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Dense communities of carotenoid-rich members of the Halobacteria (Euryarchaeota), the bacterium Salinibacter (Bacteroidetes) and the eukaryotic alga Dunaliella color the brines of most saltern crystallizer ponds red. The first report we found from the western world mentioning these red brines dates from 1765: the Encyclopédie of Diderot and coworkers. Earlier descriptions of solar salterns since Roman times do not mention red ponds. These include the Astronomica of Manilius, Pliny's Naturalis Historia (1st century), the description of Italian salterns in De Reditu Suo by Namatianus (5th century), Agricola's De Re Metallica (1556) and an anonymous description of French salterns (1669). This suggests that in earlier times, saltern brines may not have been red. In salterns which are operated today in the traditional way as practiced in the Middle Ages, no red brines are observed. Prokaryotic densities in the salterns of Sečovlje (Slovenia) and Ston (Croatia) are an order of magnitude lower than in modern saltern crystallizers. This is probably due to the much shorter residence time of the brine in the traditionally operated salterns. In China, red saltern brines were documented earlier: in Li Shizhen's compendium of Materia Medica Ben Cao Kang Mu, completed in 1578 and based on older sources., (© FEMS 2019.)

The dynamic changes that take place along the phases of microbial growth (lag, exponential, stationary, and death) have been widely studied in bacteria at the molecular and cellular levels, but little is known for archaea. In this study, a high-throughput approach was used to analyze and compare the proteomes of two haloarchaea during exponential and stationary growth: the neutrophilic Haloferax volcanii and the alkaliphilic Natrialba magadii. Almost 2000 proteins were identified in each species (≈50% of the predicted proteome). Among them, 532 and 432 were found to be differential between growth phases in H. volcanii and N. magadii, respectively. Changes upon entrance into stationary phase included an overall increase in proteins involved in the transport of small molecules and ions, stress response, and fatty acid catabolism. Proteins related to genetic processes and cell division showed a notorious decrease in amount. The data reported in this study not only contributes to our understanding of the exponential-stationary growth phase transition in extremophilic archaea but also provides the first comprehensive analysis of the proteome composition of N. magadii. The MS proteomics data have been deposited in the ProteomeXchange Consortium with the dataset identifier JPST000395., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Environmental temperature is one of the most important factors for the growth and survival of microorganisms. Here we describe a novel extremely halophilic archaeon (haloarchaea) designated as strain CBA1119 T isolated from solar salt. Strain CBA1119 T had the highest maximum and optimal growth temperatures (66 °C and 55 °C, respectively) and one of the largest genome sizes among haloarchaea (5.1 Mb). It also had the largest number of strain-specific pan-genome orthologous groups and unique pathways among members of the genus Natrinema in the class Halobacteria. A dendrogram based on the presence/absence of genes and a phylogenetic tree constructed based on OrthoANI values highlighted the particularities of strain CBA1119 T as compared to other Natrinema species and other haloarchaea members. The large genome of strain CBA1119 T may provide information on genes that confer tolerance to extreme environmental conditions, which may lead to the discovery of other thermophilic strains with potential applications in industrial biotechnology.

The halophilic archaea (haloarchaea) live in hyersaline environments such as salt lakes, salt ponds and marine salterns. To cope with the salt stress conditions, haloarchaea have developed two fundamentally different strategies: the "salt-in" strategy and the "compatible-solute" strategy. Although investigation of the molecular mechanisms underlying the tolerance to high salt concentrations has made outstanding achievements, experimental study from the aspect of transcription is rare. In the present study, we monitored cellular physiology of Natrinema sp. J7-2 cells incubated in different salinity media (15%, 25% and 30% NaCl) from several aspects, such as cellular morphology, growth, global transcriptome and the content of intracellular free amino acids. The results showed that the cells were polymorphic and fragile at a low salt concentration (15% NaCl) but had a long, slender rod shape at high salt concentrations (25% and 30% NaCl). The cells grew best in 25% NaCl, mediocre in 30% NaCl and struggled in 15% NaCl. An RNA-seq analysis revealed differentially expressed genes (DEGs) in various salinity media. A total of 1,148 genes were differentially expressed, consisting of 719 DEGs (348 up-regulated and 371 down-regulated genes) between cells in 15% vs 25% NaCl, and 733 DEGs (521 up-regulated and 212 down-regulated genes) between cells in 25% vs 30% NaCl. Moreover, 304 genes were commonly differentially expressed in both 15% vs 25% and 25% vs30% NaCl. The DEGs were enriched in different KEGG metabolic pathways, such as amino acids, glycerolipid, ribosome, nitrogen, protoporphyrin, porphyrin and porhiniods. The intracellular predominant free amino acids consisted of the glutamate family (Glu, Arg and Pro), aspartate family (Asp) and aromatic amino acids (Phe and Trp), especially Glu and Asp.

Halophilic archaea are extremophiles, adapted to high-salt environments, showing a big biotechnological potential as enzyme, lipids and pigments producers. Four inert supports (perlite, vermiculite, polyurethane foam and glass fiber) were employed for solid-state fermentation (SSF) of the halophilic archaeon Natronococcus sp. TC6 to investigate biomass and esterase production. A very low esterase activity and high water activity were observed when perlite, vermiculite and polyurethane were used as supports. When glass fiber was employed, an important moisture loss was observed (8.6%). Moreover, moisture retention was improved by mixing polyurethane and glass fiber, resulting in maximal biomass and esterase production. Three halophilic archaea: Natronococcus sp. TC6, Halobacterium sp. NRC-1 and Haloarcula marismortui were cultured by submerged fermentation (SmF) and by SSF; an improvement of 1.3- to 6.2-fold was observed in the biomass and esterase production when SSF was used. Growth was not homogeneous in the mixture, but was predominant in the glass fiber thus was probably because the glass fiber provides a holder to the cells, while the polyurethane acts as an impregnation medium reservoir. To the best of our knowledge, this work is the first report on haloarchaea cultivation by SSF aiming biomass and esterase/lipase activity production.

Pulp mill wastewater generated from wheat straw is characterized as high alkalinity and very high COD pollution load. A naturally developed microbial community in a pulp mill wastewater storage pool that had been disused were investigated in this study. Owing to natural evaporation and a huge amount of lignocellulose's deposition, the wastewater sediment contains high concentrations of organic matters and sodium ions, but low concentrations of chloride and carbonate. The microbiota inhabiting especially anaerobic community, including methanogenic arhcaea and cellulolytic species, was studied. All archaeal sequences fall into 2 clusters of family Halobacteriaceae and methanogenic archaeon in the phylum Euryarchaeota. In the methanogenic community, phylogenetic analysis of methyl coenzyme M reductase A (mcrA) genes targeted to novel species in genus Methanoculleus or novel genus of order Methanomicrobiales. The predominance of Methanomicrobiales suggests that methanogenesis in this system might be driven by the hydrogenotrophic pathway. As the important primary fermenter for methane production, the cellulolytic community of enzyme GHF48 was found to be dominated by narrower breadth of novel clostridial cellulase genes. Novel anoxic functional members in such extreme sediment provide the possibility of enhancing the efficiency of anoxic treatment of saline and alkaline wastewaters, as well as benefiting to the biomass transformation and biofuel production processes., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Natrinema sp. J7-2 is an extreme haloarchaeon capable of growing on synthetic media without amino acid supplements. Here we report the complete genome sequence of Natrinema sp. J7-2 which is composed of a 3,697,626-bp chromosome and a 95,989-bp plasmid pJ7-I. This is the first complete genome sequence of a member of the genus Natrinema. We demonstrate that Natrinema sp. J7-2 can use gluconate, glycerol, or acetate as the sole carbon source and that its genome encodes complete metabolic pathways for assimilating these substrates. The biosynthetic pathways for all 20 amino acids have been reconstructed, and we discuss a possible evolutionary relationship between the haloarchaeal arginine synthetic pathway and the bacterial lysine synthetic pathway. The genome harbors the genes for assimilation of ammonium and nitrite, but not nitrate, and has a denitrification pathway to reduce nitrite to N(2)O. Comparative genomic analysis suggests that most sequenced haloarchaea employ the TrkAH system, rather than the Kdp system, to actively uptake potassium. The genomic analysis also reveals that one of the three CRISPR loci in the Natrinema sp. J7-2 chromosome is located in an integrative genetic element and is probably propagated via horizontal gene transfer (HGT). Finally, our phylogenetic analysis of haloarchaeal genomes provides clues about evolutionary relationships of haloarchaea.

Natronomonas pharaonis is an archaeon adapted to two extreme conditions: high salt concentration and alkaline pH. It has become one of the model organisms for the study of extremophilic life. Here, we present a genome-scale, manually curated metabolic reconstruction for the microorganism. The reconstruction itself represents a knowledge base of the haloalkaliphile's metabolism and, as such, would greatly assist further investigations on archaeal pathways. In addition, we experimentally determined several parameters relevant to growth, including a characterization of the biomass composition and a quantification of carbon and oxygen consumption. Using the metabolic reconstruction and the experimental data, we formulated a constraints-based model which we used to analyze the behavior of the archaeon when grown on a single carbon source. Results of the analysis include the finding that Natronomonas pharaonis, when grown aerobically on acetate, uses a carbon to oxygen consumption ratio that is theoretically near-optimal with respect to growth and energy production. This supports the hypothesis that, under simple conditions, the microorganism optimizes its metabolism with respect to the two objectives. We also found that the archaeon has a very low carbon efficiency of only about 35%. This inefficiency is probably due to a very low P/O ratio as well as to the other difficulties posed by its extreme environment.

Halophilic Archaea cultured from ancient fluid inclusions in a 90-m-long (0- to 100,000-year-old) salt core from Death Valley, California, demonstrate survival of bacterial cells in subsurface halite for up to 34,000 years. Five enrichment cultures, representing three genera of halophilic Archaea (Halorubrum, Natronomonas and Haloterrigena), were obtained from five surface-sterilized halite crystals exclusively in one section of the core (13.0-17.8 m; 22,000-34,000 years old) containing perennial saline lake deposits. Prokaryote cells were observed microscopically in situ within fluid inclusions from every layer that produced culturable cells. Another 876 crystals analysed from depths of 8.1-86.7 m (10,000-100,000 years old) failed to yield live halophilic Archaea. Considering the number of halite crystals tested (culturing success of 0.6%), microbial survival in fluid inclusions in halite is rare and related to the paleoenvironment, which controls the distribution and abundance of trapped microorganisms. Two cultures from two crystals at 17.8 m that yielded identical 16S rRNA sequences (genus: Haloterrigena) demonstrate intra-laboratory reproducibility. Inter-laboratory reproducibility is shown by two halophilic Archaea (genus: Natronomonas), with 99.3% similarity of 16S rRNA sequences, cultured from the same core interval, but at separate laboratories.

Optimization of media components for the growth and biomass production of Halobacterium salinarum VKMM 013 was carried out using response surface methodology. A second order quadratic model was estimated and media components were determined based on quadratic regression equation generated by model. These were 6.35 g L(-1) of KCl, 9.70 g L(-1) of MgSO(4), 13.38 g L(-1) of gelatin and 12.00 g L(-1) of soluble starch in nutrient broth supplemented with artificial seawater with 20% (w/v) of NaCl. In these optimal conditions, the obtained cell concentration of 0.746 g L(-1) dry weight was in agreement with the predicted cell concentration. The optimized media significantly shortened the time required for cell culture to reach the stationary phase while providing a nearly 2.4-fold increase in biomass production. Furthermore, in cell cultures of three other halophilic archaea the use of optimized media enhanced growth rate and provided high-cell density.

Antibiotics and bile salts have been used to differentiate between heterotrophic activity of halophilic Archaea and Bacteria in saltern ponds. In NaCl-saturated brines of crystallizer ponds, most activity was attributed to Archaea. Following the recent isolation of Haloquadratum, the dominant archaeon in the salterns (reported to be sensitive to chloramphenicol and erythromycin), and the discovery of Salinibacter, a representative of the Bacteria, in the same ecosystem, reevaluation of the earlier data is required. The authors measured amino acid incorporation by Haloquadratum and Salinibacter suspended in crystallizer brine to investigate the suitability of antibiotics and bile salts to distinguish between archaeal and bacterial activities. The amino acid uptake rate per cell in Salinibacter was two orders of magnitude lower than that of Haloquadratum under the same conditions. Salinibacter was inhibited by chloramphenicol, erythromycin, and deoxycholate, but not by taurocholate. Erythromycin did not inhibit incorporation by Haloquadratum, but moderate inhibition was found by chloramphenicol at 10-50 microg mL(-1). Deoxycholate was highly inhibitory, but only partial inhibition was obtained in the presence of 25 microg mL(-1) taurocholate. Inhibition by chloramphenicol and taurocholate increased with increasing salt concentration. Erythromycin and taurocholate proved most valuable to differentiate between archaeal and bacterial activities in saltern brines.

An extremely halophilic archaeon was isolated from a sample of the brine-sediment interface of the Shaban Deep in the northern Red Sea. Phylogenetic analysis of the 16S rRNA gene sequence revealed a close proximity to Halorhabdus utahensis (99.3%), the sole species of the genus Halorhabdus. Strain SARL4B(T) formed non-pigmented colonies and showed optimum growth at 45 degrees C, in 27% (w/v) NaCl and at pH 6.5-7.0. This organism utilized a few complex substrates, such as yeast extract and starch, for growth. Strain SARL4B(T) grew under anaerobic and microaerophilic conditions but grew extremely poorly under aerobic conditions. The ether lipids were diphytanyl derivatives. The DNA G+C content of the type strain was 61.7 mol%. On the basis of the phylogenetic data and physiological and biochemical characteristics, strain SARL4B(T) represents a novel species of the genus Halorhabdus, for which the name Halorhabdus tiamatea is proposed. The type strain is SARL4B(T) (=DSM 18392(T)=JCM 14471(T)). An emended description of the genus Halorhabdus is also proposed.

Aims: The effect of various nitrogen sources and nutritional starvation was examined on the production of an extracellular protease secreted by the haloalkaliphilic archaeon Natrialba magadii., Methods and Results: Cell growth and proteolytic activity were measured in cells grown with different nitrogen sources. Proteolytic activity was produced in complex and easily metabolized nitrogen sources such as yeast extract, casein and casamino acids; meanwhile, ammonium repressed enzyme production. The time course and amount of protease accumulated showed an inverse correlation with growth rate and nutrient concentration. Starvation did not induce extracellular protease production., Conclusion: The accumulation of Nab. magadii extracellular protease is stimulated by nutrient limitation and slow growth rate indicating that it is probably induced in response to a deficit in the energetic status of the cells. Nutritional starvation did not induce protease accumulation suggesting that de novo synthesis of this protease and/or factor/s necessary for its activation are required. This enzyme may be regulated by nitrogen catabolite repression and it does not require protein substrates for induction., Significance and Impact of the Study: These results contribute to the basic knowledge on protease regulation in haloalkaliphilic archaea and will help to optimize the production of this extremozyme for biotechnological applications such as protease-catalysed peptide synthesis.

A total of forty-five halophilous microorganisms were isolated from the sediment of saline lake in Qinghai Province, among which, filamentous fungus F16 showed the highest activity of anti-microorganism and anti-tumor. The ethyl acetate extract of F16 culture filtrate showed a strong cytotoxicity, and could inhibit the growth of four kinds of bacteria, especially Escherichia coli. When the concentration of the crude extract was 50 microg x ml(-1), the inhibition rate to liver cancer cell BEL7402 reached 76. 91%. The optimal temperature for F16 growth was 15 degrees C , and the increase of salt concentration in media would inhibit its growth. When the concentration of salt surpassed 15% , F16 could not survive. F16 grew well when the pH value ranged from 5 to 9.

Aims: Isolation and screening of extreme halophilic archaeon producing extracellular haloalkaliphilic protease and optimization of culture conditions for its maximum production., Methods and Results: Halogeometricum sp. TSS101 was isolated from salt samples and screened for the secretion of protease on gelatin and casein plates containing 20% NaCl. The archaeon was grown aerobically in a 250 ml flask containing 50 ml of (w/v) NaCl 20%; MgCl(2) 1%; KCl 0.5%; trisodium citrate 0.3%; and peptone 1%; pH 7.2 at 40 degrees C on rotary shaker. The production of enzyme was investigated at various pH, temperatures, NaCl concentrations, metal ions and different carbon and nitrogen sources. The partially purified protease had activity in a broad pH range (7.0-10.0) with optimum activity at pH 10.0 and a temperature (60 degrees C). The enzyme was thermostable and retained 70% initial activity at 80 degrees C. Maximum protease production occurred at 40 degrees C in a medium containing 20% NaCl (w/v) and 1% skim milk powder after 84 h in shaking culture. Enzyme secretion was observed at a broad pH range of 7.0-10.0. Addition of CaCl(2) (200 mmol) to the culture medium enhanced the production of protease. Protein rich flours proved to be cheap and good alternative source for enzyme production. Different osmolytes were tested for the growth and production of haloalkaliphilc protease and found that betaine and glycerol enhanced growth without secretion of the protease. Immobilization studies showed that whole cells immobilized in 2% alginate beads were stable up to 10 batches and able to secrete the protease, which attained maximum production within 60 h under shaking conditions., Conclusions: Halogeometricum sp. TSS101 secreted an extracellular haloalkaliphilic and thermostable protease. The optimum conditions required for maximum production are 20% NaCl, 1% skim milk powder and temperature at 40 degrees C. Addition of CaCl(2) (200 mmol) enhanced the enzyme production. Immobilization of whole cells in absence of NaCl proved to be useful for continuous production of haloalkaliphilic protease., Significance and Impact of the Study: The low cost protein rich flours were used as an alternative carbon and nitrogen sources for enzyme production. Immobilization of halophilic cells in alginate beads can be used in continuous production of halophilic enzyme. The halophilic and thermostable protease from Halogeometricum sp. TSS101 is good source for industrial applications and can be a suitable source for preparation of fish sauce.

Haloarchaea have developed into model organisms that are utilized to study many biological processes. Examples are the mechanisms of chromosome maintenance, gene expression and its regulation, protein export and degradation, and motility and sensing. In addition to the analysis of model species like Halobacterium salinarum and Haloferax volcanii, natural communities have been characterized. Halophilic Archaea were found in low-salt environments and are thus more widespread than previously thought.

Members of the family Halobacteriaceae in the domain Archaea are obligate extreme halophiles. They occupy a variety of hypersaline environments, and their cellular biochemistry functions in a nearly saturated salty milieu. Despite extensive study, a detailed analysis of their growth kinetics is missing. To remedy this, Arrhenius plots for 14 type species of the family were generated. These organisms had maximum growth temperatures ranging from 49 to 58 degrees C. Nine of the organisms exhibited a single temperature optimum, while five grew optimally at more than one temperature. Generation times at these optimal temperatures ranged from 1.5 h (Haloterrigena turkmenica) to 3.0 h (Haloarcula vallismortis and Halorubrum saccharovorum). All shared an inflection point at 31 +/- 4 degrees C, and the temperature characteristics for 12 of the 14 type species were nearly parallel. The other two species (Natronomonas pharaonis and Natronorubrum bangense) had significantly different temperature characteristics, suggesting that the physiology of these strains is different. In addition, these data show that the type species for the family Halobacteriaceae share similar growth kinetics and are capable of much faster growth at higher temperatures than those previously reported.

The major phospholipids of Halorubrum lacusprofundi grown at 25 degrees C were archaeol phosphatidylglycerol, archaeol phosphatidylglycerylsulphate and archaeol phosphatidylglycerylphosphate methyl ester. Glycolipids included a monoglycosyl archaeol and the sulphate ester of a diglycosyl archaeol. Cultures grown at 12 degrees C contained the same suite of phospho- and glycolipids, with the addition of a series of unsaturated analogues with up to six double bonds. The patterns of unsaturation were similar for all the phospholipid series, but a different pattern occurred in the glycolipids. The analytical techniques used in this study allow facile detection of unsaturated archaeal cell membrane lipids that are degraded by commonly used chemical derivatization procedures.

Haloarchaea are the dominant microbial flora in hypersaline waters with near-saturating salt levels. The haloarchaeal diversity of an Australian saltern crystallizer pond was examined by use of a library of PCR-amplified 16S rRNA genes and by cultivation. High viable counts (10(6) CFU/ml) were obtained on solid media. Long incubation times (> or =8 weeks) appeared to be more important than the medium composition for maximizing viable counts and diversity. Of 66 isolates examined, all belonged to the family Halobacteriaceae, including members related to species of the genera Haloferax, Halorubrum, and Natronomonas. In addition, isolates belonging to a novel group (the ADL group), previously detected only as 16S rRNA genes in an Antarctic hypersaline lake (Deep Lake), were cultivated for the first time. The 16S rRNA gene library identified the following five main groups: Halorubrum groups 1 and 2 (49%), the SHOW (square haloarchaea of Walsby) group (33%), the ADL group (16%), and the Natronomonas group (2%). There were two significant differences between the organisms detected in cultivation and 16S rRNA sequence results. Firstly, Haloferax spp. were frequently isolated on plates (15% of all isolates) but were not detected in the 16S rRNA sequences. Control experiments indicated that a bias against Haloferax sequences in the generation of the 16S rRNA gene library was unlikely, suggesting that Haloferax spp. readily form colonies, even though they were not a dominant group. Secondly, while the 16S rRNA gene library identified the SHOW group as a major component of the microbial community, no isolates of this group were obtained. This inability to culture members of the SHOW group remains an outstanding problem in studying the ecology of hypersaline environments.

Organic solvent tolerance was tested in type strains of type species of the sixteen genera of Halobacteriaceae, the halophilic archaea. Most of the strains were observed to grow in the presence of hexylether (log Pow=5.1), but none grew in the presence of n-octane (log Pow=4.9) except Halogeometricum borinquense JCM 10706T and Halorubrum saccharovorum JCM 8865T. On the other hand, two strains, Haloarcula spp. OHF-1 and 2 isolated from a French solar salt were found to show stronger tolerance even to isooctane (log Pow=4.8). Growth of some strains was retarded by the presence of n-decane but reached to the same cell densities at late stationary phase. Final cell densities of some strains were greatly repressed by the presence of the solvent.

Proteolytic activity and a subtilisin inhibitor (NSI) were detected in Natrialba magadii cells. The proteolytic activity was due to two different proteases: a approximately 90-kDa metallo protease (NMP) produced during exponential growth and a 246-kDa serine protease (NSP) detected in the stationary phase. Both proteases were detected in the cytosolic fraction. NSI activity was maximal during early stages of growth and decreased in the stationary phase. NSI is a 35-kDa thermosensitive protein; it inhibits NSP activity but has no effect on NMP, and it was detected as a soluble or membrane-bound protein depending on the growth phase. Our results suggest that NSI may regulate NSP activity in vivo and that this protease may have a role in stationary phase cells. To our knowledge, this is the first report on the occurrence of protease inhibitors in Archaea.

The extremely halophilic archaeon, Halorhabdus utahensis, isolated from the Great Salt Lake, Utah, produced beta-xylanase and beta-xylosidase activities. Both enzymes were active over a broad NaCl range from near zero to 30% NaCl when tested with culture broth. A broad NaCl optimum was observed for beta-xylanase activity between 5% and 15% NaCl, while beta-xylosidase activity was highest at 5% NaCl. Almost half of the maximum activities remained at 27%-30% NaCl for both enzyme activities. When dialyzed culture supernatant and culture broth were employed for determination of beta-xylanase and beta-xylosidase stabilities, approximately 55% and 83% of the initial beta-xylanase and beta-xylosidase activities, respectively, remained after 24 h incubation at 20% NaCl. The enzymes were also shown to be slightly thermophilic; beta-xylanase activity exhibiting two optima at 55 degrees and 70 degrees C, while beta-xylosidase activity was optimal at 65 degrees C. SDS-PAGE and zymogram techniques revealed the presence of two xylan-degrading proteins of approximately 45 and 67 kDa in culture supernatants. To our knowledge, this paper is the first report on hemicellulose-degrading enzymes produced by an extremely halophilic archaeon.

A halophilic archaeon has been isolated from unsterilized salt crystals taken from the 250-million-year-old Salado formation in southeastern New Mexico. This microorganism grows only on defined media supplemented with either a combination of acetate and glycerol, glycerol and pyruvate, or pyruvate alone. The archaeon is unable to grow on complex media or to use carbohydrates, amino acids, fats, proteins, or nucleic acids for growth. Unlike other halophilic microbes, this organism possesses four glycolipids, two of which may be novel. The microbe is unique in that it has three dissimilar 16S rRNA genes. Two of the three genes show only 97% similarity to one another, while the third gene possesses only 92%-93% similarity to the other two. Inferred phylogenies indicate that the organism belongs to a deep branch in the line of Haloarcula and Halorhabdus. All three lines of taxonomic evidence: phenotype, lipid patterns, and phylogeny, support creation of a new genus and species within the halophilic Archaea. The name suggested for this new genus and species is Halosimplex carlsbadense. The type strain is 2-9-1(T) (= ATCC BAA-75 and JCM 11222) as written in the formal description.

A novel extremely halophilic archaeon was isolated from Lake Tebenquiche, situated in the northern part of the Atacama Saltern, Chile. The cells of these micro-organisms were mostly irregularly disc-shaped. They grew in medium containing saturated concentrations of NaCl and did not require magnesium for optimal growth. The polar lipid composition revealed the presence of mannosyl-2-sulfate-(1-4)-glycosyl-archaeol, the main glycolipid of the genus Halorubrum, and two new glycolipids. The G+C content of the DNA was 63.2 mol%. Phylogenetic analysis of the 16S rRNA gene placed strain ALT6-92T within the Halorubrum cluster. The low DNA-DNA hybridization value justified classification in a new species for which the name Halorubrum tebenquichense sp. nov. is proposed. The type strain is ALT6-92T (= CECT 5317T = DSM 14210T).

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.

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.

Many representatives of the family Halobacteriaceae ("halobacteria") excrete halophilic bacteriocins (halocins) that inhibit the growth of other halobacteria. In spite of the fact that halocin production is widespread among the Halobacteriaceae, no information is available on their ecological significance. To test whether halocins may play a role in the interspecies competition between different types of halobacteria in saltern crystallizer ponds inhabited by dense communities of these red halophiles, we assayed for halocins active against a variety of halobacteria in salterns from different locations worldwide. Detection of halocin activity was based on the inhibition of growth of indicator organisms on agar plates, the decreased incorporation of radiolabeled substrates, and microscopic examinations. No halocin activity was detected in any of the brines examined, in spite of the fact that halocin production was demonstrated in cultures of most microorganisms isolated from these brines. Thus, the contribution of halocins in the competition between different halobacteria in hypersaline aquatic environments is probably negligible.

Moderately halophilic bacteria are microorganisms that grow optimally in media containing 3%-15% (w/v) salt. They are represented by a heterogeneous group of microorganisms included in many different genera. Gram-negative moderately halophilic bacteria have been studied in more detail, but studies on gram-positive species are more scarce. Recent studies carried out by our research group on gram-positive moderate halophiles have permitted clarifying their taxonomic and phylogenetic position and describing new species. Thus, we have isolated six strains from ponds of salterns that show phenotypic and genotypic characteristics similar to those of Nesterenkonia halobia (formerly Micrococcus halobius), a moderately halophilic gram-positive coccus that was described on the basis of a single strain. Our data demonstrate quite clearly that they are members of this species and contribute to a better description of these moderately halophilic cocci. Similarly, a study of a large number of gram-positive moderately halophilic rods that were able to produce endospores led us to describe a new species, designated Bacillus salexigens. Further, isolates grouped in other three phenons, obtained by numerical taxonomy analysis and showing phenotypic features quite similar to those of this species, represent different genomovars, with very low DNA-DNA homology. Although they might represent additional new species, it will be necessary to determine new phenotypic features to differentiate them from previously described Bacillus species. We have also studied the viability of some old enrichments provided by B.E. Volcani, which were set up in 1936. We isolated 31 gram-positive motile endospore-forming rods that, according to their phenotypic characteristics, could represent a new species of the genus Bacillus.

A novel 1-->1 alpha-linked glucose disaccharide with sulfate at C-2 of one of the glucose moieties, 1-(2-O-sulfo-alpha-D-glucopyranosyl)-alpha-D-glycopyranose, was found to be the major organic solute accumulated by a Natronococcus sp. and several Natronobacterium species. The concentration of this novel disaccharide, termed sulfotrehalose, increased with increasing concentrations of external NaCl, behavior consistent with its identity as an osmolyte. A variety of noncharged disaccharides (trehalose, sucrose, cellobiose, and maltose) were added to the growth medium to see if they could suppress synthesis and accumulation of sulfotrehalose. Sucrose was the most effective in suppressing biosynthesis and accumulation of sulfotrehalose, with levels as low as 0.1 mM being able to significantly replace the novel charged osmolyte. Other common osmolytes (glycine betaine, glutamate, and proline) were not accumulated or used for osmotic balance in place of the sulfotrehalose by the halophilic archaeons.

As a first step in the development of a reporter system for gene expression in halophilic archaea, a beta-galactosidase was purified 140-fold from Haloferax alicantei (previously phenon K, strain Aa2.2). An overproducing mutant was first isolated by UV mutagenesis and screening on agar plates containing X-Gal substrate. Cytoplasmic extracts of the mutant contained 25-fold higher enzyme levels than the parent. Purification of the active enzyme was greatly facilitated by the ability of sorbitol to stabilise enzyme activity in the absence of salt, which allowed conventional purification methods (e.g., ion-exchange chromatography) to be utilised. The enzyme was optimally active at 4 M NaCl and was estimated to be 180 +/- 20 kDa in size, consisting of two monomers (each 78 +/- 3 kDa). It cleaves several different beta-galactoside substrates such as ONP-Gal, X-Gal and lactulose, but not lactose, and also has beta-D-fucosidase activity. No beta-glucosidase, beta-arabinosidase or beta-xylosidase activity could be detected. The amino-acid sequence at the N-terminus and of four proteolytic products has been determined.

The transcription of the 14 gvp genes of the gas-vesicle-encoding mc-vac region was investigated, using RNA from 25% and 15% (w/v) salt cultures of the moderately halophilic archaeon Haloferax mediterranei. Transcription occurred only from two promoters, located in front of the mc-gvpA and mc-gvpD genes. In both cultures transcripts spanning the entire mc-gvpDEFGHIJKLM transcription unit were formed only during the exponential growth phase. Amounts of these transcripts were larger in the 25% salt culture, in which the 2.0 kb mc-gvpD transcripts were also synthesized during the stationary phase. The levels of the mc-gvpD transcripts and of the 324 nt mc-gvpA mRNA increased in parallel during the stationary phase of the 25% salt culture. Only under these conditions were mRNAs spanning the entire mc-gvpACNO transcription unit observed, and gas-vesicles were formed. Investigation of the influence of the mc-gvpDE genes on both mc-vac promoters in transformants revealed that by themselves they were nearly inactive. The addition of mc-gvpE, however, resulted in a high level of constitutively produced mc-gvpA and mc-gvpD mRNA, indicating a transcriptional activator function for the mc-gvpE product.

Many members of the Halobacteriaceae are inhibited by quinolone compounds, which inhibit type II DNA topoisomerase. Ciprofloxacin was the most potent inhibitor, followed by ofloxacin and norfloxacin. Ciprofloxacin concentration between 25 and 60 micrograms/ml caused 50% inhibition of the growth of most Haloferax and Haloarcula species. Halobacterium species were less sensitive, At sublethal concentrations, formation of elongated and/or swollen cells was observed in many species. The alkaliphilic Natronobacterium pharaonis was very sensitive (50% inhibition by ciprofloxacin, ofloxacin, and norfloxacin at concentrations between 4 and 15 micrograms/ml). The resistance of many members of the Halobacteriaceae to high concentrations of quinolone compounds may in part be due to the high magnesium concentrations present in the growth media. Haloferax volcanii was sensitive to 40 micrograms/ml ciprofloxacin when grown at suboptimal magnesium concentrations (0.1 M), but was hardly affected by 100 micrograms/ml of the inhibitor when grown in the presence of 0.5-0.75 M MgCl2. It is suggested that the putative archaeal type II DNA topoisomerase has properties similar to those of the enzyme from Bacteria, although its sensitivity to quinolone antimicrobial compounds may be lower.

We report the presence of long stretches of tandem repeats in the genome of the halophilic Archaea Haloferax mediterranei and Haloferax volcanii. A 30 bp sequence with dyad symmetry (including 5 bp inverted repeats) was repeated in tandem, interspersed with 33-39 bp unique sequences. This structure extends for long stretches--1.4 kb at one location in H. mediterranei chromosome and about 3 kb in the H. volcanii chromosome. The tandem repeats (designated TREPs) show a similar distribution in both organisms, appearing once or twice in the H. volcanii and H. mediterranei chromosomes, and once in the largest, probably essential megaplasmid of each organism but not in the smaller replicons. Sequencing of the structures in both H. volcanii replicons revealed an extremely high sequence conservation in both replicons within the species, as well as in the different organisms. Homologous sequences have also been found in other more distantly related halophilic members of the Archaea. Transformation of H. volcanii with a recombinant plasmid containing a 1.1 kb fragment of the TREPs produced significant alterations in the host cells, particularly in terms of cell viability. The introduction of extra copies of TREPs within the vector significantly alters the distribution of the genome among the daughter cells, as observed by DAPI staining. Although the precise biological role cannot be completely ascertained, all the data conform with the tandem repeats being involved in replicon partitioning in halobacteria.

Ketohexokinase (ATP:D-fructose 1-phosphotransferase [EC 2.7.1.3]), detected for the first time in a prokaryote, i.e., the extreme halophile Haloarcula vallismortis, was isolated and characterized from the same archaebacterium. This enzyme was characterized with respect to its molecular mass, amino acid composition, salt dependency, immunological cross-reactivity, and kinetic properties. Gel filtration and sucrose density gradient centrifugation revealed a native molecular mass of 100 kDa for halobacterial ketohexokinase, which is larger than its mammalian counterpart. The enzyme could be labeled by UV irradiation in the presence of [ gamma-32P]ATP, suggesting the involvement of a phosphoenzyme intermediate. Other catalytic features of the enzyme were similar to those of its mammalian counterparts. No antigenic cross-reactivity could be detected between the H. vallismortis ketohexokinase and the ketohexokinases from different rat tissues.

The CO2-fixing enzyme of Calvin cycle ribulose-1,5-bisphosphate-carboxylase/oxygenase has been isolated from a halophilic bacterium, Haloferax mediterranei grown heterotrophically. A homogeneous preparation was obtained from sonicated extract of the cells by three steps, resulting in a specific activity of 52 nmol.min-1.mg protein-1. The physicochemical and catalytic properties of the enzyme were studied. The halobacterial ribulose-bisphosphate carboxylase is an oligomer of 54-kDa and 14-kDa subunits as detected by SDS/PAGE. By sucrose-density-gradient centrifugation, the molecular mass of the enzyme was estimated as approximately 500 kDa indicating a hexadecameric nature. No evidence for an additional form of the enzyme devoid of small subunits was obtained. The enzyme required Mg2+ for activity, KCl for activity and stability, and an optimal pH of 7.8. In contrast to many halophilic proteins, ribulose-bisphosphate carboxylase from H. mediterranei is not an acidic protein. From the comparison of amino acid composition of halobacterial enzyme with its counterparts from a few eukaryotic and eubacterial sources, the S delta Q values showed that these proteins share some compositional similarities.

The moderately halophilic, obligately anaerobic eubacteria Haloanaerobium praevalens DSM 2228 and Sporohalobacter marismortui ATCC 35420 are able to reduce a variety of nitrosubstituted aromatic compounds at a high rate to the corresponding amines. Compounds degraded included nitrobenzene, o-nitrophenol, m-nitrophenol, p-nitrophenol, nitroanilines, 2,4-dinitrophenol, and 2,4-dinitroaniline. Most of these compounds, when added at concentrations of 50 to 100 mg/liter, were completely transformed within 24 h, but at the highest concentrations growth rates were somewhat lowered. Growth of H. praevalens in the presence of 14C-labeled p-nitrophenol showed that the compound was not incorporated by the cells or degraded to acid-volatile compounds.

Two main physiological groups of bacteria are known which are adapted to high saline environments, moderate and extreme halophiles. In order to clarify some aspects of the competition between these two groups in their natural habitats, continuous cultures were used to provide a changing spectrum of conditions of salt concentration, temperature, and nutrient concentration (dilution rate). The effects of these parameters on natural solar saltern populations were studied. Complex media were used to increase the range of competing microorganisms. Nineteen strains of halophilic bacteria were isolated and studied with respect to their growth response at different salt concentrations. The temperature seemed to be the decisive factor within the range of salt concentrations studied (20-30%, w/v), the moderate halophiles being favored by low temperatures. Within this group, motile, gram-negative rods, and spiral forms were the predominant morphological types. In general, microorganisms that showed high growth rates in batch cultures predominated in continuous cultures with high dilution rates (high nutrient concentrations); those that grew slowly in batch cultures predominated in cultures with low dilution rates.