Natronococcus pandeyae sp. nov., a Novel Haloarchaeon from Sambhar Salt Lake.

A halophilic archaeon, designated strain LS1_42T, was isolated from Sambhar Salt Lake, Rajasthan, India. Cells were non-motile, coccoid, Gram-stain-variable and present in irregular clusters with light pink pigmented colonies. The strain was strictly aerobic and able to grow without Mg2+. Growth of the strain LS1_42T was observed at 25-45 °C, pH 7.0-11.0 and NaCl concentrations of 10-35% (w/v). The nearest phylogenetic neighbor of strain LS1_42T was Natronococcus amylolyticus Ah-36T based on 16S rRNA and rpoB' genes with similarity of 95.4% and 91.9%, respectively. Phylogenetic analysis based on 16S rRNA gene, rpoB' gene and whole-genome sequences indicate that the strain LS1_42T belongs to the genus Natronococcus and is closely related to N. amylolyticus. The genome size was 5.38 Mb with 98.9% completeness. The DNA G + C content of the strain LS1_42T was 63.0 mol%. The average nucleotide identity, average amino acid identity and DNA-DNA hybridization values between LS1_42T and N. amylolyticus Ah-36T were 81.3%, 77.7% and 24.8%, respectively. The major polar lipids detected were phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester. On the basis of phenotypic, chemotaxonomic and genome-based analysis, strain LS1_42T represents a novel species within the genus Natronococcus, for which the name Natronococcus pandeyae sp. nov. is proposed. The type strain is LS1_42T (MCC 3654T = JCM 33003T = KCTC 4280T = CGMCC 1.16738T).

The Cell Membrane of Sulfolobus spp.-Homeoviscous Adaption and Biotechnological Applications.

The microbial cell membrane is affected by physicochemical parameters, such as temperature and pH, but also by the specific growth rate of the host organism. Homeoviscous adaption describes the process of maintaining membrane fluidity and permeability throughout these environmental changes. Archaea, and thereby, Sulfolobus spp. exhibit a unique lipid composition of ether lipids, which are altered in regard to the ratio of diether to tetraether lipids, number of cyclopentane rings and type of head groups, as a coping mechanism against environmental changes. The main biotechnological application of the membrane lipids of Sulfolobus spp. are so called archaeosomes. Archaeosomes are liposomes which are fully or partly generated from archaeal lipids and harbor the potential to be used as drug delivery systems for vaccines, proteins, peptides and nucleic acids. This review summarizes the influence of environmental parameters on the cell membrane of Sulfolobus spp. and the biotechnological applications of their membrane lipids.

Natronococcus roseus sp. nov., a haloalkaliphilic archaeon from a hypersaline lake.

A novel halophilic archaeon, strain CG-1(T), belonging to the genus Natronococcus was isolated from sediment of the soda lake Chagannor in Inner Mongolia, China. The colonies of this strain were pink pigmented, the intensity of the colour decreased when the cells grew at salt saturation levels. The cells were non-motile cocci and strictly aerobic. Hypotonic treatment did not cause cell lysis, even in distilled water. Strain CG-1(T) grew at 15-30.0 % (w/v) NaCl and at 30-50 °C and pH 8.0-11.0, with optimal growth occurring at 25-30 % (w/v) NaCl, 37-45 °C and pH 9-9.5. MgCl(2) was not required for growth. Strain CG-1(T) was most closely related to the type strains of Natronococcus amylolyticus Ah-36(T), Natronococcus jeotgali B1(T) and Natronococcus occultus SP4(T), with which it shared 98.4 %, 96.2 and 95.7 % 16S rRNA gene sequence similarity, respectively. The polar lipids consisted of C(20)C(20) and C(20)C(25) derivatives of phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me) and minor phospholipid components. No glycolipids were detected. The DNA G+C content of strain CG-1(T) was 62.1 mol%. DNA-DNA hybridization with N. amylolyticus DSM 10524(T), phylogenetically the most closely related species, was 39 %; this value showed that strain CG-1(T) constituted a different genospecies. The comparison of 16S rRNA gene sequences, detailed phenotypic characterization, polar lipid profile and DNA-DNA hybridization studies revealed that strain CG-1(T) belongs to the genus Natronococcus and constitutes a novel species for which the name Natronococcus roseus sp. nov. is proposed. The type strain is CG-1(T) (=CECT 7984(T)=IBRC-M 10656(T)=JCM 17958(T)).

Novel ether lipid cardiolipins in archaeal membranes of extreme haloalkaliphiles.

The lipidome of two extremely haloalkaliphilic archaea, Natronococcus occultus and Natronococcus amylolyticus, has been examined by means of combined thin-layer chromatography and MALDI-TOF/MS analyses. The detailed investigation of lipid profiles has confirmed the presence of i) ether lipid phosphatidylglycerol and phosphatidylglycerophosphate methyl ester as main lipid components, ii) both C(20) and C(25) isopranoid chains in the lipid core and yielded new findings on membrane lipids of these unusual organisms. Besides some novel minor or trace phospholipids and glycolipids, data indicate the presence of ether lipid cardiolipin variants constituted by different combinations of C(20) and C(25) isopranoid chains, never before described in archaea. The role of C(25) isopranoid chains in the adaptation to high pH gradients in the presence of very high salt concentrations is discussed.

Natronococcus jeotgali sp. nov., a halophilic archaeon isolated from shrimp jeotgal, a traditional fermented seafood from Korea.

A novel halophilic archaeon (strain B1(T)) belonging to the genus Natronococcus was isolated from shrimp jeotgal, a traditional fermented food from Korea. Colonies of this strain were orange-red and cells were non-motile cocci that stained Gram-variable. Strain B1(T) grew in 7.5-30.0 % (w/v) NaCl and at 21-50 degrees C and pH 7.0-9.5, with optimal growth occurring in 23-25 % (w/v) NaCl and at 37-45 degrees C and pH 7.5. Strain B1(T) was most closely related to the type strain of Natronococcus occultus, with which it shared 97.91 % 16S rRNA gene sequence similarity. Within the phylogenetic tree, this novel strain shared a branching point with N. occultus and occupied a phylogenetic position that was distinct from the main Natronococcus branch. The degree of DNA-DNA hybridization with the type strain of N. occultus, the most closely related species phylogenetically, was 16.4 %. On the basis of these results, it is concluded that strain B1(T) represents a novel species of the genus Natronococcus, for which the name Natronococcus jeotgali is proposed. The type strain is B1(T) (=KCTC 4018(T)=DSM 18795(T)=JCM 14583(T)=CECT 7216(T)).

Control of ribosome turnover during growth of the haloalkaliphilic archaeon Natronococcus occultus.

The metabolism of ribosomes during growth of the haloalkaliphilic archaeon Natronococcus occultus was examined. The ribosome content was higher during exponential growth and diminished to 35% of the maximum in the stationary stage. The incorporation of H3-orotic acid and C14-uracil into rRNA was higher during exponential growth. After that, it decreased to 39% of the maximum in the stationary stage. The labeling of non-ribosomal RNA took place almost exclusively in the exponential stage. From loss of radioactivity, the half-life of rRNA was 11.43, 14.85, 5.28 and 7.14 h during the initial, exponential, late exponential and stationary growth stages, respectively. These results suggested that increased synthesis combined with diminished degradation were responsible for the high ribosome content displayed by Ncc. occultus during exponential growth. In contrast, diminished synthesis together with increased degradation provoked its posterior loss.

Lipolytic activity from Halobacteria: screening and hydrolase production.

Strains of Halobacteria from an Algerian culture collection were screened for their lipolytic activity against p-nitrophenyl butyrate (PNPB) and p-nitrophenyl palmitate (PNPP). Most strains were active on both esters and 12% hydrolyzed olive oil. A strain identified as Natronococcus sp. was further studied. It grew optimally at 3.5 M NaCl, pH 8 and 40 degrees C. An increase in temperature shifted the optimum salt concentration range for growth from a wider range of 2-4 M, obtained at 25-30 degrees C, to a narrower range of 3.5-4 M, obtained at 35-40 degrees C. At 45 degrees C the optimum salt concentration was 2 M. These results show a clear correlation between salt and temperature requirement. The optimum conditions for the production of hydrolytic activity during growth were: 3.5 M NaCl and pH 8 for PNPB hydrolytic activity and 4 M NaCl and pH 7.5 for PNPP hydrolytic activity; both at 40 degrees C. The clear supernatant of cells grown at 4 M NaCl showed olive oil hydrolysis activity (in presence of 4 M NaCl) demonstrating the occurrence of a lipase activity in this strain. To our knowledge, this is the first report of a lipase activity at such high salt concentration.

[Analysis of partial sequence of the gene for a novel Br protein].

A strain of halophilic archaeum AB1 was isolated and purified from Aibi Lake located in the north of Xinjiang Uygur Autonomous Region. Partial DNA fragment encoding a bacteriorhodopsin (Br) protein as well as 16S rRNA of AB1 was amplified by PCR, and their nucleotide sequences were determined subsequently. On the basis of homology and phylognetic analysis about 16S rRNA gene (16S rDNA), it could be speculated that the strain AB1 is a novel member of the genus Natronococcus. The hydropathy analysis of Br fragment revealed that the AB1 Br had a transmembrane heptahelical structure similar to that of other Brs. On the other hand, homology alignment using the deduced partial amino acid sequence of Br protein of AB1 with other Br proteins showed that AB1 Br protein is obviously different to others. These facts indicated that the Br in halophilic archaeum AB1 is a new Br protein.

Detection of quorum sensing signals in the haloalkaliphilic archaeon Natronococcus occultus.

Bacteria communicate at high cell density through quorum sensing, however, there are no reports about this mechanism in archaea. The archaeon Natronococcus occultus produces an extracellular protease at the end of growth. Early production of protease activity was observed when a low density culture was incubated with late exponential conditioned medium suggesting the presence of factor(s) inducing this activity. Conditioned medium and ethyl acetate extracts corresponding to the transition from exponential to stationary phase showed a positive signal in Agrobacterium biosensor. We report the detection of potential autoinducer molecules of the acylated homoserine lactone type in the archaeon N. occultus. These molecules may be responsible for the production/activation of extracellular protease.

Ubiquitin-like proteins in halobacteria.

Within our studies of protein degradation, the presence of ubiquitinylated proteins in haloalkaliphilic archaea was investigated. We found that Natronococcus occultus proteins that react with antibodies raised against ubiquitin appear in different growth phases, particularly in the initial and exponential ones. The expression of these proteins is increased when the cells are either treated with puromycin or starved for nutrients. Dot blot analysis of cell extracts with antibody against ubiquitin shows the presence of either ubiquitinylated or ubiquitin-like proteins not only in Natronococcus occultus, but also in various genera of halobacteria.

Autoproteolytic activation of the haloalkaliphilic archaeon Natronococcus occultus extracellular serine protease.

The haloalkaliphilic archaeon Natronococcus occultus produces an extracellular serine protease in the stationary growth phase and upon starvation. Two proteins immunologically related to the extracellular protease were detected into the cells: P200 and P190. P200 was detected at early stages of growth and its relative amount decreased as the culture reached the stationary growth phase, concomitantly with the appearance of P190 and proteolytic activity, suggesting that P200 may be the precursor of the secreted protease and P190 the mature enzyme. Both proteins were also detected in the culture medium. Conversion of inactive P200 into active P190 was attained in cell-free culture medium from stationary phase but not from exponential phase. This process was prevented in the presence of PMSF and could be attained by addition of purified mature extracellular protease to P200. Altogether these results indicate that activation of Natronococcus occultus extracellular protease may be autoproteolytic and that factor/s present in stationary phase culture medium may be required for this process.

Purification and biochemical characterization of the haloalkaliphilic archaeon Natronococcus occultus extracellular serine protease.

A serine protease was purified from Natronococcus occultus stationary phase culture medium (328-fold, yield 19%) and characterized at the biochemical level. The enzyme has a native molecular mass of 130 kDa, has chymotrypsin-like activity, is stable and active in a broad pH range (5.5-12), is rather thermophilic (optimal activity at 60 degrees C in 1-2 M NaCl) and is dependent on high salt concentrations for activity and stability (1-2 M NaCl or KCl). Polyclonal antibodies were raised against the purified protease. In Western blots, they presented no cross-reactivity with culture medium from other halobacteria nor with commercial proteases except subtilisin. The amino acid sequences of three tryptic peptides obtained from Natronococcus occultus protease did not show significant similarity to other known proteolytic enzymes. This fact, in addition to its high molecular mass suggests that Natronococcus occultus extracellular protease may be a novel enzyme.

Purification and characterization of a membrane-associated ATPase from Natronococcus occultus, a haloalkaliphilic archaeon.

Isolated membranes of the extreme haloalkaliphilic archaeon Natronococcus occultus were able to hydrolyze ATP via an ATPase, which required the presence of Mg(2+), high concentrations of NaCl, and a pH value of 9. The native molecular mass of the purified ATPase was 130 kDa and was composed of 74- and 61-kDa subunits. Enzyme activity was specific for the hydrolysis of ATP with slight activity towards GTP, CTP, and ITP. The enzyme required NaCl for maximal activity but Na(2)SO(4) and (NH(4))(2)SO(4) could substitute. The enzyme showed no activity if Na(2)SO(3) or sodium citrate was substituted for NaCl. The ATPase from N. occultus was inhibited by NBD-Cl, NaN(3), and ouabain, and was sensitive to nitrate, vanadate, DCCD, and bafilomycin A(1). It was not inhibited by NEM in contrast to other previously characterized halophile ATPases. The ATPase had a K(M) of 0.5 mM and appeared to be non-competitively inhibited by NaN(3) with a K(I) of 3.1 mM.

Extracellular protease of Natrialba magadii: purification and biochemical characterization.

A serine protease secreted by the haloalkaliphilic archaeon Natrialba magadii at the end of the exponential growth phase was isolated. This enzyme was purified 83 fold with a total yield of 25% by ethanol precipitation, affinity chromatography, and gel filtration. The native molecular mass of the enzyme determined by gel filtration was 45 kDa. Na. magadii extracellular protease was dependent on high salt concentrations for activity and stability, and it had an optimum temperature of 60 degrees C in the presence of 1.5M NaCl. The enzyme was stable and had a broad pH profile (6-12) with an optimum pH of 8-10 for azocasein hydrolysis. The protease was strongly inhibited by diisopropyl fluorophosphate (DFP), phenylmethyl sulfonylfluoride (PMSF), and chymostatin, indicating that it is a serine protease. It was sensitive to denaturing agents such as SDS, urea, and guanidine HCl and activated by thiol-containing reducing agents such as dithiotreitol (DTT) and 2-mercaptoethanol. This protease degraded casein and gelatin and showed substrate specificity for synthetic peptides containing Phe, Tyr, and Leu at the carboxyl terminus, showing that it has chymotrypsin-like activity. Na. magadii protease presented no cross-reactivity with polyclonal antibodies raised against the extracellular protease of Natronococcus occultus, suggesting that although these proteases share several biochemical traits, they might be antigenically unrelated.

Microorganismos halófitos: potencial biotecnológico e interés farmacéutico / Biotechnological potential and pharmaceutical interest of Halophilic Microorganisms

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The cell wall polymer of the extremely halophilic archaeon Natronococcus occultus.

The cell wall polymer of Natronococcus occultus (DSM 3396) consists of L-glutamate, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, D-galacturonic acid, D-glucuronic acid and D-glucose in a molar ratio of 5:7:1:8:0.5:0.3. Partial acid hydrolysis of the cell wall polymer produced soluble fragments that could be separated by HPLC. A gamma-glutamyl dipeptide was isolated. In the intact cell wall polymer, the glutamate residues form a poly-(gamma-glutamine) chain with a length of about 60 monomers, which corresponds to a relative molecular mass of approximately 7700 Da. Two other soluble dimeric fragments, composed of glutamate and either glucosamine or galactosamine in a molar ratio of 1:1, were purified from the hydrolysate, suggesting the presence of two different oligosaccharides linked to the poly-(gamma-glutamine) chain of the intact polymer. The analysis of additional fragments, which were composed of an amino sugar and galacturonic acid or glucose indicated that one oligosaccharide consisted of a glucosamine pentamer in an alpha-1,3 linkage at the reducing end and an oligomer with at least five beta-1,4-linked galacturonic acid residues at the non-reducing end. The second oligosaccharide was comprised of a galactosamine dimer in a beta-1,3 linkage at the reducing end and a maltose unit at the non-reducing end. Both oligosaccharides were linked to the alpha-amide group of the glutamine residues of the poly-(gamma-glutamine) chain. The whole cell wall polymer, which represents a novel type of natural glycoconjugate, has a relative molecular mass of 54 kDa.

Intracellular proteolytic activity of the haloalkaliphilic archaeon Natronococcus occultus. Effect of starvation.

Intracellular proteolytic activity was detected in the haloalkaliphilic archaeon Natronococcus occultus during the stationary phase of cultures grown in complete medium and during carbon and nitrogen starvation. Puromycin prevented the occurrence of proteolytic activity in starved cells, suggesting that de novo synthesis of proteolytic enzymes might be required for protein degradation during starvation. Intracellular proteolytic activity degraded casein and gelatin. It had a temperature optimum of 60 degree centigrade in 2 M NaCl and depended on high salt concentration (NaCl or KCl) for activity and stability. Gelatin zymography of cell extracts from stationary phase or starved cells showed a complex pattern of proteolytic bands ranging from ∼ 20 to 120 kDa. All these proteolytic bands were inhibited by PMSF1) and chymostatin. However, they showed differences in stability to temperature and salt concentration.