Halophilic Organisms Research Articles

Fluorescence microscopy visualization of halomucin, a secreted 927 kDa protein surrounding Haloquadratum walsbyi cells

At the time of its first publication, halomucin from Haloquadratum walsbyi strain HBSQ001 was the largest archaeal protein known (9159 aa). It has a predicted signal sequence, making it likely to be an extracellular or secreted protein. Best BLAST matches were found to be mammalian mucins that protect tissues to dehydration and chemical stress. It was hypothesized that halomucin participates in protection against desiccation by retaining water in a hull around the halophilic organisms that live at the limits of water activity. We visualized Haloquadratum cells by staining their intracellular polyhydroxybutyrate granules using Nile Blue. Halomucin was stained by immunofluorescence with antibodies generated against synthetic peptides derived from the halomucin amino acid sequence. Polyhydroxybutyrate stained cells were reconstructed in 3D which highlights not only the highly regular square shape but also the extreme flatness of Haloquadratum. Double-staining proves halomucin to be extracellular but to be only loosely associated to cells in agreement with its hypothesized function.

Electrical stimulation improves microbial salinity resistance and organofluorine removal in bioelectrochemical systems.

Fed batch bioelectrochemical systems (BESs) based on electrical stimulation were used to treat p-fluoronitrobenzene (p-FNB) wastewater at high salinities. At a NaCl concentration of 40 g/liter, p-FNB was removed 100% in 96 h in the BES, whereas in the biotic control (BC) (absence of current), p-FNB removal was only 10%. By increasing NaCl concentrations from 0 g/liter to 40 g/liter, defluorination efficiency decreased around 40% in the BES, and in the BC it was completely ceased. p-FNB was mineralized by 30% in the BES and hardly in the BC. Microorganisms were able to store 3.8 and 0.7 times more K(+) and Na(+) intracellularly in the BES than in the BC. Following the same trend, the ratio of protein to soluble polysaccharide increased from 3.1 to 7.8 as the NaCl increased from 0 to 40 g/liter. Both trends raise speculation that an electrical stimulation drives microbial preference toward K(+) and protein accumulation to tolerate salinity. These findings are in accordance with an enrichment of halophilic organisms in the BES. Halobacterium dominated in the BES by 56.8% at a NaCl concentration of 40 g/liter, while its abundance was found as low as 17.5% in the BC. These findings propose a new method of electrical stimulation to improve microbial salinity resistance.

Potassium stress growth characteristics and energetics in the haloarchaeon Haloarcula marismortui.

Growth characteristics surrounding halophilic archaeal organisms are extremely limited in the scientific literature, with studies tending toward observing changes in cellular generation times under growth conditions limited to changes in temperature and sodium chloride concentrations. Currently, knowledge of the ionic stress experienced by haloarchaeal species through an excess or depletion of other required ions is lacking at best. The halophilic archaeon, Haloarcula marismortui, was analyzed under extreme ionic stress conditions with a specific focus on induced potassium ion stress using growth curves and analysis of the intracellular ion concentrations. Generation times were determined under potassium chloride concentrations ranging from 8 to 720 mM, and also in the presence of the alternative monovalent cations of lithium, rubidium, and cesium under limiting potassium conditions. Intracellular ion concentrations, as determined by inductively coupled mass spectrometry (ICP-MS), indicate a minimum intracellular total ion requirement of 1.13 M while tolerating up to 2.43 M intracellular concentrations. The presence of intracellular rubidium and cesium indicates that monovalent ion transport is important for energy production. Comparison of eight archaeal genomes indicates an increased diversity of potassium transport complex subunits in the halophilic organisms. Analysis of the generation times, intracellular concentrations and genome survey shows Har. marismortui exhibits an ability to cope with monovalent cation concentration changes in its native environment and provides insight into the organisms ion transport capability and specificity.Electronic supplementary materialThe online version of this article (doi:10.1007/s00792-014-0716-z) contains supplementary material, which is available to authorized users.

Characterization of halophilic bacterial communities in Turda Salt Mine (Romania).

Halophilic organisms are having adaptations to extreme salinity, the majority of them being Archaean, which have the ability to grow at extremely high salt concentrations, (from 3 % to 35 %). Level of salinity causes natural fluctuations in the halophilic populations that inhabit this particular habitat, raising problems in maintaining homeostasis of the osmotic pressure. Samples such as salt and water taken from Turda Salt Mine were analyzed in order to identify the eco-physiological bacterial groups. Considering the number of bacteria of each eco-physiological group, the bacterial indicators of salt quality (BISQ) were calculated and studied for each sample. The phosphatase, catalase and dehydrogenases enzymatic activities were quantitatively determined and the enzymatic indicators of salt quality (EISQ) were calculated. Bacterial isolates were analyzed using 16S rRNA gene sequence analysis. Universal bacterial primers, targeting the consensus region of the bacterial 16S rRNA gene were used. Analysis of a large fragment, of 1499 bp was performed to improve discrimination at the species level.

Protective role of salt in catalysis and maintaining structure of halophilic proteins against denaturation.

Search for new industrial enzymes having novel properties continues to be a desirable pursuit in enzyme research. The halophilic organisms inhabiting under saline/ hypersaline conditions are considered as promising source of useful enzymes. Their enzymes are structurally adapted to perform efficient catalysis under saline environment wherein n0n-halophilic enzymes often lose their structure and activity. Haloenzymes have been documented to be polyextremophilic and withstand high temperature, pH, organic solvents, and chaotropic agents. However, this stability is modulated by salt. Although vast amount of information have been generated on salt mediated protection and structure function relationship in halophilic proteins, their clear understanding and correct perspective still remain incoherent. Furthermore, understanding their protein architecture may give better clue for engineering stable enzymes which can withstand harsh industrial conditions. The article encompasses the current level of understanding about haloadaptations and analyzes structural basis of their enzyme stability against classical denaturants.

New insights into microbial adaptation to extreme saline environments

Extreme halophiles are microorganisms adapted to low water activity living at the upper salt concentration that life can tolerate. We review here recent data that specify the main factors, which determine their peculiar salt-dependent biochemistry. The data suggested that evolution proceeds by stage to modify the molecular dynamics properties of the entire proteome. Extreme halophiles therefore represent tractable models to understand how fast and to what extent microorganisms adapt to environmental changes. Halophiles are also robust organisms, capable to resist multiple stressors. Preliminary studies indicated that they have developed a cellular response specifically aimed to survive when the salt condition fluctuates. Because of these properties halophilic organisms deserve special attention in the search for traces of life on other planets.

Characterization of Halophilic Acyl-CoA Thioesterase from Chromohalobacter salexigens for Use in Biofuel Production

Acyl-CoA thioesterases are enzymes which catalyze the hydrolysis of acyl-CoAs at thioester linkages to form free fatty acids and coenzyme A. These enzymes, along with lipases, have a wide spectrum of use in industrial applications including the production of fatty acids from oil-producing microorganisms for use as biofuel feed-stocks. This technology coupled with an increasing interest in sustainable biofuels has led researchers to investigate algae based systems of fuel production. Since a number of marine algae that are being evaluated for biofuel production prefer moderately halophilic environments, it follows that thioesterases/lipases originating from a halophilic organism may be best suited for use with a marine algae-based biofuel system. Recent studies have shown increased production of free fatty acids in Escherichia coli and cyanobacteria following overexpression of recombinant, leaderless thioesterase I (TesA) from E. coli. A homolog of TesA from the moderate halophilic bacterium Chromohalobacter salexigens has been identified, cloned, and recombinantly expressed in E. coli strains BL21 and M15. Because previous studies indicated that histidine tag position alters TesA substrate specificity, three different recombinant versions of the C. salexigens TesA were produced for this study; an N-terminal histidine-tagged enzyme, a C-terminal histidine-tagged enzyme, and one in which the N-terminal histidine-tag was removed via DAPase digestion. Introduction of a C-terminal histidine tag shifted substrate preference to shorter (<C10) carbon chain lengths, while introduction of an N-terminal tag resulted in overall reduced activity. Finally, removal of the N-terminal tag restored overall activity levels while slightly increasing enzyme preference for longer (C10/C12) carbon chain substrates. Given the requirement of lipolytic activity to generate free fatty acids for biofuel synthesis, this study has important implications for the use of thioesterases/lipases in marine algae based biofuel systems.

Ferredoxin-dependent glutamate synthase: involvement in ammonium assimilation in Haloferax mediterranei

Glutamate synthase (GOGAT) is one of the two important enzymes involved in the ammonium assimilation pathway glutamine synthetase (GS)/GOGAT, which enables Hfx. mediterranei to thrive in media with low ammonium concentration or containing just nitrate as single nitrogen source. The gene coding for this enzyme, gltS, has been sequenced, analysed and compared with other GOGATs from different organisms from the three domains of life. According to its amino acid sequence, Hfx. mediterranei GOGAT displays high homology with those from other archaeal halophilic organisms and with the bacterial alpha-like subunit. Hfx. mediterranei GOGAT and GS expression was induced under conditions of ammonium restriction. The GOGAT protein was found to be a monomer with a molecular mass of 163.78 kDa, which is consistent with that estimated by gel filtration, 198 ± 30 kDa. The enzyme is highly ferredoxin dependent: activity was only observed with one of the two different 2Fe-2S ferredoxins chromatographically isolated from Hfx. mediterranei. The enzyme also displayed typical halophilic behaviour, being fully stable, and producing maximal activity, at salt concentrations from 3 to 4 M NaCl, pH 7.5 and a temperature of 50 °C.

Stoichiometric and kinetic analysis of extreme halophilic Archaea on various substrates in a corrosion resistant bioreactor

Extreme halophilic Archaea are extremophile species which can thrive in hypersaline environments of up to 3-5 M sodium chloride concentration. Although their ecology and physiology are widely identified on the microbiological level, little emphasis has been laid on quantitative bioprocess development with extreme halophiles. The goal of this study was to establish, on the one hand, a methodological basis for quantitative bioprocess analysis of extreme halophilic Archaea with an extreme halophilic strain as an example. Firstly, as a novel usage, a corrosion resistant bioreactor setup for extreme halophiles has been implemented. Then, paying special attention to total bioprocess quantification approaches, an indirect method for biomass quantification using on-line process signals was introduced. Subsequently, robust quantitative data evaluation methods for halophiles could be developed, providing defined and controlled cultivation conditions in the bioreactor and therefore obtaining suitable quality of on-line as well as off-line datasets. On the other hand, new physiological results of extreme halophiles in bioreactor have also been obtained based on the quantitative methodological tools. For the first time, quantitative data on stoichiometry and kinetics were collected and evaluated on different carbon sources. The results on various substrates were interpreted, with proposed metabolic mechanisms, by linking to the reported primary carbon metabolism of extreme halophilic Archaea. Moreover, results of chemostat cultures demonstrated that extreme halophilic organisms show Monod-kinetics on different sole carbon sources. A diauxic growth pattern was described on a mixture of substrates in batch cultivations. In addition, the methodologies presented here enable one to characterize the utilized strain Haloferax mediterranei (HFX) as a potential new host organism. Thus, this study offers a strong methodological basis as well as a fundamental physiological assessment for bioreactor quantification of extreme halophiles that can serve as primary knowledge for applications of extreme halophiles in biotechnology.

A role for programmed cell death in the microbial loop.

The microbial loop is the conventional model by which nutrients and minerals are recycled in aquatic eco-systems. Biochemical pathways in different organisms become metabolically inter-connected such that nutrients are utilized, processed, released and re-utilized by others. The result is that unrelated individuals end up impacting each others' fitness directly through their metabolic activities. This study focused on the impact of programmed cell death (PCD) on a population's growth as well as its role in the exchange of carbon between two naturally co-occurring halophilic organisms. Flow cytometric, biochemical, 14C radioisotope tracing assays, and global transcriptomic analyses show that organic algal photosynthate released by Dunalliela salina cells undergoing PCD complements the nutritional needs of other non-PCD D. salina cells. This occurs in vitro in a carbon limited environment and enhances the growth of the population. In addition, a co-occurring heterotroph Halobacterium salinarum re-mineralizes the carbon providing elemental nutrients for the mixoheterotrophic chlorophyte. The significance of this is uncertain and the archaeon can also subsist entirely on the lysate of apoptotic algae. PCD is now well established in unicellular organisms; however its ecological relevance has been difficult to decipher. In this study we found that PCD in D. salina causes the release of organic nutrients such as glycerol, which can be used by others in the population as well as a co-occurring halophilic archaeon. H. salinarum also re-mineralizes the dissolved material promoting algal growth. PCD in D. salina was the mechanism for the flow of dissolved photosynthate between unrelated organisms. Ironically, programmed death plays a central role in an organism's own population growth and in the exchange of nutrients in the microbial loop.

Complete genome sequence of Halorhodospira halophila SL1

Halorhodospira halophila is among the most halophilic organisms known. It is an obligately photosynthetic and anaerobic purple sulfur bacterium that exhibits autotrophic growth up to saturated NaCl concentrations. The type strain H. halophila SL1 was isolated from a hypersaline lake in Oregon. Here we report the determination of its entire genome in a single contig. This is the first genome of a phototrophic extreme halophile. The genome consists of 2,678,452 bp, encoding 2,493 predicted genes as determined by automated genome annotation. Of the 2,407 predicted proteins, 1,905 were assigned to a putative function. Future detailed analysis of this genome promises to yield insights into the halophilic adaptations of this organism, its ability for photoautotrophic growth under extreme conditions, and its characteristic sulfur metabolism.

Metabolic engineering for enhanced hydrogen production: a review

Hydrogen gas exhibits potential as a sustainable fuel for the future. Therefore, many attempts have been made with the aim of producing high yields of hydrogen gas through renewable biological routes. Engineering of strains to enhance the production of hydrogen gas has been an active area of research for the past 2 decades. This includes overexpression of hydrogen-producing genes (native and heterologous), knockout of competitive pathways, creation of a new productive pathway, and creation of dual systems. Interestingly, genetic mutations in 2 different strains of the same species may not yield similar results. Similarly, 2 different studies on hydrogen productivities may differ largely for the same mutation and on the same species. Consequently, here we analyzed the effect of various genetic modifications on several species, considering a wide range of published data on hydrogen biosynthesis. This article includes a comprehensive metabolic engineering analysis of hydrogen-producing organisms, namely Escherichia coli, Clostridium, and Enterobacter species, and in addition, a short discussion on thermophilic and halophilic organisms. Also, apart from single-culture utilization, dual systems of various organisms and associated developments have been discussed, which are considered potential future targets for economical hydrogen production. Additionally, an indirect contribution towards hydrogen production has been reviewed for associated species.

Investigation of element distribution between components of a salt-lake system by SR-XRF

Numerous bitter lakes, largely closed and shallow, are concentrated in a small area of the South of Western Siberia (Kulunda and Barabinsk steppes). Most lakes are marked by the development of thick black or grayish blue silt organogenic sediments formed due to the dying-off of local halophilic organisms. Salt lakes are of great interest for scientists. The investigation was conducted in the context of complex biological and biochemical studies aimed at identifying regularities in the redistribution of elements between components of a salt-lake system by SR-XRF (Synchrotron Radiation X-ray Fluorescence).

Meiobenthos composition and structure in highly mineralized tributaries of Lake El’ton

The meiobenthos of six tributaries of the hypergaline Lake El’ton (the Volgograd region) in the low-flow period (August) has been researched for the first time. New species have been found for science, Russia, and the region. Widespread halophilic and halobiontic organisms formed the basis of the community, which was characterized by the relatively low diversity and an extremely wide range of abundance and biomass with the strong domination of some species. The tendency of the species richness to decrease with increasing mineralization from the mesoto eu- and hypergaline level was observed.

Nmag_2608, an extracellular ubiquitin-like domain-containing protein from the haloalkaliphilic archaeon Natrialba magadii

Ubiquitin-like proteins (Ubls) and ubiquitin-like domain-containing proteins (Ulds) found in both eukaryotes and prokaryotes display an ubiquitin fold. We previously characterized a 124-amino acid polypeptide (P400) from the haloalkaliphilic archaeon Natrialba magadii having structural homology with ubiquitin family proteins. The reported N. magadii's genome allowed the identification of the Nmag_2608 gene for the protein containing P400, which belongs to specific orthologs of halophilic organisms. It was found that Nmag_2608 has an N-terminal signal peptide with a lipobox motif characteristic of bacterial lipoproteins. Also, it presents partial identity with the ubiquitin-like domain-containing proteins, soluble ligand binding β-grasp proteins. Western blots and heterologous expression tests in E. coli evidenced that Nmag_2608 is processed and secreted outside the cell, where it could perform its function. The analysis of Nmag_2608 expression in N. magadii's cells suggests a co-transcription with the adjoining Nmag_2609 gene encoding a protein of the cyclase family. Also, the transcript level decreased in cells grown in low salinity and starved. To conclude, this work reports for the first time an extracellular archaeal protein with an ubiquitin-like domain.

Characterization of a Novel Overexpression System in Haloferax volcanii

A novel inducible protein expression system has been developed in the halophilic archaea, Haloferax volcanii (Hvo.) The expression system is based on the histidine utilization (Hut) operon and can be induced by histidine or urocanate, a histidine metabolite. This study characterizes the 88 base promoter region between the gene that encodes a newly discovered Hut regulatory protein (Hut R) and the first gene in the Hut operon, Hut U. Plasmids have been constructed that contain the gene for Hut R, the promoter region, the first three codons of Hut U and the gene for L7ae, an RNA binding protein, used as an expression reporter (see figure). The plasmid containing the wildtype promoter yields a high level of inducible gene expression when transformed into Hvo. Two specific mutations have been constructed in the promoter near the Hut U start sequence, deleting either 5 base (Del 1) or 20 bases (Del 2) immediately upstream of the Hut U start codon. These mutants yield decreased gene expression as measured by Northern and Western analysis. The new system appears to be generally useful for overexpression of halophilic proteins and has the potential for protein overexpression from a variety of halophilic organisms. This work was supported by Capital University.

Actibacterium mucosum gen. nov., sp. nov., a marine alphaproteobacterium from Mediterranean seawater

Strain R46(T), a marine alphaproteobacterium, was isolated from Mediterranean seawater at Malvarrosa beach, Valencia, Spain. It is an aerobic chemo-organotrophic, mesophilic and slightly halophilic organism, with complex ionic requirements. Phylogenetic analyses based on the 16S rRNA and gyrB gene sequences showed that strain R46(T) formed a separate branch within the family Rhodobacteraceae, bearing similarities below 94.7 and 80.3%, respectively, to any other recognized species. It contained Q10 as the predominant isoprenoid quinone and C(18:1)ω7c/C(18:1)ω6c as the major cellular fatty acid. Phosphatidylglycerol was the only identified polar lipid, although other lipids were also detected. The DNA G+C content of the genomic DNA was 61.3 mol%. On the basis of extensive phenotypic and phylogenetic comparative analysis, it is concluded that the strain represents a novel genus and species, for which the name Actibacterium mucosum gen. nov., sp. nov. is proposed. The type strain of the type species is Actibacterium mucosum R46(T) ( = CECT 7668(T) = KCTC 23349(T)).

Effects of different ion compositions on growth of obligately halophilic protozoan Halocafeteria seosinensis

Substantial halophilic organisms have been found in 100-200‰ salinities. These ranges represent a highly specialized halophilic environment to which only a few halotolerant species have adapted. Recent studies have underlined the existence of diverse obligately halophilic protozoa in the salinity ranges of 100-200‰. The ranges of salinity under which these organisms can grow have been examined to some extent, but the balance of specific ions that will support growth has not been investigated. The heterotrophic nanoflagellate Halocafeteria, the type strain of which grows optimally at 150‰ salinity and 35°C, is a commonly encountered obligate halophile found in very hypersaline environments. These extreme environments can vary in their Mg:Ca ratios (i.e. weight ratios) and sulfate concentrations. To examine growth response of Halocafeteria to the different chemical compositions, densities of Halocafeteria seosinensis strain EHF34 were monitored in seven different ion composition media for 9days at 1- to 2-day intervals (at 150‰ salinity and 35°C, with no prey limitation). Halocafeteria does not grow at Mg:Ca ratios of 35 and 100 and at high sulfate concentrations of 11.6 and 31.6gl(-1). It grows well in 0.6gl(-1) sulfate media at Mg:Ca ratios of 2, 10 or 35, but not 100. The present study demonstrates that the growth of the obligate halophile Halocafeteria can be affected by different ion compositions in hypersaline environments. Therefore, Halocafeteria may not be ubiquitous in hypersaline environments due to its ionic requirements.

Unveiling microbial life in the new deep‐sea hypersaline Lake Thetis. Part II: a metagenomic study

So far only little is known about the microbial ecology of Mediterranean deep-sea hypersaline anoxic lakes (DHALs). These brine lakes were formed by evaporite dissolution/brine seeps and are important model environments to provide insights into possible metabolisms and distributions of microorganisms on the early Earth. Our study on the Lake Thetis, a new thalassohaline DHAL located South-East of the Medriff Corridor, has revealed microbial communities of contrasting compositions with a high number of novel prokaryotic candidate divisions. The major finding of our present work is co-occurrence of at least three autotrophic carbon dioxide fixation pathways in the brine-seawater interface that are likely fuelled by an active ramified sulphur cycle. Genes for the reductive acetyl-CoA and reductive TCA pathways were also found in the brine suggesting that these pathways are operational even at extremely elevated salinities and that autotrophy is more important in hypersaline environments than previously assumed. Surprisingly, genes coding for RuBisCo were found in the highly reduced brine. Three types of sulphide oxidation pathways were found in the interface. The first involves a multienzyme Sox complex catalysing the complete oxidation of reduced sulphur compounds to sulphate, the second type recruits SQR sulphide:quinone reductase for oxidation of sulphide to elemental sulphur, which, in the presence of sulphide, could further be reduced by polysulphide reductases in the third pathway. The presence of the latter two allows a maximal energy yield from the oxidation of sulphide and at the same time prevents the acidification and the accumulation of S(0) deposits. Amino acid composition analysis of deduced proteins revealed a significant overrepresentation of acidic residues in the brine compared with the interface. This trait is typical for halophilic organisms as an adaptation to the brine's extreme hypersalinity. This work presents the first metagenomic survey of the microbial communities of the recently discovered Lake Thetis whose brine constitutes one of saltiest water bodies ever reported.

Protein attributes contribute to halo-stability, bioinformatics approach

Halophile proteins can tolerate high salt concentrations. Understanding halophilicity features is the first step toward engineering halostable crops. To this end, we examined protein features contributing to the halo-toleration of halophilic organisms. We compared more than 850 features for halophilic and non-halophilic proteins with various screening, clustering, decision tree, and generalized rule induction models to search for patterns that code for halo-toleration. Up to 251 protein attributes selected by various attribute weighting algorithms as important features contribute to halo-stability; from them 14 attributes selected by 90% of models and the count of hydrogen gained the highest value (1.0) in 70% of attribute weighting models, showing the importance of this attribute in feature selection modeling. The other attributes mostly were the frequencies of di-peptides. No changes were found in the numbers of groups when K-Means and TwoStep clustering modeling were performed on datasets with or without feature selection filtering. Although the depths of induced trees were not high, the accuracies of trees were higher than 94% and the frequency of hydrophobic residues pointed as the most important feature to build trees. The performance evaluation of decision tree models had the same values and the best correctness percentage recorded with the Exhaustive CHAID and CHAID models. We did not find any significant difference in the percent of correctness, performance evaluation, and mean correctness of various decision tree models with or without feature selection. For the first time, we analyzed the performance of different screening, clustering, and decision tree algorithms for discriminating halophilic and non-halophilic proteins and the results showed that amino acid composition can be used to discriminate between halo-tolerant and halo-sensitive proteins.