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1.  Culturable halophilic archaea at the initial and crystallization stages of salt production in a natural solar saltern of Goa, India 
Aquatic Biosystems  2012;8:15.
Background
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.
Results
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.
Conclusion
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.
doi:10.1186/2046-9063-8-15
PMCID: PMC3444409  PMID: 22747590
Archaea; Haloarchaea; Hypersaline; Solar saltern
2.  Responses of Haloarchaea to Simulated Microgravity 
Astrobiology  2011;11(3):199-205.
Abstract
Various effects of microgravity on prokaryotes have been recognized in recent years, with the focus on studies of pathogenic bacteria. No archaea have been investigated yet with respect to their responses to microgravity. For exposure experiments on spacecrafts or on the International Space Station, halophilic archaea (haloarchaea) are usually embedded in halite, where they accumulate in fluid inclusions. In a liquid environment, these cells will experience microgravity in space, which might influence their viability and survival. Two haloarchaeal strains, Haloferax mediterranei and Halococcus dombrowskii, were grown in simulated microgravity (SMG) with the rotary cell culture system (RCCS, Synthecon). Initially, salt precipitation and detachment of the porous aeration membranes in the RCCS were observed, but they were avoided in the remainder of the experiment by using disposable instead of reusable vessels. Several effects were detected, which were ascribed to growth in SMG: Hfx. mediterranei's resistance to the antibiotics bacitracin, erythromycin, and rifampicin increased markedly; differences in pigmentation and whole cell protein composition (proteome) of both strains were noted; cell aggregation of Hcc. dombrowskii was notably reduced. The results suggest profound effects of SMG on haloarchaeal physiology and cellular processes, some of which were easily observable and measurable. This is the first report of archaeal responses to SMG. The molecular mechanisms of the effects induced by SMG on prokaryotes are largely unknown; haloarchaea could be used as nonpathogenic model systems for their elucidation and in addition could provide information about survival during lithopanspermia (interplanetary transport of microbes inside meteorites). Key Words: Haloferax mediterranei—Halococcus dombrowskii—Simulated microgravity—Rotary cell culture system—Antibiotic resistance—Lithopanspermia. Astrobiology 11, 199–205.
doi:10.1089/ast.2010.0536
PMCID: PMC3079168  PMID: 21417742
3.  Identification of polyhydroxyalkanoates in Halococcus and other haloarchaeal species 
Polyhydroxyalkanoates (PHAs) are accumulated in many prokaryotes. Several members of the Halobacteriaceae produce poly-3-hydroxybutyrate (PHB), but it is not known if this is a general property of the family. We evaluated identification methods for PHAs with 20 haloarchaeal species, three of them isolates from Permian salt. Staining with Sudan Black B, Nile Blue A, or Nile Red was applied to screen for the presence of PHAs. Transmission electron microscopy and 1H-nuclear magnetic resonance spectroscopy were used for visualization of PHB granules and chemical confirmation of PHAs in cell extracts, respectively. We report for the first time the production of PHAs by Halococcus sp. (Halococcus morrhuae DSM 1307T, Halococcus saccharolyticus DSM 5350T, Halococcus salifodinae DSM 8989T, Halococcus dombrowskii DSM 14522T, Halococcus hamelinensis JCM 12892T, Halococcus qingdaonensis JCM 13587T), Halorubrum sp. (Hrr. coriense DSM 10284T, Halorubrum chaoviator DSM 19316T, Hrr. chaoviator strains NaxosII and AUS-1), haloalkaliphiles (Natronobacterium gregoryi NCMB 2189T, Natronococcus occultus DSM 3396T) and Halobacterium noricense DSM 9758T. No PHB was detected in Halobacterium salinarum NRC-1 ATCC 700922, Hbt. salinarum R1 and Haloferax volcanii DSM 3757T. Most species synthesized PHAs when growing in synthetic as well as in complex medium. The polyesters were generally composed of PHB and poly-ß-hydroxybutyrate-co-3-hydroxyvalerate (PHBV). Available genomic data suggest the absence of PHA synthesis in some haloarchaea and in all other Euryarchaeota and Crenarchaeota. Homologies between haloarchaeal and bacterial PHA synthesizing enzymes had indicated to some authors probable horizontal gene transfer, which, considering the data obtained in this study, may have occurred already before Permian times.
Electronic supplementary material
The online version of this article (doi:10.1007/s00253-010-2611-6) contains supplementary material, which is available to authorized users.
doi:10.1007/s00253-010-2611-6
PMCID: PMC2895300  PMID: 20437233
Polyhydroxybutyrate; Haloarchaea; Halococcus; Halobacterium; Haloalkaliphile
4.  Phylogenetically Driven Sequencing of Extremely Halophilic Archaea Reveals Strategies for Static and Dynamic Osmo-response 
PLoS Genetics  2014;10(11):e1004784.
Organisms across the tree of life use a variety of mechanisms to respond to stress-inducing fluctuations in osmotic conditions. Cellular response mechanisms and phenotypes associated with osmoadaptation also play important roles in bacterial virulence, human health, agricultural production and many other biological systems. To improve understanding of osmoadaptive strategies, we have generated 59 high-quality draft genomes for the haloarchaea (a euryarchaeal clade whose members thrive in hypersaline environments and routinely experience drastic changes in environmental salinity) and analyzed these new genomes in combination with those from 21 previously sequenced haloarchaeal isolates. We propose a generalized model for haloarchaeal management of cytoplasmic osmolarity in response to osmotic shifts, where potassium accumulation and sodium expulsion during osmotic upshock are accomplished via secondary transport using the proton gradient as an energy source, and potassium loss during downshock is via a combination of secondary transport and non-specific ion loss through mechanosensitive channels. We also propose new mechanisms for magnesium and chloride accumulation. We describe the expansion and differentiation of haloarchaeal general transcription factor families, including two novel expansions of the TATA-binding protein family, and discuss their potential for enabling rapid adaptation to environmental fluxes. We challenge a recent high-profile proposal regarding the evolutionary origins of the haloarchaea by showing that inclusion of additional genomes significantly reduces support for a proposed large-scale horizontal gene transfer into the ancestral haloarchaeon from the bacterial domain. The combination of broad (17 genera) and deep (≥5 species in four genera) sampling of a phenotypically unified clade has enabled us to uncover both highly conserved and specialized features of osmoadaptation. Finally, we demonstrate the broad utility of such datasets, for metagenomics, improvements to automated gene annotation and investigations of evolutionary processes.
Author Summary
The ability to adjust to changing osmotic conditions (osmoadaptation) is crucial to the survival of organisms across the tree of life. However, significant gaps still exist in our understanding of this important phenomenon. To help fill some of these gaps, we have produced high-quality draft genomes for 59 osmoadaptation “experts” (extreme halophiles of the euryarchaeal family Halobacteriaceae). We describe the dispersal of osmoadaptive protein families across the haloarchaeal evolutionary tree. We use this data to suggest a generalized model for haloarchaeal ion transport in response to changing osmotic conditions, including proposed new mechanisms for magnesium and chloride accumulation. We describe the evolutionary expansion and differentiation of haloarchaeal general transcription factor families and discuss their potential for enabling rapid adaptation to environmental fluxes. Lastly, we challenge a recent high-profile proposal regarding the evolutionary origins of the haloarchaea by showing that inclusion of additional genomes significantly reduces support for a proposed large-scale horizontal gene transfer into the ancestral haloarchaeon from the bacterial domain. This result highlights the power of our dataset for making evolutionary inferences, a feature which will make it useful to the broader evolutionary community. We distribute our genomic dataset through a user-friendly graphical interface.
doi:10.1371/journal.pgen.1004784
PMCID: PMC4230888  PMID: 25393412
5.  Taxonomic study of the genera Halogeometricum and Halosarcina: transfer of Halosarcina limi and Halosarcina pallida to the genus Halogeometricum as Halogeometricum limi comb. nov. and Halogeometricum pallidum comb. nov., respectively 
Members of the haloarchaeal genera Halosarcina and Halogeometricum (family Halobacteriaceae) are closely related to each other and show 96.6–98 % 16S rRNA gene sequence similarity. This is higher than the accepted threshold value (95 %) to separate two genera, and a taxonomic study using a polyphasic approach of all four members of the two genera was conducted to clarify their relationships. Polar lipid profiles indicated that Halogeometricum rufum RO1-4T, Halosarcina pallida BZ256T and Halosarcina limi RO1-6T are related more to each other than to Halogeometricum borinquense CGMCC 1.6168T. Phylogenetic analyses using the sequences of three different genes (16S rRNA gene, rpoB′ and EF-2) strongly supported the monophyly of these four species, showing that they formed a distinct clade, separate from the related genera Halopelagius, Halobellus, Haloquadratum, Haloferax and Halogranum. The results indicate that the four species should be assigned to the same genus, and it is proposed that Halosarcina pallida and Halosarcina limi be transferred to the genus Halogeometricum as Halogeometricum pallidum comb. nov. (type strain, BZ256T = KCTC 4017T = JCM 14848T) and Halogeometricum limi comb. nov. (type strain, RO1-6T = CGMCC 1.8711T = JCM 16054T).
doi:10.1099/ijs.0.055038-0
PMCID: PMC3799225  PMID: 24097833
6.  Nitrogen metabolism in haloarchaea 
Saline Systems  2008;4:9.
The nitrogen cycle (N-cycle), principally supported by prokaryotes, involves different redox reactions mainly focused on assimilatory purposes or respiratory processes for energy conservation. As the N-cycle has important environmental implications, this biogeochemical cycle has become a major research topic during the last few years. However, although N-cycle metabolic pathways have been studied extensively in Bacteria or Eukarya, relatively little is known in the Archaea. Halophilic Archaea are the predominant microorganisms in hot and hypersaline environments such as salted lakes, hot springs or salted ponds. Consequently, the denitrifying haloarchaea that sustain the nitrogen cycle under these conditions have emerged as an important target for research aimed at understanding microbial life in these extreme environments.
The haloarchaeon Haloferax mediterranei was isolated 20 years ago from Santa Pola salted ponds (Alicante, Spain). It was described as a denitrifier and it is also able to grow using NO3-, NO2- or NH4+ as inorganic nitrogen sources. This review summarizes the advances that have been made in understanding the N-cycle in halophilic archaea using Hfx mediterranei as a haloarchaeal model. The results obtained show that this microorganism could be very attractive for bioremediation applications in those areas where high salt, nitrate and nitrite concentrations are found in ground waters and soils.
doi:10.1186/1746-1448-4-9
PMCID: PMC2483277  PMID: 18593475
7.  Extremely halophilic archaea and the issue of long-term microbial survival 
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.
doi:10.1007/s11157-006-0007-y
PMCID: PMC3188376  PMID: 21984879
Extreme halophiles; Haloarchaea; Life detection; Microbial longevity; Salt mines; Salt sediments; Space missions; Subterranean; Taxonomy of halobacteriaceae
8.  The effects of extremes of pH on the growth and transcriptomic profiles of three haloarchaea 
F1000Research  2014;3:168.
The halophilic archaea (haloarchaea) live in saline environments, which are found across the globe.  In addition to salinity, these niches can be quite dynamic and experience extreme conditions such as low oxygen content, radiation (gamma and UV), pH and temperature.  However, of all the naturally occurring stresses faced by the haloarchaea, only one, pH, has not been previously investigated in regard to the changes induced in the transcriptome. Therefore, we endeavored to determine the responses in three haloarchaea: Halorubrum lacusprofundi (Hla), Haloferax volcanii (Hvo), and Halobacterium sp. NRC-1 (NRC-1) to growth under acidic and alkaline pH. Our observations showed that the transcriptomes of Hvo and NRC-1 regulated stress, motility, and ABC transporters in a similar manner, which is in line with previous reports from other prokaryotes when grown in an acidic environment.  However, the pattern for Hla was more species specific. For alkaline stress, all three haloarchaea responded in a manner similar to well-studied archaea and bacteria showing the haloarchaeal response was general to prokaryotes. Additionally, we performed an analysis on the changes in the transcriptomes of the three haloarchaea when shifting from one pH extreme to the other. The results showed that the transcriptomes of all three haloarchaea respond more similarly when moving from alkaline to acidic conditions compared to a shift in the opposite direction. Interestingly, our studies also showed that individual genes of multiple paralogous gene families ( tbp, tfb, orc/ cdc6, etc.) found in the haloarchaea were regulated under specific stresses thereby providing evidence that they modulate the response to various environmental stresses. The studies described here are the first to catalog the changes in the haloarchaeal transcriptomes under growth in extreme pH and help us understand how life is able to thrive under all conditions present on Earth and, if present, on extraterrestrial bodies as well.
doi:10.12688/f1000research.4789.2
PMCID: PMC4176423  PMID: 25285207
9.  Complete genome sequence of Halogeometricum borinquense type strain (PR3T) 
Standards in Genomic Sciences  2009;1(2):150-159.
Halogeometricum borinquense Montalvo-Rodríguez et al. 1998 is the type species of the genus, and is of phylogenetic interest because of its distinct location between the halobacterial genera Haloquadratum and Halosarcina. H. borinquense requires extremely high salt (NaCl) concentrations for growth. It can not only grow aerobically but also anaerobically using nitrate as electron acceptor. The strain described in this report is a free-living, motile, pleomorphic, euryarchaeon, which was originally isolated from the solar salterns of Cabo Rojo, Puerto Rico. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first complete genome sequence of the halobacterial genus Halogeometricum, and this 3,944,467 bp long six replicon genome with its 3937 protein-coding and 57 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.
doi:10.4056/sigs.23264
PMCID: PMC3035229  PMID: 21304651
halophile; free-living; non-pathogenic; aerobic; pleomorphic cells; euryarchaeon
10.  Gene cloning and heterologous synthesis of a haloalkaliphilic extracellular protease of Natrialba magadi (Nep) 
The gene encoding the protease Nep secreted by the haloalkaliphilic archaeon Natrialba magadii was cloned and sequenced. Upstream of the nep gene, a region related to haloarchaeal TATA-box and BRE-like consensus sequences was identified. The nep-encoded polypeptide had a molecular mass of 56.4-kDa, a pI of 3.77 and included a 121-amino acid propeptide not present in the mature Nep. A Tat motif (GRRSVL) was also identified at residues 10 to 15 suggesting it is a substrate of the Tat pathway. The primary sequence of Nep was closely related to serine proteases of the subtilisin family from archaea and bacteria (50 to 85% similarity). The nep gene was expressed in Escherichia coli and Haloferax volcanii resulting in production of active Nep protease. In contrast to the recombinant E. coli strains in which Nep activity was only detected in cell lysate, high levels of Nep protein and activity were detected in the culture medium of stationary phase recombinant Hfx. volcanii strains. The Hfx. volcanii synthesized protease was active in high salt, high pH and high DMSO. This study provides the first molecular characterization of a halolysin-like protease from alkaliphilic haloarchaea and is the first description of a recombinant system that facilitates high-level secretion of a haloarchaeal protease.
doi:10.1007/s00792-008-0174-6
PMCID: PMC2614922  PMID: 18553052
Natrialba magadii; Haloalkaliphilic protease; Gene cloning and expression; Solvent tolerance; Tat pathway
11.  The core and unique proteins of haloarchaea 
BMC Genomics  2012;13:39.
Background
Since the first genome of a halophilic archaeon was sequenced in 2000, biologists have been advancing the understanding of genomic characteristics that allow for survival in the harsh natural environments of these organisms. An increase in protein acidity and GC-bias in the genome have been implicated as factors in tolerance to extreme salinity, desiccation, and high solar radiation. However, few previous attempts have been made to identify novel genes that would permit survival in such extreme conditions.
Results
With the recent release of several new complete haloarchaeal genome sequences, we have conducted a comprehensive comparative genomic analysis focusing on the identification of unique haloarchaeal conserved proteins that likely play key roles in environmental adaptation. Using bioinformatic methods, we have clustered 31,312 predicted proteins from nine haloarchaeal genomes into 4,455 haloarchaeal orthologous groups (HOGs). We assigned likely functions by association with established COG and KOG databases in NCBI. After identifying homologs in four additional haloarchaeal genomes, we determined that there were 784 core haloarchaeal protein clusters (cHOGs), of which 83 clusters were found primarily in haloarchaea. Further analysis found that 55 clusters were truly unique (tucHOGs) to haloarchaea and qualify as signature proteins while 28 were nearly unique (nucHOGs), the vast majority of which were coded for on the haloarchaeal chromosomes. Of the signature proteins, only one example with any predicted function, Ral, involved in desiccation/radiation tolerance in Halobacterium sp. NRC-1, was identified. Among the core clusters, 33% was predicted to function in metabolism, 25% in information transfer and storage, 10% in cell processes and signaling, and 22% belong to poorly characterized or general function groups.
Conclusion
Our studies have established conserved groups of nearly 800 protein clusters present in all haloarchaea, with a subset of 55 which are predicted to be accessory proteins that may be critical or essential for success in an extreme environment. These studies support core and signature genes and proteins as valuable concepts for understanding phylogenetic and phenotypic characteristics of coherent groups of organisms.
doi:10.1186/1471-2164-13-39
PMCID: PMC3287961  PMID: 22272718
12.  The effects of extremes of pH on the growth and transcriptomic profiles of three haloarchaea 
F1000Research  2014;3:168.
The halophilic archaea (haloarchaea) live in saline environments which are found across the globe.  In addition to salinity, these niches can be quite dynamic and experience extreme conditions such as low oxygen content, radiation (gamma and UV), pH and temperature.  However, of all the naturally occurring stresses faced by the haloarchaea, only one, pH, has not been previously reported on. Therefore, we endeavored to determine the responses of the transcriptomes of three haloarchaea (Hla, Hvo, and NRC-1) to growth under acidic and alkaline pH. Our observations showed that the transcriptomes of Hvo and NRC-1 respond in a similar manner to each other as well as other prokaryotes when grown in an acidic environment, while the pattern for Hla was dissimilar. For alkaline stress, all three haloarchaea responded in a manner similar to well-studied archaea and bacteria and had four-times more significantly regulated transcripts in common, compared to acidic growth. Additionally, we performed an analysis on the changes in the transcriptomes of the three haloarchaea when shifting from one pH extreme to the other. The results showed that the transcriptomes of all three haloarchaea respond more similarly when moving from alkaline to acidic conditions compared to moving from an acidic to alkaline environment. Interestingly, our studies also showed that individual genes of multiple paralogous gene families ( tbp, tfb, orc/ cdc6, etc.) found in the haloarchaea were regulated under specific stresses thereby providing evidence that they modulate the response to various environmental stresses. The studies described here are the first to catalog the changes in the haloarchaeal transcriptomes under growth in extreme pH and help us understand how life is able to thrive under all conditions present on Earth and, if present, on extraterrestrial bodies as well.
doi:10.12688/f1000research.4789.1
PMCID: PMC4176423  PMID: 25285207
13.  Effect of Salinity on Rhizobium Growth and Survival † 
This study examines the effect of salinity on the growth and survival of Rhizobium spp. in culture media and soil. Eleven isolates from saline and nonsaline environments were compared. The growth (mean doubling time) of all strains and species tested decreased when the electrical conductivity of the culture medium (yeast extract-mannitol) was raised from 1.2 mS cm−1 to 6.7 mS cm−1 (15% seawater equivalent) or to 13.1 mS cm−1 (28% seawater equivalent). Three of eleven strains failed to grow at 13.1 mS cm−1. Although growth was affected by salinity, four strains selected from the growth rate study could survive in extremely high concentrations of salt. Two strains with growth rates sensitive to salt and two strains with growth rates relatively unaffected by salt were inoculated into solutions with electrical conductivities of up to 43.0 mS cm−1 (92% seawater equivalent). Not only did all four strains survive the initial osmotic shock (at 5 h after inoculation), but it was not until 27 days after inoculation that the sensitive strains exhibited a significant reduction in viable numbers. The salt-tolerant strains survived for more than 65 days with no reduction in viable counts. The interaction between soil moisture tension and soil salinity in relation to Rhizobium survival in gamma-irradiated soil was also examined. Six treatment combinations were used, ranging from −0.1 bars and 0.2 mS cm−1 to −15 bars and 12 mS cm−1. Sensitive strains declined from 107 to 105 organisms per g of soil after 84 days of incubation at −15 bars and 12 mS cm−1. Tolerant strains survived for the same period with no loss in viable numbers. The results of these experiments indicate that many strains of Rhizobium can grow and survive at salt concentrations which are inhibitory to most agricultural legumes. The emphasis of research concerning the effects of salinity on symbiotic nitrogen fixation should, therefore, be directed to aspects of the symbiosis other than the survival of the Rhizobium spp.
PMCID: PMC242113  PMID: 16346113
14.  Analysis of the Transcriptional Regulator GlpR, Promoter Elements, and Posttranscriptional Processing Involved in Fructose-Induced Activation of the Phosphoenolpyruvate-Dependent Sugar Phosphotransferase System in Haloferax mediterranei 
Among all known archaeal strains, the phosphoenolpyruvate-dependent phosphotransferase system (PTS) for fructose utilization is used primarily by haloarchaea, which thrive in hypersaline environments, whereas the molecular details of the regulation of the archaeal PTS under fructose induction remain unclear. In this study, we present a comprehensive examination of the regulatory mechanism of the fructose PTS in the haloarchaeon Haloferax mediterranei. With gene knockout and complementation, microarray analysis, and chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR), we revealed that GlpR is the indispensable activator, which specifically binds to the PTS promoter (PPTS) during fructose induction. Further promoter-scanning mutation indicated that three sites located upstream of the H. mediterranei PPTS, which are conserved in most haloarchaeal PPTSs, are involved in this induction. Interestingly, two PTS transcripts (named T8 and T17) with different lengths of 5′ untranslated region (UTR) were observed, and promoter or 5′ UTR swap experiments indicated that the shorter 5′ UTR was most likely generated from the longer one. Notably, the translation efficiency of the transcript with this shorter 5′ UTR was significantly higher and the ratio of T8 (with the shorter 5′ UTR) to T17 increased during fructose induction, implying that a posttranscriptional mechanism is also involved in PTS activation. With these insights into the molecular regulation of the haloarchaeal PTS, we have proposed a working model for haloarchaea in response to environmental fructose.
doi:10.1128/AEM.03372-13
PMCID: PMC3911062  PMID: 24334671
15.  Combined Use of Cultivation-Dependent and Cultivation-Independent Methods Indicates that Members of Most Haloarchaeal Groups in an Australian Crystallizer Pond Are Cultivable 
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 (106 CFU/ml) were obtained on solid media. Long incubation times (≥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.
doi:10.1128/AEM.70.9.5258-5265.2004
PMCID: PMC520848  PMID: 15345408
16.  Life at low water activity. 
Two major types of environment provide habitats for the most xerophilic organisms known: foods preserved by some form of dehydration or enhanced sugar levels, and hypersaline sites where water availability is limited by a high concentration of salts (usually NaCl). These environments are essentially microbial habitats, with high-sugar foods being dominated by xerophilic (sometimes called osmophilic) filamentous fungi and yeasts, some of which are capable of growth at a water activity (a(w)) of 0.61, the lowest a(w) value for growth recorded to date. By contrast, high-salt environments are almost exclusively populated by prokaryotes, notably the haloarchaea, capable of growing in saturated NaCl (a(w) 0.75). Different strategies are employed for combating the osmotic stress imposed by high levels of solutes in the environment. Eukaryotes and most prokaryotes synthesize or accumulate organic so-called 'compatible solutes' (osmolytes) that have counterbalancing osmotic potential. A restricted range of bacteria and the haloarchaea counterbalance osmotic stress imposed by NaCl by accumulating equivalent amounts of KCl. Haloarchaea become entrapped and survive for long periods inside halite (NaCl) crystals. They are also found in ancient subterranean halite (NaCl) deposits, leading to speculation about survival over geological time periods.
doi:10.1098/rstb.2004.1502
PMCID: PMC1693405  PMID: 15306380
17.  Novel Insights into the Diversity of Catabolic Metabolism from Ten Haloarchaeal Genomes 
PLoS ONE  2011;6(5):e20237.
Background
The extremely halophilic archaea are present worldwide in saline environments and have important biotechnological applications. Ten complete genomes of haloarchaea are now available, providing an opportunity for comparative analysis.
Methodology/Principal Findings
We report here the comparative analysis of five newly sequenced haloarchaeal genomes with five previously published ones. Whole genome trees based on protein sequences provide strong support for deep relationships between the ten organisms. Using a soft clustering approach, we identified 887 protein clusters present in all halophiles. Of these core clusters, 112 are not found in any other archaea and therefore constitute the haloarchaeal signature. Four of the halophiles were isolated from water, and four were isolated from soil or sediment. Although there are few habitat-specific clusters, the soil/sediment halophiles tend to have greater capacity for polysaccharide degradation, siderophore synthesis, and cell wall modification. Halorhabdus utahensis and Haloterrigena turkmenica encode over forty glycosyl hydrolases each, and may be capable of breaking down naturally occurring complex carbohydrates. H. utahensis is specialized for growth on carbohydrates and has few amino acid degradation pathways. It uses the non-oxidative pentose phosphate pathway instead of the oxidative pathway, giving it more flexibility in the metabolism of pentoses.
Conclusions
These new genomes expand our understanding of haloarchaeal catabolic pathways, providing a basis for further experimental analysis, especially with regard to carbohydrate metabolism. Halophilic glycosyl hydrolases for use in biofuel production are more likely to be found in halophiles isolated from soil or sediment.
doi:10.1371/journal.pone.0020237
PMCID: PMC3102087  PMID: 21633497
18.  Investigating the Effects of Simulated Martian Ultraviolet Radiation on Halococcus dombrowskii and Other Extremely Halophilic Archaebacteria 
Astrobiology  2009;9(1):104-112.
The isolation of viable extremely halophilic archaea from 250-million-year-old rock salt suggests the possibility of their long-term survival under desiccation. Since halite has been found on Mars and in meteorites, haloarchaeal survival of martian surface conditions is being explored. Halococcus dombrowskii H4 DSM 14522T was exposed to UV doses over a wavelength range of 200–400 nm to simulate martian UV flux. Cells embedded in a thin layer of laboratory-grown halite were found to accumulate preferentially within fluid inclusions. Survival was assessed by staining with the LIVE/DEAD kit dyes, determining colony-forming units, and using growth tests. Halite-embedded cells showed no loss of viability after exposure to about 21 kJ/m2, and they resumed growth in liquid medium with lag phases of 12 days or more after exposure up to 148 kJ/m2. The estimated D37 (dose of 37 % survival) for Hcc. dombrowskii was ≥ 400 kJ/m2. However, exposure of cells to UV flux while in liquid culture reduced D37 by 2 orders of magnitude (to about 1 kJ/m2); similar results were obtained with Halobacterium salinarum NRC-1 and Haloarcula japonica. The absorption of incoming light of shorter wavelength by color centers resulting from defects in the halite crystal structure likely contributed to these results. Under natural conditions, haloarchaeal cells become embedded in salt upon evaporation; therefore, dispersal of potential microscopic life within small crystals, perhaps in dust, on the surface of Mars could resist damage by UV radiation.
doi:10.1089/ast.2007.0234
PMCID: PMC3182532  PMID: 19215203
Halococcus dombrowskii; Simulated martian UV radiation; LIVE/DEAD staining; Halite fluid inclusions; UV transmittance and reflectance; Desiccation
19.  Raman spectroscopy as a potentialmethod for the detection of extremely halophilic archaea embedded in halite in terrestrial and possibly extraterrestrial samples 
Journal of Raman spectroscopy : JRS  2009;40(12):1996-2003.
Evidence for the widespread occurrence of extraterrestrial halite, particularly on Mars, has led to speculations on the possibility of halophilic microbial forms of life; these ideas have been strengthened by reports of viable haloarchaea from sediments of geological age (millions of years). Raman spectroscopy, being a sensitive detection method for future astrobiological investigations onsite, has been used in the current study for the detection of nine different extremely halophilic archaeal strains which had been embedded in laboratory-made halite crystals in order to simulate evaporitic conditions. The cells accumulated preferentially in tiny fluid inclusions, in simulation of the precipitation of salt in natural brines. FT-Raman spectroscopy using laser excitation at 1064 nm and dispersive micro Raman spectroscopy at 514.5 nm were applied. The spectra showed prominent peaks at 1507, 1152 and 1002 cm−1 which are attributed to haloarchaeal C50 carotenoid compounds (mainly bacterioruberins). Their intensity varied from strain to strain at 1064-nm laser excitation. Other distinguishable features were peaks due to peptide bonds (amide I, amide III) and to nucleic acids. No evidence for fatty acids was detected, consistent with their general absence in all archaea.
These results contribute to a growing database on Raman spectra of terrestrial microorganisms from hypersaline environments and highlight the influence of the different macromolecular composition of diverse strains on these spectra.
doi:10.1002/jrs.2357
PMCID: PMC3207228  PMID: 22058585
Raman spectroscopy; extremely halophilic archaea; halite; astrobiology; fluid inclusions; carotenoids; bacterioruberins; Martian subsurface
20.  Biodiversity of Archaea and floral of two inland saltern ecosystems in the Alto Vinalopó Valley, Spain 
Saline Systems  2010;6:10.
Background
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.
Results
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.
Conclusions
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.
doi:10.1186/1746-1448-6-10
PMCID: PMC2984398  PMID: 20942947
21.  Growth and physiological adaptation of whole plants and cultured cells from a halophyte turf grass under salt stress 
AoB Plants  2014;6:plu041.
Soil salinization is a serious problem in agricultural lands worldwide. Understanding the mechanisms of salt-tolerant plants will contribute to knowledge necessary to genetically engineer salt-tolerant crops that grow on these saline lands. We identified a genotype of Sporobolus virginicus, a salt-tolerant turf grass, that showed a salinity tolerance to up to a three-fold higher NaCl concentration than seawater salinity. In addition to salt secretion from salt glands on the leaves, this genotype accumulated K+ and proline, a compatible solute, to higher levels than other genotypes under salinity. These properties must contribute to the advanced salt tolerance of this genotype.
Understanding the mechanisms used by halophytic members of the Poaceae to cope with salt stress will contribute to the knowledge necessary to genetically engineer salt-tolerant crops. In this study, we identified a genotype of Sporobolus virginicus, a halophytic turf grass collected in Japan, and investigated its growth rate, ion concentration and secretion, and proline concentration in comparison with the reported properties of genotypes collected from the USA, South Africa and Egypt. Surprisingly, the Japanese genotype showed a salinity tolerance up to 1.5 M NaCl, a 3-fold higher concentration than seawater salinity. Shoot growth was stimulated by 100 mM NaCl and root growth was stimulated at salinities of up to 1 M NaCl. Accumulation of Na+ and CI− in shoots and roots was rapidly elevated by salinity stress but did not exceed the levels required for osmotic adjustment, due in part to ion secretion by salt glands, which are present in genotypes of S. virginicus. However, the Japanese genotypes accumulated K+ to a higher level than other genotypes, resulting in a relatively high K+/Na+ ratio even under salinity stress. An increase in proline concentration was observed that was proportional to the NaCl concentration in the culture solution and might partially account for osmotic adjustment in the shoots. We also generated and characterized cultured cells of S. virginicus. In 500 mM NaCl, the cultured cells showed an enhanced growth compared with cultured cells of rice. The concentration of Na+ and CI− in the cultured cells in 300–500 mM NaCl was lower than in 100 mM NaCl. Cultured cells of S. virginicus accumulated proline to higher levels than rice cells cultured under salinity stress. The active regulation of Na+, Cl− and K+ influx/efflux and proline accumulation might be involved in salt tolerance mechanisms at the cellular level as well as in planta.
doi:10.1093/aobpla/plu041
PMCID: PMC4137795  PMID: 25024277
Chloride ion; cultured cells; halophyte; potassium ion; proline; salt tolerance; sodium ion; Sporobolus virginicus
22.  Analysis of bacterial flora in dohyo soil 
Objectives
Sumo wrestling is one of the most popular sports in Japan. Injuries are not uncommon as this is a vigorous contact sport. Sumo wrestlers have little in the way of protective clothing; their main garb is the mawashi, making them prone to exposure to any microorganisms in the dohyo. The bacterial flora of the dohyo has received little attention. If the constituent flora is identified, then appropriate treatment or prevention of any bacterial lesions or infections incurred by the wrestlers is possible.
Methods
The Vitek AMS system used in this study was developed by McDonnell Douglas Corporation. In this system, the physiological and biochemical properties of Gram-positive and negative bacilli, Gram-positive and-negative cocci, and fungi isolated from clinical materials and environments are examined using test cards specifically for each microorganism group, and the results are automatically read by a computer and encoded. Obtained codes are compared with a built-in database, and bacterial species of test strains are identified.
Results
In this study, using the automatic identification kit VITEK or ATB, we describe the aerobic bacterial flora found in the dohyo over the four seasons of the year. We also investigated the effect of salt on the bacterial flora as sumo wrestlers toss salt on the dohyo before each match. We show the relationship between salinity changes and variations in the flora observed upon the addition of salt. Without salt, at the beginning of a match, Gram-negative bacteria predominate. When salt is added, there is a transient decrease in the incidence of flora followed by an increase in the incidence Grampositive cocci.
Conclusions
Sixteen bacterial genera were identified using the bacterial identification systems in dohyo soil samples during the year. The number of identified bacterial species was 32. Even in the presence of salt, there is a measurable amount of bacterial flora in dohyo soil; salt does not act as an antibacterial agent.
doi:10.1007/BF02898187
PMCID: PMC2723622  PMID: 21432076
aerobic bacterial flora; identification; sumo wrestler; salt tossed on dohyo
23.  Bacterial Growth at the High Concentrations of Magnesium Sulfate Found in Martian Soils 
Astrobiology  2012;12(2):98-106.
Abstract
The martian surface environment exhibits extremes of salinity, temperature, desiccation, and radiation that would make it difficult for terrestrial microbes to survive. Recent evidence suggests that martian soils contain high concentrations of MgSO4 minerals. Through warming of the soils, meltwater derived from subterranean ice-rich regolith may exist for an extended period of time and thus allow the propagation of terrestrial microbes and create significant bioburden at the near surface of Mars. The current report demonstrates that halotolerant bacteria from the Great Salt Plains (GSP) of Oklahoma are capable of growing at high concentrations of MgSO4 in the form of 2 M solutions of epsomite. The epsotolerance of isolates in the GSP bacterial collection was determined, with 35% growing at 2 M MgSO4. There was a complex physiological response to mixtures of MgSO4 and NaCl coupled with other environmental stressors. Growth also was measured at 1 M concentrations of other magnesium and sulfate salts. The complex responses may be partially explained by the pattern of chaotropicity observed for high-salt solutions as measured by agar gelation temperature. Select isolates could grow at the high salt concentrations and low temperatures found on Mars. Survival during repetitive freeze-thaw or drying-rewetting cycles was used as other measures of potential success on the martian surface. Our results indicate that terrestrial microbes might survive under the high-salt, low-temperature, anaerobic conditions on Mars and present significant potential for forward contamination. Stringent planetary protection requirements are needed for future life-detection missions to Mars. Key Words: Analogue—Mars—Planetary protection—Salts—Life in extreme environments. Astrobiology 12, 98–106.
doi:10.1089/ast.2011.0720
PMCID: PMC3277918  PMID: 22248384
24.  Amino Acid Substitutions in Cold-Adapted Proteins from Halorubrum lacusprofundi, an Extremely Halophilic Microbe from Antarctica 
PLoS ONE  2013;8(3):e58587.
The halophilic Archaeon Halorubrum lacusprofundi, isolated from the perennially cold and hypersaline Deep Lake in Antarctica, was recently sequenced and compared to 12 Haloarchaea from temperate climates by comparative genomics. Amino acid substitutions for 604 H. lacusprofundi proteins belonging to conserved haloarchaeal orthologous groups (cHOGs) were determined and found to occur at 7.85% of positions invariant in proteins from mesophilic Haloarchaea. The following substitutions were observed most frequently: (a) glutamic acid with aspartic acid or alanine; (b) small polar residues with other small polar or non-polar amino acids; (c) small non-polar residues with other small non-polar residues; (d) aromatic residues, especially tryptophan, with other aromatic residues; and (e) some larger polar residues with other similar residues. Amino acid substitutions for a cold-active H. lacusprofundi β-galactosidase were then examined in the context of a homology modeled structure at residues invariant in homologous enzymes from mesophilic Haloarchaea. Similar substitutions were observed as in the genome-wide approach, with the surface accessible regions of β-galactosidase displaying reduced acidity and increased hydrophobicity, and internal regions displaying mainly subtle changes among smaller non-polar and polar residues. These findings are consistent with H. lacusprofundi proteins displaying amino acid substitutions that increase structural flexibility and protein function at low temperature. We discuss the likely mechanisms of protein adaptation to a cold, hypersaline environment on Earth, with possible relevance to life elsewhere.
doi:10.1371/journal.pone.0058587
PMCID: PMC3594186  PMID: 23536799
25.  Solar salt lake as natural environmental source for extraction halophilic pigments 
Iranian Journal of Microbiology  2010;2(2):103-109.
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.
Results
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.
Conclusion
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.
PMCID: PMC3279771  PMID: 22347558
Halorubrum sodomense; haloarchaea; salt lake; metarhodopsin II; pigment

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