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Recommended design for more accurate duplication of natural conditions in salt marsh creation.

Construction of 653 ha of salt marsh habitat from dredged material near the Aransas National Wildlife Refuge, Texas, has been proposed, with the goal of increasing the area of habitat available to endangered whooping cranes ( Grus americana). We assessed prototype created wetlands, and their similarity to natural reference sites, in terms of topography, vegetation, and hydrology. The created sites were steeply sloped relative to natural sites and were dominated by monotypic stands of Spartina alterniflora. Natural sites were dominated by vegetation more tolerant of desiccation and hypersalinity and by unvegetated salt pans. Differences in vegetation communities and distributions of habitat types resulted from efforts to enhance habitat diversity in created marsh cells through manipulation of marsh topography. However, the scale at which this diversity occurred in natural marsh of the study area was not considered. When constructing wetlands in cellular configurations, we recommend creation of large complexes of adjoining, hydrologically linked, cells wherein the desired habitat diversity is created at the scale of the entire complex, rather than within a single cell. Suggested design modifications would increase the similarity of created marshes to natural reference sites, potentially improving habitat function.

Animals↗

Salt-dependent expression of ammonium assimilation genes in the halotolerant yeast, Debaryomyces hansenii.

Debaryomyces hansenii is adapted to grow in saline environments, accumulating high intracellular Na(+) concentrations. Determination of the DhGDH1-encoded NADP-glutamate dehydrogenase enzymatic activity showed that it increased in a saline environment. Thus, it was proposed that, in order to overcome Na(+) inhibition of enzyme activity, this organism possessed salt-dependent mechanisms which resulted in increased activity of enzymes pertaining to the central metabolic pathways. However, the nature of the mechanisms involved in augmented enzyme activity were not analyzed. To address this matter, we studied the expression of DhGDH1 and DhGLN1 encoding glutamine synthetase, which constitute the central metabolic circuit involved in ammonium assimilation. It was found that: (1) expression of DhGDH1 is increased when D. hansenii is grown in the presence of high NaCl concentrations, while that of DhGLN1 is reduced, (2) DhGDH1 expression in Saccharomyces cerevisiae takes place in a GLN3- and HAP2,3-dependent manner and (3) salt-dependent DhGDH1 and DhGLN1 expression involves mechanisms which are limited to D. hansenii and are not present in S. cerevisiae. Thus, salt-dependent regulation of the genes involved in central metabolic pathways could form part of a strategy leading to the ability to grow under hypersaline conditions.

Base Sequence↗

Intraspecific comparative analysis of the species Salinibacter ruber.

Salinibacter ruber is the first extremely halophilic member of the Bacteria domain of proven environmental relevance in hypersaline brines at or approaching NaCl saturation, that has been brought to pure culture. A collection of 17 strains isolated from five different geographical locations (Mallorca, Alicante, Ebro Delta, Canary Islands, and Peruvian Andes) were studied following the currently accepted taxonomic approach. Additionally, random amplification of genomic DNA led to the phenetic analysis of the intraspecific diversity. Altogether the taxonomic study indicated that S. ruber remained highly homogeneous beyond any geographical barrier. However, genomic fingerprints indicated that populations from different isolation sites could still be discriminated.

Bacterial Physiological Phenomena↗

Physiological responses of the halophilic archaeon Halobacterium sp. strain NRC1 to desiccation and gamma irradiation.

We report that the halophilic archaeon Halobacterium sp. strain NRC-1 is highly resistant to desiccation, high vacuum and 60Co gamma irradiation. Halobacterium sp. was able to repair extensive double strand DNA breaks (DSBs) in its genomic DNA, produced both by desiccation and by gamma irradiation, within hours of damage induction. We propose that resistance to high vacuum and 60Co gamma irradiation is a consequence of its adaptation to desiccating conditions. Gamma resistance in Halobacterium sp. was dependent on growth stage with cultures in earlier stages exhibiting higher resistance. Membrane pigments, specifically bacterioruberin, offered protection against cellular damages induced by high doses (5 kGy) of gamma irradiation. High-salt conditions were found to create a protective environment against gamma irradiation in vivo by comparing the amount of DSBs induced by ionizing radiation in the chromosomal DNA of Halobacterium sp. to that of the more radiation-sensitive Escherichia coli that grows in lower-salt conditions. No inducible response was observed after exposing Halobacterium sp. to a nonlethal dose (0.5 kGy) of gamma ray and subsequently exposing the cells to either a high dose (5 kGy) of gamma ray or desiccating conditions. We find that the hypersaline environment in which Halobacterium sp. flourishes is a fundamental factor for its resistance to desiccation, damaging radiation and high vacuum.

Cobalt Radioisotopes↗

Metabolic versatility of haloalkaliphilic bacteria from soda lakes belonging to the Alkalispirillum-Alkalilimnicola group.

Four new isolates were obtained from denitrifying enrichments with various electron donors using sediment samples from hypersaline soda lakes. Based on 16S rRNA gene analysis and DNA-DNA hybridization results, they were all identified as members of the Gammaproteobacteria closely associated with the Alkalispirillum-Alkalilimnicola group. Two isolates were obtained from samples enriched with nitrate as electron acceptor and H2 or polysulfide as electron donors, and another two strains were obtained with N2O as the electron acceptor and sulfide or acetate as electron donors. All four new isolates, together with the type strains of the genera Alkalispirillum and Alkalilimnicola originally described as obligate aerobes, were capable of anaerobic growth with acetate using either nitrate or N2O as electron acceptors. Their denitrification pathway, however, was disrupted at the level of nitrite. RuBisCO form I gene was detected and sequenced in the new isolates and in Alkalilimnicola halodurans but not in Alkalispirillum mobile. These data, together with the evidence of Oremland et al. (Appl Environ Microbiol 68:4795-4802, 2002) on the potential of Alkalilimnicola sp. MLHE-1 for autotrophic growth with arsenite as electron donor and nitrate as electron acceptor, demonstrate much higher metabolic diversity of this specific group of haloalkaliphilic Gammaproteobacteria than was originally anticipated.

Carbonates↗

Interrelationships between Dunaliella and halophilic prokaryotes in saltern crystallizer ponds.

Thanks to their often very high population densities and their simple community structure, saltern crystallizer ponds form ideal sites to study the behavior of halophilic microorganisms in their natural environment at saturating salt concentrations. The microbial community is dominated by square red halophilic Archaea, recently isolated and described as Haloquadratum walsbyi, extremely halophilic red rod-shaped Bacteria of the genus Salinibacter, and the unicellular green alga Dunaliella as the primary producer. We review here, the information available on the microbial community structure of the saltern crystallizer brines and the interrelationships between the main components of their biota. As Dunaliella produces massive amounts of glycerol to provide osmotic stabilization, glycerol is often postulated to be the most important source of organic carbon for the heterotrophic prokaryotes in hypersaline ecosystems. We assess here, the current evidence for the possible importance of glycerol and other carbon sources in the nutrition of the Archaea and the Bacteria, the relative contribution of halophilic Bacteria and Archaea to the heterotrophic activity in the brines, and other factors that determine the nature of the microbial communities that thrive in the salt-saturated brines of saltern crystallizer ponds.

Adaptation, Physiological↗

Halocins: are they involved in the competition between halobacteria in saltern ponds?

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.

Archaeal Proteins↗

Occurrence of virus-like particles in the Dead Sea.

Electron-microscopic examination of water samples from the hypersaline Dead Sea showed the presence of high numbers of virus-like particles. Between 0.9 and 7.3 x 10(7) virus-like particles ml(-1) were enumerated in October 1994 in the upper 20 m of the water column during the decline of a bloom of halophilic Archaea. Virus-like particles outnumbered bacteria by a factor of 0.9-9.5 (average 4.4). A variety of viral morphologies were detected, the most often encountered being spindle-shaped, followed by polyhedral and tailed phages. In addition, other types of particles were frequently found, such as unidentified algal scales, and virus-sized star-shaped particles. Water samples collected during 1995 contained low numbers of both bacteria and virus-like particles (1.9-2.6 x 10(6) and 0.8-4.6 x 10(7) ml(-1) in April 1995), with viral numbers sharply declining afterwards (less than 10(4) ml(-1) in November 1995-January 1996). It is suggested that viruses may play a major role in the decline of halophilic archaeal communities in the Dead Sea. an environment in which protozoa and other predators are absent.

Middle East↗

Distribution and diversity of halophilic bacteria in a subsurface salt formation.

The Waste Isolation Pilot Plant (WIPP) is a salt mine constructed 650 meters below the ground surface by the United States Department of Energy. The facility will be used for permanent disposal of transuranic wastes. This underground repository has been constructed in the geologically stable Permian age Salado salt formation. Of the wastes to be placed into the facility, 85% will be biodegradable cellulose. A 3-year survey of the bacterial populations existing within the facility was conducted. Bacterial populations were found to be heterogeneously distributed throughout the mine. Populations in some mine areas reached as high as 1.0 x 10(4) colony-forming units per gram of NaCl. The heterogeneous distribution of bacteria within the mine did not follow any recognizable pattern related to either age of the workings or to human activity. A biochemical comparison between ten known species of halophilic bacteria, and strains isolated from both the mine and nearby surface hypersaline lakes, showed the presence of extreme halophiles with wide biochemical diversity, some of which could prove to represent previously undescribed groups. The halophilic bacteria isolated from the mine were found to degrade cellulose and a wide variety of other carbon compounds. When exposed to two types of common laboratory paper, the cellulose-degrading halophiles attached to the substrate within 30 minutes of inoculation. Cultures enriched directly from a brine seep in the mine easily destroyed both papers and produced detectable amounts of oxalacetic and pyruvic acids. The combination of heterogeneity in the distribution of organisms, the presence of a physiologically diverse community, and the relatively slow metabolism of cellulose may explain several long-standing debates about the existence of microorganisms in ancient underground salt formations.

Acids↗

Novel haloarchaeal 16S rRNA gene sequences from Alpine Permo-Triassic rock salt.

Prokaryotic diversity in Alpine salt sediments was investigated by polymerase chain reaction (PCR) amplification of 16S rRNA genes, sequencing of cloned products, and comparisons with culturable strains. DNA was extracted from the residue following filtration of dissolved Permo-Triassic rock salt. Fifty-four haloarchaeal sequences were obtained, which could be grouped into at least five distinct clusters. Similarity values of three clusters to known 16S rRNA genes were less than 90%-95%, suggesting the presence of uncultured novel taxa; two clusters were 98% and 99% similar to isolates from Permo-Triassic or Miocene salt from England and Poland, and to Halobacterium salinarum, respectively. Some rock salt samples, including drilling cores, yielded no amplifiable DNA and no cells or only a few culturable cells. This result suggested a variable distribution of haloarchaea within different strata, probably consistent with the known geologic heterogeneity of Alpine salt deposits. We recently reported identical culturable Halococcus salifodinae strains in Permo-Triassic salt sediments from England, Germany, and Austria; together with the data presented here, those results suggest one plausible scenario to be an ancient continuous hypersaline ocean (Zechstein sea) populated by haloarchaea, whose descendants are found today in the salt sediments. The novelty of the sequences also suggested avoidance of haloarchaeal contaminants during our isolation of strains, preparation of DNA, and PCR reactions.

Cloning, Molecular↗

Evidence of interspecies hydrogen transfer from glycerol in saline environments.

Two halanaerobic bacteria--Halanaerobium saccharolytica subsp. senegalense and Halanaerobium sp. strain FR1H--produced acetate, H2, and CO2 from glycerol fermentation, but the glycerol consumption rate was low. In contrast, in the presence of the moderately halophilic hydrogenotrophic sulfate-reducing bacterium, Desulfohalobium retbaense, used as H2 scavenger in the coculture, glycerol oxidation by both halanaerobes significantly increased. Cocultures of both halanaerobes with D. retbaense on glycerol led to acetate, hydrogen sulfide, and CO2 production, whereas glycerol fermentation by the two strains led to the production of acetate, hydrogen, and CO2. The increased glycerol oxidation by H. saccharolytica and strain FRI H in coculture with D. retbaense resulted from low H2 partial pressure caused by the hydrogen-oxidizing activity of D. retbaense. These results provide the first evidence of interspecies hydrogen transfer in saline environments and indicate that this mechanism may play an important role in organic matter mineralization in hypersaline ecosystems.

Bacteria, Anaerobic↗

Cyanobacterial diversity in extreme environments in Baja California, Mexico: a polyphasic study.

Cyanobacterial diversity from two geographical areas of Baja California Sur, Mexico, were studied: Bahia Concepcion, and Ensenada de Aripez. The sites included hypersaline ecosystems, sea bottom, hydrothermal springs, and a shrimp farm. In this report we describe four new morphotypes, two are marine epilithic from Bahia Concepcion, Dermocarpa sp. and Hyella sp. The third, Geitlerinema sp., occurs in thermal springs and in shrimp ponds, and the fourth, Tychonema sp., is from a shrimp pond. The partial sequences of the 16S rRNA genes and the phylogenetic relationship of four cyanobacterial strains (Synechococcus cf. elongatus, Leptolyngbya cf. thermalis, Leptolyngbya sp., and Geitlerinema sp.) are also presented. Polyphasic studies that include the combination of light microscopy, cultures and the comparative analysis of 16S rRNA gene sequences provide the most powerful approach currently available to establish the diversity of these oxygenic photosynthetic microorganisms in culture and in nature.

Animals↗

Tissue distribution, effects of salinity acclimation, and ontogeny of aquaporin 3 in the marine teleost, silver sea bream (Sparus sarba).

The purpose of the present study was to ascertain the tissue-specific expression of the water channel protein, aquaporin 3 (AQP3), during salinity acclimation and larval development of silver sea bream (Sparus sarba). A cDNA fragment encoding aquaporin 3 (aqp3) from silver sea bream gill was cloned and from the deduced amino acid sequence a polyclonal antibody was prepared. AQP3 was found to be present in gill, kidney, liver, brain, heart, and spleen but not in whole blood. The abundance of AQP3 was significantly highest in gills of hypoosmotic (6 ppt) and isoosmotic (12 ppt) acclimated sea bream when compared to seawater (33 ppt) and hypersaline (50 ppt)- acclimated sea bream. Spleen tissue also displayed significantly high levels of AQP3 protein in hypoosmotic and isoosmotic salinities whereas the AQP3 abundance in brain, liver, heart, and kidney remained unchanged across the range of salinities tested. The ontogenetic profile of AQP3 was also investigated from developing sea bream larvae and AQP3 was first detected at 14 days posthatch (dph) and increased steadily up to 28-46 dph. In conclusion, this study has demonstrated that AQP3 expression is modulated in gill and spleen tissue of salinity acclimated sea bream and that it can be detected relatively early during larval development.

Acclimatization↗

Taxonomy and phylogeny of the xerophilic genus Wallemia (Wallemiomycetes and Wallemiales, cl. et ord. nov.).

The genus Wallemia comprises xerophilic species. Based on parenthesome ultrastructure it has been linked to the Filobasidiales (basidiomycetes). Species show a unique type of conidiogenesis, including basauxic development of fertile hyphae, segregation of conidial units more or less basipetally, and disarticulation of conidial units into mostly four arthrospore-like conidia. Wallemia is known from air, soil, dried food (causing spoilage), and salt. It can be isolated from hypersaline water of man-made salterns on different continents. Based on analyses of the nuclear small subunit ribosomal DNA (SSU rDNA) Wallemia has been placed into a highly supported clade together with Ustilaginomycetes and Hymenomycetes (Basidiomycota). Within this clade, it possesses an isolated position distantly related to the Filobasidiales and was characterized by numerous nucleotide substitutions not shared by any other fungus. Tests on xerotolerance indicated that Wallemia presents one of the most xerophilic fungal taxa. Xerotolerance is otherwise rare in the Basidiomycota. To acknowledge its unique morphology, evolution, and xerotolerance, a new basidiomycetous class Wallemiomycetes covering an order Wallemiales, is proposed. Based on differences in conidial size, xerotolerance, and sequence data of the rDNA internal transcribed spacer regions (ITS rDNA), at least three Wallemia species are segregated, identified as Wallemia ichthyophaga, Wallemia sebi, and Torula epizoa var. muriae, for which the combination Wallemia muriae is proposed. The three species are neotypified. Wallemia ichthyophaga differs from W. sebi and W. muriae in numerous nucleotides of the SSU and ITS rDNA. This high variation within Wallemia indicates existence of at least two cryptic genera not distinguishable by morphological characters.

Basidiomycota↗

Glycinebetaine as an osmoregulant and compatible solute in the marine cyanobacterium Spirulina subsalsa.

Glycinebetaine was found to be the major organic substrate accumulating under hypersaline growth conditions in the halotolerant cyanobacterium Spirulina subsalsa. In addition to its proposed role as osmolite, glycinebetaine is shown to specifically protect enzymatic activity. Glucose-6-phosphate dehydrogenase from S. subsalsa retained full activity in the presence of NaCl at concentrations as high as 1.5 M, provided that comparable concentrations of glycinebetaine were also present in the reaction mixture. A kinetic analysis indicated that glycinebetaine protected the enzyme against both NaCl-induced decrease in Vmax and reduction in affinity to glucose 6-phosphate. The alternative osmolites, glycerol and proline, protected the enzyme against the reduction in Vmax but not against the reduction in affinity to glucose 6-phosphate.

Betaine↗

Polar lipids of non-alkaliphilic Halococci.

Until recently, only one species of Halococcus has been recognized, namely, H. morrhuae, but a large number of extremely halophilic non-alkaliphilic cocci have now been isolated from hypersaline habitats in Spain and classified into four phenons (A-D); one of the phenon D strains has been classified as a new species, Halococcus saccharolyticus. Examination of the lipids of H. saccharolyticus and four strains of phenons A-C showed the presence in all of them of C20-C20 and C20-C25 diether molecular species of phosphatidylglycerophosphate (PGP), phosphatidylglycerol (PG) and phosphatidic acid (PA); a monounsaturated isoprenoid C20-C20 (phytanyl-phytenyl) species of PGP; a sulfated diglycosyl diphytanylglycerol (S-DGD) with structure 2,3-diphytanyl-1-(6-HSO3-mannosyl-1-2-glucosyl)-glycerol, which is identical to the S-DGD-1 in Haloferax mediterranei; a phosphoglycolipid (P-TGD) tentatively identified as a phytanyl-phytenyl-(H2PO3-galactosyl-mannosyl-glycosyl)-glyce rol, and two unidentified glycolipids present only in traces. No phosphatidylglycerosulfate (PGS) was detected in any of the strains examined. This pattern of lipids appears to be characteristic of the strains of Halococcus from salterns in Spain, but studies of a larger number and variety of Haloccus are necessary to establish this conclusion with certainty.

Glycolipids↗

Diphytanyl glycerol ether distributions in sediments of the Orca Basin.

Archaebacterially produced diphytanyl glycerol ether (DPGE) was examined in core sediments from the Orca Basin, an anoxic hypersaline basin in the northwestern Gulf of Mexico, to observe its spatial variability and potential origin. A differential extraction protocol was employed to quantify the isopranyl glycerol ethers associated with unbound, intermediate-bound, and kerogen-bound lipid fractions. Archaebacterial lipids were evident at all depths for the unbound and intermediate-bound fractions. Concentrations of DPGE ranged from 0.51 to 2.91 micrograms/g dry sediment at the surface and showed secondary maxima deeper in basin sediments. Intermediate-bound DPGE concentrations exhibited an inverse relationship to unbound DPGE concentrations. Kerogen-bound DPGE concentrations were normally below detection limits. Earlier studies describing the general homogeneity of lipid components within the overlying brine and at the brine/seawater interface suggest that the large-scale sedimentary DPGE variations observed in this study result from spatial and temporal variations in in situ production by methanogenic or extremely halophilic archaebacteria.

Archaea↗

Evidence for gammacerane as an indicator of water column stratification.

A new route for the formation of gammacerane from tetrahymanol is proposed; in addition to dehydration and hydrogenation, sulphurisation and early C-S cleavage are shown to be important in the pathway of formation, especially in marine sediments. Evidence is twofold. First, relatively large amounts of the gammacerane skeleton are sequestered in S-rich macromolecular aggregates formed by natural sulphurisation of functionalised lipids. Selective cleavage of polysulphide linkages with MeLi/MeI led to formation of 3-methylthiogammacerane, indicating that the gammacerane skeleton is primarily bound via sulphur at position 3, consistent with the idea that tetrahymanol (or the corresponding ketone) is the precursor for gammacerane. Second, upon mild artificial maturation of two sediments using hydrous pyrolysis, gammacerane is released from S-rich macromolecular aggregates by cleavage of the relatively weak C-S bonds. The stable carbon isotopic compositions of gammacerane and lipids derived from primary producers and green sulphur bacteria in both the Miocene Gessoso-solfifera and Upper Jurassic Allgau Formations indicate that gammacerane is derived from bacterivorous ciliates which were partially feeding on green sulphur bacteria. This demonstrates that anaerobic ciliates living at or below the chemocline are important sources for gammacerane, consistent with the fact that ciliates only biosynthesize tetrahymanol if their diet is deprived of sterols. This leads to the conclusion that gammacerane is an indicator for water column stratification, which solves two current enigmas in gammacerane geochemistry. Firstly, it explains why gammacerane is often found in sediments deposited under hypersaline conditions but is not necessarily restricted to this type of deposits. Secondly, it explains why lacustrine deposits may contain abundant gammacerane since most lakes in the temperate climatic zones are stratified during summer.

Animals↗