Halophiles are found in all three domains of life. Within the Bacteria we know halophiles within the phyla Cyanobacteria, Proteobacteria, Firmicutes, Actinobacteria, Spirochaetes, and Bacteroidetes. Within the Archaea the most salt-requiring microorganisms are found in the class Halobacteria. Halobacterium and most of its relatives require over 100–150 g/l salt for growth and structural stability. Also within the order Methanococci we encounter halophilic species. Halophiles and non-halophilic relatives are often found together in the phylogenetic tree, and many genera, families and orders have representatives with greatly different salt requirement and tolerance. A few phylogenetically coherent groups consist of halophiles only: the order Halobacteriales, family Halobacteriaceae (Euryarchaeota) and the anaerobic fermentative bacteria of the order Halanaerobiales (Firmicutes). The family Halomonadaceae (Gammaproteobacteria) almost exclusively contains halophiles. Halophilic microorganisms use two strategies to balance their cytoplasm osmotically with their medium. The first involves accumulation of molar concentrations of KCl. This strategy requires adaptation of the intracellular enzymatic machinery, as proteins should maintain their proper conformation and activity at near-saturating salt concentrations. The proteome of such organisms is highly acidic, and most proteins denature when suspended in low salt. Such microorganisms generally cannot survive in low salt media. The second strategy is to exclude salt from the cytoplasm and to synthesize and/or accumulate organic 'compatible' solutes that do not interfere with enzymatic activity. Few adaptations of the cells' proteome are needed, and organisms using the 'organic-solutes-in strategy' often adapt to a surprisingly broad salt concentration range. Most halophilic Bacteria, but also the halophilic methanogenic Archaea use such organic solutes. A variety of such solutes are known, including glycine betaine, ectoine and other amino acid derivatives, sugars and sugar alcohols. The 'high-salt-in strategy' is not limited to the Halobacteriaceae. The Halanaerobiales (Firmicutes) also accumulate salt rather than organic solutes. A third, phylogenetically unrelated organism accumulates KCl: the red extremely halophilic Salinibacter (Bacteroidetes), recently isolated from saltern crystallizer brines. Analysis of its genome showed many points of resemblance with the Halobacteriaceae, probably resulting from extensive horizontal gene transfer. The case of Salinibacter shows that more unusual types of halophiles may be waiting to be discovered.
Strictly anaerobic halophiles, namely fermentative, sulfate-reducing, homoacetogenic, phototrophic, and methanogenic bacteria are involved in the oxidation of organic carbon in hypersaline environments. To date, six anaerobic fermentative genera, containing nine species, have been described. Two of them are homoacetogens. Six species belong to the family Haloanaerobiaceae, as indicated by their unique 16S rRNA oligonucleotide sequences. Desulfohalobium retbaense and Desulfovibrio halophilus represent the only two moderately halophilic sulfate reducers so far reported. Among anoxygenic phototrophic anaerobes, a few purple bacteria with optimal growth at salinities between 6 and 11% NaCl have been isolated from hypersaline habitats. They belong to the genera Rhodospirillum, Chromatium, Thiocapsa, and Ectothiorhodospira. The commonest organisms isolated so far are Chromatium salexigens, Thiocapsa halophila, and Rhodospirillum salinarum. Extremely halophilic purple bacteria have most commonly been isolated from alkaline brines and require about 20 to 25% NaCl for optimal growth. They belong to the family Ectothiorodhospiraceae. Their osmoregulation involves synthesis or uptake of compatible solutes such as glycine-betaine that accumulate in their cytoplasm. The existence of methanogens in hypersaline environments is related to the presence of noncompetitive substrates such as methylamines, which originate mainly from the breakdown of osmoregulatory amines. Methanogenesis probably does not contribute to the mineralization of carbohydrates at NaCl concentrations higher than 15%. Above this concentration, sulfate reduction is probably the main way to oxidize H2 (although at rates too low to use up all the H2 formed) and occupies a terminal function kn the degradation of carbohydrates. Three genera and five species of halophilic methylotrophic methanogens have been reported. A bloom of phototrophic bacteria in the marine salterns of Salins-de-Giraud, located on the Mediterranean French coast in the Rhone Delta, is also described.
All tested strains of halophilic archaebacteria of the genera Halobacterium, Haloarcula, Haloferax, and Natronobacterium lysed in 1% Bacto-Peptone (Difco) containing 25% NaCl, whereas no lysis was observed with other strains belonging to archaebacteria of the genera Halococcus, Natronococcus, and Sulfolobus, methanogenic bacteria, and moderately halophilic eubacteria. Substances in Bacto-Peptone which caused lysis of halobacteria were purified and identified as taurocholic acid and glycocholic acid. High-performance liquid chromatography analyses of peptones revealed that Bacto-Peptone contained nine different bile acids, with a total content of 9.53 mg/g, whereas much lower amounts were found in Peptone Bacteriological Technical (Difco) and Oxoid Peptone. Different kinds of peptones can be used to distinguish halophilic eubacteria and archaebacteria in mixed cultures from hypersaline environments.
Generally, extremophiles have been deemed to survive in the extreme environments to which they had adapted to grow. Recently many extremophiles have been isolated from places where they are not expected to grow. Alkaliphilic microorganisms have been isolated from acidic soil samples with pH 4.0, and thermophiles have been isolated from samples of low temperature. Numerous moderately halophilic microorganisms, defined as those that grow optimally in media containing 0.5–2.5 Molar (3–15%) NaCl, and halotolerant microorganisms that are able to grow in media without added NaCl and in the presence of high NaCl have been isolated from saline environments such as salterns, salt lakes and sea sands. It has tacitly been believed that habitats of halophiles able to grow in media containing more than 20% (3.4 M) are restricted to saline environments, and no reports have been published on the isolation of halophiles from ordinary garden soil samples.
We demonstrated that many halophilic bacteria that are able to grow in the presence of 20% NaCl are inhabiting in non-saline environments such as ordinary garden soils, yards, fields and roadways in an area surrounding Tokyo, Japan. Analyses of partial 16S rRNA gene sequences of 176 isolates suggested that they were halophiles belonging to genera of the family Bacillaceae, Bacillus (11 isolates), Filobacillus (19 isolates), Gracilibacillus (6 isolates), Halobacillus (102 isolates), Lentibacillus (1 isolate), Paraliobacillus (5 isolates) and Virgibacillus (17 isolates). Sequences of 15 isolates showed similarities less than 92%, suggesting that they may represent novel taxa within the family Bacillaceae.
The numbers of total bacteria of inland soil samples were in a range from 1.4 × 107/g to 1.1 × 106/g. One tenth of the total bacteria was occupied by endospore-forming bacteria. Only very few of the endospore-forming bacteria, roughly 1 out of 20,000, are halophilic bacteria. Most of the halophilic bacteria were surviving as endospores in the soil samples, in a range of less than 1 to about 500/g soil. Samples collected from seashore in a city confronting Tokyo Bay gave the total numbers of bacteria and endospores roughly 1000 time smaller than those of inland soil samples. Numbers of halophilic bacteria per gram, however, were almost the same as those of inland soil samples. A possible source of the halophilic endospore originating from Asian dust storms is discussed.
Inland solar salterns established in the vicinity of Sambhar Lake are extreme saline environments with high salinity and alkalinity. In view of the fact that microbes inhabiting such extreme saline environments flourish the contemporary bioprospecting, it was aimed to selectively isolate slow growing and rare actinomycetes from the unexplored solar salterns. A total of 14 slow growing actinomycetes were selectively isolated from three composite soil samples of inland solar salterns. Among the isolates, four groups were formed according to similarity of the banding patterns obtained by amplified ribosomal DNA restriction analysis (ARDRA). A subset of representative isolates for each ARDRA group was identified using 16S rDNA sequence based phylogenetic analysis and subsequently the entire isolates were assigned under three different genera; Streptomyces, Pseudonocardia, and Actinoalloteichus. The genus Streptomyces was found to be the dominant among the isolates. Furthermore, rare actinomycete genus Actinoalloteichus was isolated for the first time from solar saltern. Determination of salt-tolerance revealed that certain level of salt-tolerance and moderate halophilism occurs among the actinomycetes isolated from the inland salterns. In addition, all the acinomycetes were screened in two levels to unravel their ability to produce antimicrobial compounds. Significant antimicrobial activity was found among the actinomycetes against a range of bacteria and fungi to worth further characterization of these persuasive actinomycetes and their antimicrobial secondary metabolites. In a nutshell, this study offered a first interesting insight on occurrence of antagonistic rare actinomycetes and streptomycetes in inland solar salterns associated with Sambhar salt Lake.
solar saltern; rare actinomycetes; ARDRA; phylogeny
Maras salterns are located 3,380 m above sea level in the Peruvian Andes. These salterns consist of more than 3,000 little ponds which are not interconnected and act as crystallizers where salt precipitates. These ponds are fed by hypersaline spring water rich in sodium and chloride. The microbiota inhabiting these salterns was examined by fluorescence in situ hybridization (FISH), 16S rRNA gene clone library analysis, and cultivation techniques. The total counts per milliliter in the ponds were around 2 × 106 to 3 × 106 cells/ml, while the spring water contained less than 100 cells/ml and did not yield any detectable FISH signal. The microbiota inhabiting the ponds was dominated (80 to 86% of the total counts) by Archaea, while Bacteria accounted for 10 to 13% of the 4′,6′-diamidino-2-phenylindole (DAPI) counts. A total of 239 16S rRNA gene clones were analyzed (132 Archaea clones and 107 Bacteria clones). According to the clone libraries, the archaeal assemblage was dominated by microorganisms related to the cosmopolitan square archaeon “Haloquadra walsbyi,” although a substantial number of the sequences in the libraries (31% of the 16S rRNA gene archaeal clones) were related to Halobacterium sp., which is not normally found in clone libraries from solar salterns. All the bacterial clones were closely related to each other and to the γ-proteobacterium “Pseudomonas halophila” DSM 3050. FISH analysis with a probe specific for this bacterial assemblage revealed that it accounted for 69 to 76% of the total bacterial counts detected with a Bacteria-specific probe. When pond water was used to inoculate solid media containing 25% total salts, both extremely halophilic Archaea and Bacteria were isolated. Archaeal isolates were not related to the isolates in clone libraries, although several bacterial isolates were very closely related to the “P. halophila” cluster found in the libraries. As observed for other hypersaline environments, extremely halophilic bacteria that had ecological relevance seemed to be easier to culture than their archaeal counterparts.
Lake Vanda is a perennially ice-covered and stratified lake in the McMurdo Dry Valleys, Antarctica. The lake develops a distinct chemocline at about a 50-m depth, where the waters transition from cool, oxic, and fresh to warm, sulfidic, and hypersaline. The bottom water brine is unique, as the highly chaotropic salts CaCl2 and MgCl2 predominate, and CaCl2 levels are the highest of those in any known microbial habitat. Enrichment techniques were used to isolate 15 strains of heterotrophic bacteria from the Lake Vanda brine. Despite direct supplementation of the brine samples with different organic substrates in primary enrichments, the same organism, a relative of the halophilic bacterium Halomonas (Gammaproteobacteria), was isolated from all depths sampled. The Lake Vanda (VAN) strains were obligate aerobes and showed broad pH, salinity, and temperature ranges for growth, consistent with the physicochemical properties of the brine. VAN strains were halophilic and quite CaCl2 tolerant but did not require CaCl2 for growth. The fact that only VAN strain-like organisms appeared in our enrichments hints that the highly chaotropic nature of the Lake Vanda brine may place unusual physiological constraints on the bacterial community that inhabits it.
Background and Objectives
Halophilic bacteria produce a variety of pigments, which function as immune modulators and have prophylactic action against cancers. In this study, colorful halophilic bacteria were isolated from solar salt lake and their pigments was extracted in optimal environmental conditions and compared with the pigments of Halorubrum sodomense ATCC 33755.
Materials and Methods
Water samples from the solar salt lake in Imam Khomeini port in southwest of Iran were used as a source for isolation of pigment-producing bacteria. Halorubrum sodomense ATCC 33755 was used as control for pigment production. The conditions for optimum growth and pigment production were established for the isolated bacteria. Pigment were analyzed by spectrophotometer, TLC and NMR assay. The 16S rRNA genes were sequenced and results were used to differentiate haloarchaea from halophilic bacterial strains.
Among the isolated strains, YS and OS strains and Halorubrum sodomense were recognized as moderate and extremely halophile with maximum growth in the presence of 15% and 30% NaCl concentrations, respectively. Experiments conducted to find out the optimum conditions for growth and pigment production temperature at 25°C, pH = 7.2 and shaking conditions at 120 rpm for three strains. Without shaking, little growth with no pigment production was observed. Total pigment produced by red, yellow and orange strains was measured at 240, 880 and 560 mg per dry cell weight respectively. Amplification yielded bands of to isolated strains only observed with bacteria primers. This result suggesting the YS and OS strains were not haloarchaea.
The isolated halophilic bacteria produced much higher amounts of pigments than Halorubrum sodomense. Photo intermediates including metarhodopsin II (meta II, λmax=380 nm) were determined as major pigment in Halorubrum sodomense.
Halorubrum sodomense; haloarchaea; salt lake; metarhodopsin II; pigment
The deep-sea, hypersaline anoxic brine lakes in the Mediterranean are among the most extreme environments on earth, and in one of them, the MgCl2-rich Discovery basin, the presence of active microbes is equivocal. However, thriving microbial communities have been detected especially in the chemocline between deep seawater and three NaCl-rich brine lakes, l'Atalante, Bannock and Urania. By contrast, the microbiota of these brine-lake sediments remains largely unexplored.
Eighty nine isolates were obtained from the sediments of four deep-sea, hypersaline anoxic brine lakes in the Eastern Mediterranean Sea: l'Atalante, Bannock, Discovery and Urania basins. This culture collection was dominated by representatives of the genus Bacillus and close relatives (90% of all isolates) that were investigated further. Physiological characterization of representative strains revealed large versatility with respect to enzyme activities or substrate utilization. Two third of the isolates did not grow at in-situ salinities and were presumably present as endospores. This is supported by high numbers of endospores in Bannock, Discovery and Urania basins ranging from 3.8 × 105 to 1.2 × 106 g-1 dw sediment. However, the remaining isolates were highly halotolerant growing at salinities of up to 30% NaCl. Some of the novel isolates affiliating with the genus Pontibacillus grew well under anoxic conditions in sulfidic medium by fermentation or anaerobic respiration using dimethylsulfoxide or trimethylamine N-oxide as electron acceptor.
Some of the halophilic, facultatively anaerobic relatives of Bacillus appear well adapted to life in this hostile environment and suggest the presence of actively growing microbial communities in the NaCl-rich, deep-sea brine-lake sediments.
The prosperity of Hallstatt (Salzkammergut region, Austria) is based on the richness of salt in the surrounding mountains and salt mining, which is documented as far back as 1500 years B.C. Substantial archaeological evidence of Bronze and Iron Age salt mining has been discovered, with a wooden staircase (1108 B.C.) being one of the most impressive and well preserved finds. However, after its discovery, fungal mycelia have been observed on the surface of the staircase, most probably due to airborne contamination after its find.
As a basis for the further preservation of this valuable object, the active micro-flora was examined to investigate the presence of potentially biodegradative microorganisms.
Most of the strains isolated from the staircase showed to be halotolerant and halophilic microorganisms, due to the saline environment of the mine. Results derived from culture-dependent assays revealed a high fungal diversity, including both halotolerant and halophilic fungi, the most dominant strains being members of the genus Phialosimplex (synonym: Aspergillus). Additionally, some typical cellulose degraders, namely Stachybotrys sp. and Cladosporium sp. were detected. Numerous bacterial strains were isolated and identified as members of 12 different genera, most of them being moderately halophilic species. The most dominant isolates affiliated with species of the genera Halovibrio and Marinococcus. Halophilic archaea were also isolated and identified as species of the genera Halococcus and Halorubrum. Molecular analyses complemented the cultivation assays, enabling the identification of some uncultivable archaea of the genera Halolamina, Haloplanus and Halobacterium. Results derived from fungi and bacteria supported those obtained by cultivation methods, exhibiting the same dominant members in the communities.
The results clearly showed the presence of some cellulose degraders that may become active if the requirements for growth and the environmental conditions turn suitable; therefore, these microorganisms must be regarded as a threat to the wood.
A few extremely halophilic Archaea (Halobacterium salinarum, Haloquadratum walsbyi, Haloferax mediterranei, Halorubrum vacuolatum, Halogeometricum borinquense, Haloplanus spp.) possess gas vesicles that bestow buoyancy on the cells. Gas vesicles are also produced by the anaerobic endospore-forming halophilic Bacteria Sporohalobacter lortetii and Orenia sivashensis. We have extensive information on the properties of gas vesicles in Hbt. salinarum and Hfx. mediterranei and the regulation of their formation. Different functions were suggested for gas vesicle synthesis: buoying cells towards oxygen-rich surface layers in hypersaline water bodies to prevent oxygen limitation, reaching higher light intensities for the light-driven proton pump bacteriorhodopsin, positioning the cells optimally for light absorption, light shielding, reducing the cytoplasmic volume leading to a higher surface-area-to-volume ratio (for the Archaea) and dispersal of endospores (for the anaerobic spore-forming Bacteria). Except for Hqr. walsbyi which abounds in saltern crystallizer brines, gas-vacuolate halophiles are not among the dominant life forms in hypersaline environments. There only has been little research on gas vesicles in natural communities of halophilic microorganisms, and the few existing studies failed to provide clear evidence for their possible function. This paper summarizes the current status of the different theories why gas vesicles may provide a selective advantage to some halophilic microorganisms.
gas vesicles; Halobacterium; Haloferax; Haloquadratum; Haloplanus; Halogeometricum; bacteriorhodopsin; oxygen
Two halophilic anaerobic bacteria, one of which had chitinolytic activity, were isolated from a solar saltern in southern California. These organisms were long, gram-negative, motile, flexible rods. The biochemical and physiological characteristics of these bacteria were very similar but were different from the characteristics of other haloanaerobic bacteria. Both grew at salt concentrations ranging from 0.5 to 5 M and at temperatures ranging from 23 to 50°C. They were sensitive to chloramphenicol but resistant to penicillin, carbenicillin, d-cycloserine, streptomycin, and tetracycline. An analysis of DNAs and whole-cell proteins showed that they were closely related taxonomically and distinguishable from other halophilic anaerobic bacteria. They exhibited 92.3 to 100% DNA homology as determined by DNA-DNA hybridization. The guanine-plus-cytosine contents of their DNAs were 34.8±1 mol%. The two isolates, strains W5C8 and W3C1, differed from other halophilic anaerobic bacteria sufficiently to support establishment of a new genus and species, Haloanaerobacter chitinovorans. Strain W5C8 exhibited chitinolytic activity and is designated the type strain. Two chitin-induced extracellular proteins with molecular weights of 38 × 103 and 40 × 103 were detected in strain W5C8.
The extraction of salt from seawater by means of coastal solar salterns is a very well-described process. Moreover, the characterization of these environments from ecological, biochemical and microbiological perspectives has become a key focus for many research groups all over the world over the last 20 years. In countries such as Spain, there are several examples of coastal solar salterns (mainly on the Mediterranean coast) and inland solar salterns, from which sodium chloride is obtained for human consumption. However, studies focused on the characterization of inland solar salterns are scarce and both the archaeal diversity and the plant communities inhabiting these environments remain poorly described.
Two of the inland solar salterns (termed Redonda and Penalva), located in the Alto Vinalopó Valley (Alicante, Spain), were characterized regarding their geological and physico-chemical characteristics and their archaeal and botanical biodiversity. A preliminary eukaryotic diversity survey was also performed using saline water. The chemical characterization of the brine has revealed that the salted groundwater extracted to fill these inland solar salterns is thalassohaline. The plant communities living in this environment are dominated by Sarcocornia fruticosa (L.) A.J. Scott, Arthrocnemum macrostachyum (Moris) K. Koch, Suaeda vera Forsk. ex Gmelin (Amaranthaceae) and several species of Limonium (Mill) and Tamarix (L). Archaeal diversity was analyzed and compared by polymerase chain reaction (PCR)-based molecular phylogenetic techniques. Most of the sequences recovered from environmental DNA samples are affiliated with haloarchaeal genera such as Haloarcula, Halorubrum, Haloquadratum and Halobacterium, and with an unclassified member of the Halobacteriaceae. The eukaryote Dunaliella was also present in the samples.
To our knowledge, this study constitutes the first analysis centered on inland solar salterns located in the southeastern region of Spain. The results obtained revealed that the salt deposits of this region have marine origins. Plant communities typical of salt marshes are present in this ecosystem and members of the Halobacteriaceae family can be easily detected in the microbial populations of these habitats. Possible origins of the haloarchaea detected in this study are discussed.
Accumulation of compatible solutes, by uptake or de novo synthesis, enables bacteria to reduce the difference between osmotic potentials of the cell cytoplasm and the extracellular environment. To examine this process in the halophilic and halotolerant methanogenic archaebacteria, 14 strains were tested for the accumulation of compatible solutes in response to growth in various extracellular concentrations of NaCl. In external NaCl concentrations of 0.7 to 3.4 M, the halophilic methanogens accumulated K+ ion and low-molecular-weight organic compounds. beta-Glutamate was detected in two halotolerant strains that grew below 1.5 M NaCl. Two unusual beta-amino acids, N epsilon-acetyl-beta-lysine and beta-glutamine (3-aminoglutaramic acid), as well as L-alpha-glutamate were compatible solutes among all of these strains. De novo synthesis of glycine betaine was also detected in several strains of moderately and extremely halophilic methanogens. The zwitterionic compounds (beta-glutamine, N epsilon-acetyl-beta-lysine, and glycine betaine) and potassium were the predominant compatible solutes among the moderately and extremely halophilic methanogens. This is the first report of beta-glutamine as a compatible solute and de novo biosynthesis of glycine betaine in the methanogenic archaebacteria.
Halophilic archaebacteria (haloarchaea) thrive in environments with salt concentrations approaching saturation, such as natural brines, the Dead Sea, alkaline salt lakes and marine solar salterns; they have also been isolated from rock salt of great geological age (195–250 million years). An overview of their taxonomy, including novel isolates from rock salt, is presented here; in addition, some of their unique characteristics and physiological adaptations to environments of low water activity are reviewed. The issue of extreme long-term microbial survival is considered and its implications for the search for extraterrestrial life. The development of detection methods for subterranean haloarchaea, which might also be applicable to samples from future missions to space, is presented.
Extreme halophiles; Haloarchaea; Life detection; Microbial longevity; Salt mines; Salt sediments; Space missions; Subterranean; Taxonomy of halobacteriaceae
An extremely halophilic archaeon, strain ETD6, was isolated from a marine solar saltern in Sfax, Tunisia. Analysis of the 16S rRNA gene sequence showed that the isolate was phylogenetically related to species of the genus Halorubrum among the family Halobacteriaceae, with a close relationship to Hrr. xinjiangense (99.77%
of identity). However, value for DNA-DNA hybridization between strain ETD6 and Hrr.xinjiangense were about 24.5%. The G+C content of the genomic DNA was 65.1 mol%
(T(m)). Strain ETD6 grew in 15–35%
(w/v) NaCl. The temperature and pH ranges for growth were 20–55°C and 6–9, respectively. Optimal growth occurred at 25%
NaCl, 37°C, and pH 7.4. The results of the DNA hybridization against Hrr. xinjiangense and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain ETD6 from other Hrr. species. Therefore, strain ETD6 represents a novel species of the genus Halorubrum, for which the name Hrr. sfaxense sp. nov. is proposed. The Genbank EMBL-EBI accession number is GU724599.
The aim of the present study was to investigate indigenous fungal communities isolated from extreme environments (hypersaline waters of solar salterns and subglacial ice), for the production of metabolic compounds with selected biological activities: hemolysis, antibacterial, and acetylcholinesterase inhibition. In their natural habitats, the selected fungi are exposed to environmental extremes, and therefore the production of bioactive metabolites was tested under both standard growth conditions for mesophilic microorganisms, and at high NaCl and sugar concentrations and low growth temperatures. The results indicate that selected halotolerant and halophilic species synthesize specific bioactive metabolites under conditions that represent stress for non-adapted species. Furthermore, adaptation at the level of the chemical nature of the solute lowering the water activity of the medium was observed. Increased salt concentrations resulted in higher hemolytic activity, particularly within species dominating the salterns. The appearance of antibacterial potential under stress conditions was seen in the similar pattern of fungal species as for hemolysis. The active extracts exclusively affected the growth of the Gram-positive bacterium tested, Bacillus subtilis. None of the extracts tested showed inhibition of acetylcholinesterase activity.
hypersaline environments; black yeast; NaCl; secondary metabolites; hemolysis; antibacterial activity
Hortaea werneckii is one of the most salt-tolerant species among microorganisms. It has been isolated from hypersaline waters of salterns as one of the predominant species of a group of halophilic and halotolerant melanized yeast-like fungi, arbitrarily named as "black yeasts". It has previously been shown that H. werneckii has distinct mechanisms of adaptation to high salinity environments that are not seen in salt-sensitive and only moderately salt-tolerant fungi. In H. werneckii, the HOG pathway is important for sensing the changes in environmental osmolarity, as demonstrated by identification of three main pathway components: the mitogen-activated protein kinase (MAPK) HwHog1, the MAPK kinase HwPbs2, and the putative histidine kinase osmosensor HwHhk7.
In this study, we show that the expression of HwHOG1 in salt-adapted cells depends on the environmental salinity and that HwHOG1 transcription responds rapidly but reciprocally to the acute hyper-saline or hypo-saline stress. Molecular modelling of HwHog1 reveals an overall structural homology with other MAPKs. HwHog1 complements the function of ScHog1 in the Saccharomyces cerevisiae multistress response. We also show that hyper-osmolar, oxidative and high-temperature stresses activate the HwHog1 kinase, although under high-temperature stress the signal is not transmitted via the MAPK kinase Pbs2. Identification of HOG1-like genes from other halotolerant fungi isolated from solar salterns demonstrates a high degree of similarity and excellent phylogenetic clustering with orthologues of fungal origin.
The HOG signalling pathway has an important role in sensing and responding to hyper-osmolar, oxidative and high-temperature stresses in the halophilic fungi H. werneckii. These findings are an important advance in our understanding of the HOG pathway response to stress in H. werneckii, a proposed model organism for studying the salt tolerance of halophilic and halotolerant eukaryotes.
Archaea and Bacteria constitute a majority of life systems on Earth but have long been considered inferior to Eukarya in terms of solute tolerance. Whereas the most halophilic prokaryotes are known for an ability to multiply at saturated NaCl (water activity (aw) 0.755) some xerophilic fungi can germinate, usually at high-sugar concentrations, at values as low as 0.650–0.605 aw. Here, we present evidence that halophilic prokayotes can grow down to water activities of <0.755 for Halanaerobium lacusrosei (0.748), Halobacterium strain 004.1 (0.728), Halobacterium sp. NRC-1 and Halococcus morrhuae (0.717), Haloquadratum walsbyi (0.709), Halococcus salifodinae (0.693), Halobacterium noricense (0.687), Natrinema pallidum (0.681) and haloarchaeal strains GN-2 and GN-5 (0.635 aw). Furthermore, extrapolation of growth curves (prone to giving conservative estimates) indicated theoretical minima down to 0.611 aw for extreme, obligately halophilic Archaea and Bacteria. These were compared with minima for the most solute-tolerant Bacteria in high-sugar (or other non-saline) media (Mycobacterium spp., Tetragenococcus halophilus, Saccharibacter floricola, Staphylococcus aureus and so on) and eukaryotic microbes in saline (Wallemia spp., Basipetospora halophila, Dunaliella spp. and so on) and high-sugar substrates (for example, Xeromyces bisporus, Zygosaccharomyces rouxii, Aspergillus and Eurotium spp.). We also manipulated the balance of chaotropic and kosmotropic stressors for the extreme, xerophilic fungi Aspergillus penicilloides and X. bisporus and, via this approach, their established water-activity limits for mycelial growth (∼0.65) were reduced to 0.640. Furthermore, extrapolations indicated theoretical limits of 0.632 and 0.636 aw for A. penicilloides and X. bisporus, respectively. Collectively, these findings suggest that there is a common water-activity limit that is determined by physicochemical constraints for the three domains of life.
Recent metagenomic studies on saltern ponds with intermediate salinities have determined that their microbial communities are dominated by both Euryarchaeota and halophilic bacteria, with a gammaproteobacterium closely related to the genera Alkalilimnicola and Arhodomonas being one of the most predominant microorganisms, making up to 15% of the total prokaryotic population. Here we used several strategies and culture media in order to isolate this organism in pure culture. We report the isolation and taxonomic characterization of this new, never before cultured microorganism, designated M19-40T, isolated from a saltern located in Isla Cristina, Spain, using a medium with a mixture of 15% salts, yeast extract, and pyruvic acid as the carbon source. Morphologically small curved cells (young cultures) with a tendency to form long spiral cells in older cultures were observed in pure cultures. The organism is a Gram-negative, nonmotile bacterium that is strictly aerobic, non-endospore forming, heterotrophic, and moderately halophilic, and it is able to grow at 10 to 25% (wt/vol) NaCl, with optimal growth occurring at 15% (wt/vol) NaCl. Phylogenetic analysis based on 16S rRNA gene sequence comparison showed that strain M19-40T has a low similarity with other previously described bacteria and shows the closest phylogenetic similarity with species of the genera Alkalilimnicola (94.9 to 94.5%), Alkalispirillum (94.3%), and Arhodomonas (93.9%) within the family Ectothiorhodospiraceae. The phenotypic, genotypic, and chemotaxonomic features of this new bacterium showed that it constitutes a new genus and species, for which the name Spiribacter salinus gen. nov., sp. nov., is proposed, with strain M19-40T (= CECT 8282T = IBRC-M 10768T = LMG 27464T) being the type strain.
A Gram-negative, extremely halophilic, coccoid archaeal strain, CM5T, was isolated from a crude sea-salt sample collected near Qingdao, China. The organism grew optimally at 35–40 °C and pH 6.0 in the presence of 20 % (w/v) NaCl. Its colonies were red in colour and it could use glucose as a sole carbon source for growth. The 16S rRNA gene sequence of CM5T was most closely related to those of Halococcus species. Its pattern of antibiotic susceptibility was similar to those of other described Halococcus species. Biochemical tests revealed no sign of H2S production or gelatin liquefaction. The main polar lipids of strain CM5T were phosphatidylglycerol, phosphatidylglycerol methylphosphate and sulfated diglycosyl diether. No phosphatidylglycerol sulfate was present. The DNA G+C content of strain CM5T was 61.2 mol% and it gave DNA–DNA reassociation values of 33.7, 57.1 and 29.6 %, respectively, with Halococcus salifodinae DSM 8989T, Halococcus dombrowskii DSM 14522T and Halococcus morrhuae ATCC 17082T. Based on its morphological and chemotaxonomic properties and phylogenetic analysis of 16S rRNA gene sequence data, we propose that CM5T should be classified within a novel species, Halococcus qingdaonensis sp. nov., with strain CM5T (=CGMCC 1.4243T=JCM 13587T) as the type strain.
Halophilic bacteria are commonly found in natural environments containing significant concentration of NaCl such as inland salt lakes and evaporated sea-shore pools, as well as environments such as curing brines, salted food products and saline soils. Dependence on salt is an important phenotypic characteristic of halophilic bacteria, which can be used in the polyphasic characterization of newly discovered microorganisms. In this study the diversity of halophilic bacteria in foreshore soils of Daecheon, Chungnam, and Saemangeum, Jeonbuk, was investigated. Two types of media, namely NA and R2A supplemented with 3%, 5%, 9%, 15%, 20% and 30% NaCl were used. More than 200 halophilic bacteria were isolated and BOX-PCR fingerprinting analysis was done for the typing of the isolates. The BLAST identification results showed that isolated strains were composed of 4 phyla, Firmicutes (60%), Proteobacteria (31%), Bacteriodetes (5%) and Actinobacteria (4%). Isolates were affiliated with 16 genera and 36 species. Bacillus was the dominant genus in the phylum Firmicutes, comprising 24% of the total isolates. Halomonas (12%) and Shewanella (12%) were also found as the main genera. These findings show that the foreshore soil of Daecheon Beach and Saemangeum Sea of Korea represents an untapped source of bacterial biodiversity.
culturable diversity; halophilic bacteria; foreshore soil
Goa is a coastal state in India and salt making is being practiced for many years. This investigation aimed in determining the culturable haloarchaeal diversity during two different phases of salt production in a natural solar saltern of Ribandar, Goa. Water and sediment samples were collected from the saltern during pre-salt harvesting phase and salt harvesting phase. Salinity and pH of the sampling site was determined. Isolates were obtained by plating of the samples on complex and synthetic haloarchaeal media. Morphology of the isolates was determined using Gram staining and electron microscopy. Response of cells to distilled water was studied spectrophotometrically at 600nm. Molecular identification of the isolates was performed by sequencing the 16S rRNA.
Salinity of salt pans varied from 3-4% (non-salt production phase) to 30% (salt production phase) and pH varied from 7.0-8.0. Seven haloarchaeal strains were isolated from water and sediment samples during non-salt production phase and seventeen haloarchaeal strains were isolated during the salt production phase. All the strains stained uniformly Gram negative. The orange-red acetone extract of the pigments showed similar spectrophotometric profile with absorption maxima at 393, 474, 501 and 535 nm. All isolates obtained from the salt dilute phase were grouped within the genus Halococcus. This was validated using both total lipid profiling and 16S rRNA data sequencing. The isolates obtained from pre-salt harvesting phase were resistant to lysis. 16S rRNA data showed that organisms belonging to Halorubrum, Haloarcula, Haloferax and Halococcus genera were obtained during the salt concentrated phase. The isolates obtained from salt harvesting phase showed varied lysis on suspension in distilled water and /or 3.5% NaCl.
Salterns in Goa are transiently operated during post monsoon season from January to May. During the pre-salt harvesting phase, all the isolates obtained belonged to Halococcus sp. During the salt harvesting phase, isolates belonging to Halorubrum, Haloarcula, Haloferax and Halococcus genera were obtained. This study clearly indicates that Halococcus sp. dominates during the low salinity conditions.
Archaea; Haloarchaea; Hypersaline; Solar saltern
Hydrolases constitute a class of enzymes widely distributed in nature from bacteria to higher eukaryotes. The halotolerance of many enzymes derived from halophilic bacteria can be exploited wherever enzymatic transformations are required to function under physical and chemical conditions, such as in the presence of organic solvents and extremes in temperature and salt content. In recent years, different screening programs have been performed in saline habitats in order to isolate and characterize novel enzymatic activities with different properties to those of conventional enzymes. Several halophilic hydrolases have been described, including amylases, lipases and proteases, and then used for biotechnological applications. Moreover, the discovery of biopolymer-degrading enzymes offers a new solution for the treatment of oilfield waste, where high temperature and salinity are typically found, while providing valuable information about heterotrophic processes in saline environments. In this work, we describe the results obtained in different screening programs specially focused on the diversity of halophiles showing hydrolytic activities in saline and hypersaline habitats, including the description of enzymes with special biochemical properties. The intracellular lipolytic enzyme LipBL, produced by the moderately halophilic bacterium Marinobacter lipolyticus, showed advantages over other lipases, being an enzyme active over a wide range of pH values and temperatures. The immobilized LipBL derivatives obtained and tested in regio- and enantioselective reactions, showed an excellent behavior in the production of free polyunsaturated fatty acids (PUFAs). On the other hand, the extremely halophilic bacterium, Salicola marasensis sp. IC10 showing lipase and protease activities, was studied for its ability to produce promising enzymes in terms of its resistance to temperature and salinity.
halophiles; extremophiles; hydrolases; saline environments
An extreme halophilic bacterium was isolated from solar saltern samples and identified based on biochemical tests and 16S r RNA sequencing as Chromohalobacter sp. strain TVSP101. The halophilic protease was purified using ultrafiltration, ethanol precipitation, hydrophobic interaction column chromatography and gel permeation chromatography to 180 fold with 22% yield. The molecular mass of the protease determined by SDS PAGE was 66 kDa. The purified enzyme was salt dependent for its activity and stability with an optimum of 4.5 M NaCl. The optimum temperature for maximum protease activity was 75ºC. The protease was optimally active at pH 8 and retained more than 80% of its activity in the range of pH 7-10. Sucrose and glycine at 10% (w/v) were the most effective osmolytes, retained 100% activity in the absence of NaCl. The activity was completely inhibited by ZnCl2 (2 mM), 0.1% SDS and PMSF (1mM). The enzyme was not inhibited by 1mM of pepstatin, EDTA and PCMB. The protease was active and retained 100% it activity in 10% (v/v) DMSO, DMF, ethanol and acetone.
Chromohalobacter sp. TVSP101; halothermophilic protease; purification; organic solvents; osmolytes