Salt-tolerant Organisms Research Articles

Genomics and transcriptomics reveal β-carotene synthesis mechanism in Dunaliella salina.

Dunaliella salina is by far the most salt-tolerant organism and contains many active substances, including β-carotene, glycerol, proteins, and vitamins, using in the production of dried biomass or cell extracts for the biofuels, pharmaceutical formulations, food additives, and fine chemicals, especially β-carotene. We report a high-quality genome sequence of D. Salina FACHB435, which has a 472 Mb genome size, with a contig N50 of 458 Kb. A total of 30,752 protein-coding genes were predicted. The annotation results evaluated by BUSCO was shown that completeness was 91.0% and replication was 53.1%. The fragments were 6.3% and the deletions were 2.6%. Phylogenomic and comparative genomic analyses revealed that A. thaliana diverged from Volvocales about 448 million years ago, then Volvocales C. eustigma, D. salina, and other species diverged about 250 million years ago. High light could promote the accumulation of β-carotene in D. salina at a 13 d stage of culture. The enrichment of DEGs in KEGG, it notes that the predicted up-regulated genes of carotenoid metabolic pathway include DsCrtB, DsPDS, DsZ-ISO, DsZDS, DsCRTISO, DsLUT5, DsCrtL-B, and DsCCD8, while the predicted down-regulated genes include DsCrtF, and DsLUT1. The four genes that were both up-regulated and down-regulated were DsZEP, DsCrtR-b, DsCruA/P and DsCrtZ 4. The research results can provide scientific basis for the industrialization practice of D. salina.

INVESTIGATING THE MICROBIOTA OF SOLAR SALTERN OF MULUND, MUMBAI, INDIA

Solar salterns, located in tropical and subtropical areas worldwide, are artificial shallow ponds for the production ofhalite (NaCl) from seawater. They are often built as multipond systems. Multi-pond solar salterns have been widespreadenvironments for studies on halophilic microorganisms. In the present study Archaeal and bacterial diversity of thesalterns located at Mulund (19°10’12’’N, 72°57’18’’E), Mumbai was investigated. Three Halophiles were isolated fromthe brine samples collected. All isolated archaea were shown intensely orange pigmentation. These salt tolerantorganisms were characterized using morphological, biochemical and 16S rRNA analysis. Phylogenetic analysis of thesesequences revealed a range of identities to several group of bacteria. The clones fell into major lineages theActinobacteria and firmicutes. Presence of industrially important carotenoids producing halophiles were also recorded.

Whole-genome sequencing and genome-scale metabolic modeling of Chromohalobacter canadensis 85B to explore its salt tolerance and biotechnological use.

Salt tolerant organisms are increasingly being used for the industrial production of high‐value biomolecules due to their better adaptability compared to mesophiles. Chromohalobacter canadensis is one of the early halophiles to show promising biotechnology potential, which has not been explored to date. Advanced high throughput technologies such as whole‐genome sequencing allow in‐depth insight into the potential of organisms while at the frontiers of systems biology. At the same time, genome‐scale metabolic models (GEMs) enable phenotype predictions through a mechanistic representation of metabolism. Here, we sequence and analyze the genome of C. canadensis 85B, and we use it to reconstruct a GEM. We then analyze the GEM using flux balance analysis and validate it against literature data on C. canadensis. We show that C. canadensis 85B is a metabolically versatile organism with many features for stress and osmotic adaptation. Pathways to produce ectoine and polyhydroxybutyrates were also predicted. The GEM reveals the ability to grow on several carbon sources in a minimal medium and reproduce osmoadaptation phenotypes. Overall, this study reveals insights from the genome of C. canadensis 85B, providing genomic data and a draft GEM that will serve as the first steps towards a better understanding of its metabolism, for novel applications in industrial biotechnology.

Halophilic Algal Communities in Biological Soil Crusts Isolated From Potash Tailings Pile Areas

Mining potash for fertilizer produces vast amounts of highly saline waste that is deposited in potash tailings piles. Rainfall washes the salts out, affecting the surrounding ecosystems. Only salt-tolerant organisms occur close to the piles, since other species cannot survive in these hypersaline conditions. Halophytic plant communities around tailings piles have been well investigated. However, studies exploring the biodiversity of the space behind the vegetation line that separates suitable salinities for plant growth from hostile conditions are rare. On top of the tailings piles, only micro-organisms thrive. This study, for the first time, explored the microalgae and cyanobacteria in biocrusts that in-habit potash tailings pile areas between the vegetation line and the pile body. Two biocrust types were studied: young biocrusts at or close to the tailings piles, and established biocrusts situated in the near surroundings. The estimated abundance of green algae, cyanobacteria and diatoms was studied using a direct microscopy approach, followed by the isolation and establishment of unialgal strains and morphological species determinations. Soil environmental characteristics were analyzed. Soil samples showed ECSat (electrical conductivity) values that drastically exceeded the scales commonly used to describe soil salinity, indicating extremely saline conditions. Indeed, the isolate composition was shaped by soil salinity parameters. Soil ECSat of young biocrusts tended to be higher than in estab-lished biocrusts, and filamentous green algae were most abundant. In contrast, established biocrusts tended to have a lower ECSat and were mostly dominated by filamentous cyanobacteria. Algal and cyanobacteria isolate composition differed significantly in young and established biocrusts, although the species number did not. Some of the salt-tolerant algal strains are assumed to be candidates for the formation of artificial biocrusts on the surface of the tailings. Attempts to “green” the piles by the establishment of higher plants to trap rainwater and therefore reduce the salt-output are difficult to apply. Plants require a thick layer of substrate to reduce the salinity, and the substrate easily erodes on the steep slopes. However, microalgae isolated from potash tailings pile areas seem promising, since they can survive on a thinner substrate layer and are already adapted to these hypersaline conditions.

Toxicity of various road-deicing salts to Asian clams (Corbicula fluminea).

Humans are altering environments by destroying habitats, introducing species, and releasing pollution. One emergent pollutant is the salinization of freshwater habitats from road-deicing salts. Government agencies have set thresholds to protect freshwater ecosystems, yet these values are exceeded in many systems. The present study investigated the tolerance of Asian clams (Corbicula fluminea), a common invasive bivalve, to the common road salt (sodium chloride [NaCl]) and 2 alternatives (magnesium chloride [MgCl2 ] and calcium chloride [CaCl2 ]). Experiments conducted at 4 and 8 d revealed that Asian clams are very salt tolerant. The median lethal concentration after 4 d of exposure (LC504-d ) estimate was 2162 mg Cl- /L for MgCl2 , 3554 mg Cl- /L for CaCl2 , and more than 22 581 mg Cl- /L for NaCl, which were all significantly different from each other (p ≤ 0.05). The LC508-d values were significantly different (p ≤ 0.05) from each other and from the LC504-d values, and were estimated to be 1769 mg Cl- /L for MgCl2 , 2235 Cl- /L for CaCl2 , and 10 069 mg Cl- /L for NaCl. Mortality was determined using 2 methods: either no response after exposure or no response after being in freshwater following exposure. For the majority of the LC50s, these methods were not significantly different (p > 0.05). The high salt tolerance of Asian clams is a concern because of their transportation in ballast water between aquatic ecosystems. Furthermore, salt-tolerant organisms may outcompete sensitive organisms in salinized ecosystems, which may alter ecosystem services. Environ Toxicol Chem 2018;37:1839-1845. © 2018 SETAC.

Identification of two different chemosensory pathways in representatives of the genus Halomonas

BackgroundSpecies of the genus Halomonas are salt-tolerant organisms that have a versatile metabolism and can degrade a variety of xenobiotic compounds, utilizing them as their sole carbon source. In this study, we examined the genome of a Halomonas isolate from a hydrocarbon-contaminated site to search for chemosensory genes that might be responsible for the observed chemotactic behavior of this organism as well as for other responses to environmental cues.ResultsUsing genome-wide comparative tools, our isolate was identified as a strain of Halomonas titanicae (strain KHS3), together with two other Halomonas strains with available genomes that had not been previously identified at a species level.The search for the main components of chemosensory pathways resulted in the identification of two clusters of chemosensory genes and a total of twenty-five chemoreceptor genes.One of the gene clusters is very similar to the che cluster from Escherichia coli and, presumably, it is responsible for the chemotactic behavior towards a variety of compounds. This gene cluster is present in 47 out of 56 analyzed Halomonas strains with available genomes.A second che-like cluster includes a gene coding for a diguanylate cyclase with a phosphotransfer and two receiver domains, as well as a gene coding for a chemoreceptor with a longer cytoplasmic domain than the other twenty-four. This seemingly independent pathway resembles the wsp pathway from Pseudomonas aeruginosa although it also presents several differences in gene order and domain composition. This second chemosensory gene cluster is only present in a sub-group within the genus Halomonas. Moreover, remarkably similar gene clusters are also found in some orders of Proteobacteria phylogenetically more distant from the Oceanospirillales, suggesting the occurrence of lateral transfer events.ConclusionsChemosensory pathways were investigated within the genus Halomonas. A canonical chemotaxis pathway, controlled by a variable number of chemoreceptors, is widespread among Halomonas species. A second chemosensory pathway of unique organization that involves some type of c-di-GMP signaling was found to be present only in one branch of the genus, as well as in other proteobacterial lineages.

Analysis of carotenogenic genes promoters and WRKY transcription factors in response to salt stress in Dunaliella bardawil

The unicellular alga Dunaliella bardawil is a highly salt-tolerant organism, capable of accumulating glycerol, glycine betaine and β-carotene under salt stress, and has been considered as an excellent model organism to investigate the molecular mechanisms of salt stress responses. In this study, several carotenogenic genes (DbCRTISO, DbZISO, DbLycE and DbChyB), DbBADH genes involved in glycine betaine synthesis and genes encoding probable WRKY transcription factors from D. bardawil were isolated, and promoters of DbCRTISO and DbChyB were cloned. The promoters of DbPSY, DbLycB, DbGGPS, DbCRTISO and DbChyB contained the salt-regulated element (SRE), GT1GMSCAM4, while the DbGGPS promoter has another SRE, DRECRTCOREAT. All promoters of the carotenogenic genes had light-regulated elements and W-box cis-acting elements. Most WRKY transcription factors can bind to the W-box, and play roles in abiotic stress. qRT-PCR analysis showed that salt stress up-regulated both carotenogenic genes and WRKY transcription factors. In contrast, the transcription levels of DbBADH showed minor changes. In D. bardawil, it appears that carotenoid over-accumulation allows for the long-term adaptation to salt stress, while the rapid modulation of glycine betaine biosynthesis provides an initial response.

Rational engineering of a mesohalophilic carbonic anhydrase to an extreme halotolerant biocatalyst.

Enzymes expressed by highly salt-tolerant organisms show many modifications compared with salt-affected counterparts including biased amino acid and lower α-helix content, lower solvent accessibility and negative surface charge. Here, we show that halotolerance can be generated in an enzyme solely by modifying surface residues. Rational design of carbonic anhydrase II is undertaken in three stages replacing 18 residues in total, crystal structures confirm changes are confined to surface residues. Catalytic activities and thermal unfolding temperatures of the designed enzymes increase at high salt concentrations demonstrating their shift to halotolerance, whereas the opposite response is found in the wild-type enzyme. Molecular dynamics calculations reveal a key role for sodium ions in increasing halotolerant enzyme stability largely through interactions with the highly ordered first Na+ hydration shell. For the first time, an approach to generate extreme halotolerance, a trait with broad application in industrial biocatalysis, in a wild-type enzyme is demonstrated.

Identifying novel salt-tolerant genes from Dunaliella salina using a Haematococcus pluvialis expression system

Dunaliella salina (Dunal) Teod, a unicellular eukaryotic green alga, is a highly salt-tolerant organism. To identify novel genes with potential roles in salinity tolerance, a salt stress-induced D. salina cDNA library was screened based on the expression in Haematococcus pluvialis, an alga also from Volvocales but one that is hypersensitive to salt. Five novel salt-tolerant clones were obtained from the library. Among them, Ds-26-16 and Ds-A3-3 contained the same open reading frame (ORF) and encoded a 6.1 kDa protein. Transgenic tobacco overexpressing Ds-26-16 and Ds-A3-3 exhibited increased leaf area, stem height, root length, total chlorophyll, and glucose content, but decreased proline content, peroxidase activity, and ascorbate content, and enhanced transcript level of Na+/H+ antiporter salt overly sensitive 1 gene (NtSOS1) expression, compared to those in the control plants under salt condition, indicating that Ds-26-16 enhanced the salt tolerance of tobacco plants. The transcript of Ds-26-16 in D. salina was upregulated in response to salt stress. The expression of Ds-26-16 in Escherichia coli showed that the ORF contained the functional region and changed the protein(s) expression profile. A mass spectrometry assay suggested that the most abundant and smallest protein that changed is possibly a DNA-binding protein or Cold shock-like protein. Subcellular localization analysis revealed that Ds-26-16 was located in the nuclei of onion epidermal cells or nucleoid of E. coli cells. In addition, the possible use of shoots regenerated from leaf discs to quantify the salt tolerance of the transgene at the initial stage of tobacco transformation was also discussed.

Assays optimized for detection and quantification of antibacterial activity in shark cell lysates under high salt conditions

To assess non-cellular innate immune mechanisms that play a role in the antimicrobial defense of an organism several assay systems have been devised to screen for such factors. Most assays, however, have been developed to measure activity against clinical isolates of medical importance. There is scant information on methods optimal for assaying material from sharks and other marine fish for antimicrobial activity particularly against salt tolerant organisms that are likely to be encountered in the marine environment. We have modified and optimized agar diffusion and broth dilution assays for detection and quantification of antibacterial activity of shark leukocyte lysates. By replacing marine agar, typically used for marine organisms, with artificial sea water complete medium (SCM) enriched with tryptone and yeast extract has resulted in an improved inhibition zone assay that uses Planococcus citreus, a salt-tolerant organism as the target organism. Antibacterial activity is correlated to the size of zone of no bacterial growth around wells containing bioactive test sample. An alternative broth based microdilution growth assay uses the 96 well format and the antibacterial effect of the sample on growth of P. citreus, the target organism, is measured spectrophotometrically as percent inhibition of bacterial growth when compared to the growth of P. citreus grown in medium alone that represents 100% growth. The assay can also be used to titrate antibacterial activity and express the level of growth inhibition as a titer.

Molecular assessment of salt-tolerant, perchlorate- and nitrate-reducing microbial cultures

The microbial ecology of enrichment cultures adapted to the removal of perchlorate and nitrate from high salt solutions and ion-exchange brines was examined over a period of four years using denaturing gradient gel electrophoresis and total DNA extraction with cloning and in each case partial sequencing of the 16S rDNA genes. The cultures studied were a result of enrichment from marine sediment inoculum initiated in 2001. The resulting enrichment cultures were fed perchlorate, or perchlorate and nitrate, in a 3% (w/v) NaCl defined medium or ion-exchange brines (5.6% NaCl) containing perchlorate and nitrate with acetate as the electron donor. All of the sequences' closest matches in the NCBI GenBank database were to marine or salt-tolerant organisms. Strains belonging to the genera Halomonas or Marinobacter were found to dominate in cultures that were fed nitrate in addition to perchlorate, but were effectively absent from cultures fed perchlorate alone. The cultures fed perchlorate as the sole electron acceptor were relatively diverse with the dominant sequences belonging to the genera Dechloromarinus and Denitromonas. A study examining the effects of growing the cultures on different electron acceptors to the cultures revealed that Denitromonas may be more dominant than Dechloromarinus as the salt-tolerant, perchlorate-reducing organism.

Specific nutrient removal rates in saline wastewater treatment using sequencing batch reactor

Effects of salt concentration (0–6%, w/v) on specific nutrient removal rates from saline wastewater in a sequencing batch reactor (SBR) were investigated. The sequencing batch operation consisted of anaerobic, oxic, anoxic and oxic phases with hydraulic residence times (HRT) of 1/3/1/1 h and a settling phase of 3/4 h. Solids retention time (SRT) was kept constant at 10 days in all experiments. Specific nutrient (COD, NH 4-N and PO 4-P) removal rates decreased with increasing salt concentration due to adverse effects of salt on microorganisms. A salt tolerant organism, Halobacter halobium was added to the activated sludge culture (1/1, v/v) in order to improve the nutrient removal performance of the SBR. Nutrient removal performances of Halobacter-free and Halobacter-added activated sludge cultures were compared for all salt contents tested. Specific rates of nutrient removal obtained with the Halobacter-added culture were higher that those of Halobacter-free activated sludge, especially at high salt contents.

Enhanced biological treatment of saline wastewater by using halophilic bacteria

Biological treatment of saline wastewater by conventional activated sludge culture usually results in low removal of chemical oxygen demand (COD) because of plasmolysis of the organisms at high salt concentrations. Since salt removal operations by physicochemical processes before biological treatment are costly, a salt-tolerant organism (Halobacter halobium) was used for effective biological treatment of saline wastewater in this study. Halobacter halobium was used in activated sludge culture for COD removal from saline wastewater (1-5% salt) by fed-batch operation of an aeration tank. Inclusion of Halobacter halobium into activated sludge culture improved the rate and extent of COD removals especially with salt above 2% (w/v).

Mutagenicity of pyrolysates of salt-tolerant bacteria from food and cigarettes

Smoke condensates obtained by pyrolysis of the cells of microorganisms isolated from food and cigarettes and of laboratory cultures were shown to be mutagenic to Salmonella typhimurium TA98 and TA100 in incorporation with liver microsomal fraction S-9 mix. The smoke condensates from salttolerant bacteria, which belonged to Micrococcus, Bacillus and Corynebacterium spp., showed higher mutagenic activity than those from other less salt-tolerant organisms, fish flesh or pork. Smoke deriving from microorganisms, especially salt-tolerant ones, in food or cigarettes as well as from food components might take part in human carcinogenesis because of the correlation between mutagenicity and carcinogenicity.

The physical state of osmoregulatory solutes in unicellular algae. A natural-abundance carbon-13 nuclear-magnetic-resonance relaxation study.

Natural-abundance 13C n.m.r. spin-lattice relaxation-time measurements have been carried out on intact cells of the unicellular blue--green alga Synechococcus sp. and the unicellular green alga Dunaliella salina, with the aim of characterizing the environments of the organic osmoregulatory solutes in these salt-tolerant organisms. In Synechococcus sp., all of the major organic osmoregulatory solute, 2-O-alpha-D-glucopyranosylglycerol, is visible in spectra of intact cells. Its rotational motion in the cell is slower by a factor of approx. 2.4 than in aqueous solution, but the molecule is still freely mobile and therefore able to contribute to the osmotic balance. In D. salina, only about 60% of the osmoregulatory solute glycerol is visible in spectra of intact cells. The rotational mobility of this observable fraction is approximately half that found in aqueous solution, but the data also indicate that there is a significant concentration of some paramagnetic species in D. salina which contributes to the overall spin-lattice relaxation of the glycerol carbon atoms. The non-observable fraction, which must correspond to glycerol molecules that have very broad 13C resonances and that are in slow exchange with bulk glycerol, has not been properly characterized as yet, but may represent glycerol in the chloroplast. The implications of these findings in relation to the physical state of the cytoplasm and the mechanism of osmoregulation in these cells are discussed.