaea belongs to the genus Halorobrum, as deduced from the analysis of its 16S rRNA encoding gene and has been designated as Halorubrum sp. SH1. The growth conditions for the new strain were optimized studying temperature, NaCl concentration, agitation rate and light intensity. The C50-carotenoids, bacterioruberin, and its derivatives bisanhydrobacterioruberin and trisanhydrobacterioruberin, were found to be the predominant pigments produced by this strain of Halorubrum, as determined using HPLC-DAD and UHPLC-ESI-MS/MS techniques. This extremely halophilic archaeon could be a good candidate for the production of bacterioruberins of high added-value due to their coloring, antioxidant, and possible anticancer properties. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:592-600, 2016.

Agar-supported cultivation of Halorubrum sp. SSR, and production of halocin C8 on the scale-up prototype Platotex

Halorubrum sp. SSR was isolated from a solar saltern in Algeria. The strain exhibited a high antibiotic activity against the indicator strain Natronorubrum aibiense G23, and the bioactive compound showed thermal, acid and alkali stability. SSR was grown on agar-supported cultivation (AgSF) to compare yields and applicability with traditional submerged cultivation. AgSF scale-up was implemented taking benefit from the solid-state cultivation prototype Platotex. This technology leads to high amounts of the target Halocin and facilitate the downstream steps. The antibiotic compound was purified according to a fast efficient procedure including ion exchange chromatography followed by a fractionation on C18 Sep-Pack cartridge. The compound was identified as Halocin C8 according to N-terminal amino acid sequencing and high-resolution mass spectrometry.

Optimization of Total Carotenoid Production by Halorubrum Sp. TBZ126 Using Response Surface Methodology

Carotenoids are one of the most diverse and broadly distributed classes of pigments in nature with a high number of biotechnological applications. Carotenoids have a broad range of functions, especially in relation to human health and their role as biological antioxidants. The increasing demand for consumption of natural carotenoids has raised interest in their bio-production. The objective of the present study was the analysis of environmental factors (temperature, pH and salinity) through response surface methodology (RSM) on the total carotenoid production of Halorubrum sp. TBZ126. In addition the effect of light was evaluated. Five levels of temperature, pH, and salinity were selected based on central composite design (CCD) and RSM to reach the optimum values for the cell growth and carotenoid production. Bio-production was carried out in an orbital shaker using a 10% (v/v) inoculum, and agitation at 120 rpm for 9 days in a non-illuminated environment. Dry cell weight was determined and total carotenoid was estimated by spectrophotometer. The production of biomass ranged from 0.04 to 0.84 g/l and the total carotenoid from 0.15 to 10.78 mg/l. The optimum conditions for cell growth and total carotenoid production in Halorubrum sp. TBZ126 cultures, were temperature 31oC and 32oC, pH 7.51 and 7.94 and NaCl (w/v) 18.33% and 20.55%, respectively. In conclusion, employing RSM design and under the light as an inducing factor, carotenoid production by Halorubrum sp. TBZ126 was elevated to about 145%. Additionally, TBZ126 could produce carotenoids at lower concentrations of NaCl (as low as 2.5%), in the absence of sodium acetate without elevating magnesium sulfate concentration.

Molecular characterization of pHRDV1, a new virus-like mobile genetic element closely related to pleomorphic viruses in haloarchaea

A novel haloarchaeal plasmid, pHRDV1 (13,053bp), was isolated from the haloarchaeal isolate Halorubrum sp. T3. Molecular and bioinformatics analyses showed that this element is a double-stranded circular DNA molecule containing two putative transcripts with opposite directions. The amino acid sequences of six of the nineteen predicted open reading frames were similar to those found in haloarchaeal pleomorphic viruses, such as Halorubrum pleomorphic virus 3 and Halogeometricum pleomorphic virus 1. There was also a strong conservation in gene order between the plasmid and these viruses. All three conserved viral proteins (VPs), which are characteristic of haloarchaeal pleomorphic viruses VP3, VP4 and VP8, were found in pHRDV1. Furthermore, a typical repressor-operator system similar to haloarchaeal myovirus φCh1, was found on the genome of pHRDV1. However, no viral particles were detected in the supernatants of Halorubrum sp. T3, either in the presence or absence of mitomycin C. These results imply that plasmid pHRDV1 is a distinctive virus-like mobile genetic element that harbors some unique properties that make it different from all of the known haloarchaeal plasmids or viruses.

Analysis of Carotenoid Production by Halorubrum sp. TBZ126; an Extremely Halophilic Archeon from Urmia Lake.

Carotenoids are of great interest in many scientific disciplines because of their wide distribution, diverse functions and interesting properties. The present report describes a new natural source for carotenoid production. Halorubrum sp., TBZ126, an extremely halophilic archaeon, was isolated from Urmia Lack following culture of water sample on marine agar medium and incubation at 30 °C. Then single colonies were cultivated in broth media. After that the cells were collected and carotenoids were extracted with acetone-methanol (7:3 v/v). The identification of carotenoids was performed by UV-VIS spectroscopy and confirmed by thin layer chromatography (TLC) in the presence of antimony pentachloride (SbCl5). The production profile was analyzed using liquid-chromatography mass spectroscopy (LC-MS) techniques. Phenotypic characteristics of the isolate were carried out and the 16S rRNA gene was amplified using polymerase chain reaction (PCR). LC-MS analytical results revealed that produced carotenoids are bacterioruberin, lycopene and β-carotene. Bacterioruberin was found to be the predominant produced carotenoid. 16S rRNA analysis showed that TBZ126 has 100% similarity with Halorubrum chaoviator Halo-G*T (AM048786). Halorubrum sp. TBZ126, isolated from Urmia Lake has high capacity in the production of carotenoids. This extremely halophilic archaeon could be considered as a prokaryotic candidate for carotenoid production source for future studies.

Cardiolipin is associated with the terminal oxidase of an extremely halophilic archaeon

Membranes having an a high content of cardiolipin were isolated from an extremely halophilic archaeon Halorubrum sp. Absorbance difference spectra of detergent-solubilized plasma membranes reduced by dithionite suggested the presence of b-type cytochromes. Non-denaturing gel electrophoresis revealed only one fraction having TMPD-oxidase activity in which cardiolipin was the major lipid component. The electroeluted fraction showed a cytochrome c oxidase activity characterized by the reduced minus oxidized difference spectra as a terminal heme-copper oxidase. The cytochrome c oxidase activity of the archaeal cardiolipin-rich membranes was inhibited by the cardiolipin-specific fluorescent marker 10- N-nonyl acridine orange (NAO) in a dose-dependent manner. The results indicate that an archaeal analogue of cardiolipin is tightly associated to archaeal terminal oxidases and is required for its optimal functioning.