The marine cyanobacterium Crocosphaera watsonii WH8501 synthesizes the compatible solute trehalose by a laterally acquired OtsAB fusion protein

Compatible solutes are small organic molecules that are involved in the acclimation to various stresses such as temperature and salinity. Marine or moderate halotolerant cyanobacteria accumulate glucosylglycerol, while cyanobacteria with low salt tolerance (freshwater strains) usually accumulate sucrose or trehalose as the main compatible solutes. The screening of the genome of the marine, unicellular N(2) -fixing cyanobacterium Crocosphaera watsonii WH8501 revealed that instead of genes for glucosylglycerol biosynthesis, a fusion protein for the synthesis of trehalose was found that displayed similarities to trehalose-phosphate-synthase and -phosphatase (OtsAB pathway) from enterobacteria. Accordingly, cells of Crocosphaera showed salt-stimulated expression of the otsAB gene as well as a salt-dependent trehalose accumulation. The biochemical characterization of recombinant full-length OtsAB and truncated OtsB versions revealed that the otsAB gene in Crocosphaera encodes for an active trehalose-phosphate-synthase/phosphatase fusion protein. Genes coding for such proteins were not found in the genomes of other cyanobacteria but were present in many other, non-related marine bacteria, suggesting that otsAB might have been acquired by lateral gene transfer into the Crocosphaera genome.     

The compatibility of osmotica in cyanobacteria

Abstract. The solutes accumulated by cyanobacteria in response to hyper-osmotic stress include Na⁺, K⁺, sucrose, trehalose, glucosyl-glycerol, glyeine betaine and glutamate betaine. The compatibility of several of these solutes with glutamine synthetase activity has been examined using cell-free extracts from a range of freshwater, marine and halotolerant cyanobacteria. All of the solutes tested were compatible with (i.e. non-inhibitory to) enzymic activity at physiological concentrations and the results demonstrate a rank order of compatibility which correlates with the concentrations at which the organic solutes occur in cyanobacteria, i.e. glycine betaine > polyol-derivatives > disaccharides and with the upper salinity limit for growth. The protection against inhibition by NaCl (halo-protection) afforded by these solutes to enzymic activity was also examined. Only glycine betaine was found to exert a significant halo-protective effect and this may be explained by differences in the mechanism of compatible solute function between small charged molecules and sugars/polyols.     

Role of oxidative stress in the extremely salt-tolerant yeast Hortaea werneckii

Eukaryotic halotolerant microorganisms are important as model organisms to understand the general mechanisms of resistance to environmental salinity. The ability of the extremely halotolerant black yeast Hortaea werneckii to combat oxidative stress was addressed, using hydrogen peroxide to generate the reactive oxygen species. Increasing environmental salinity was found to have no effect on its high ability to degrade hydrogen peroxide but resulted in a decrease in viability in response to externally added hydrogen peroxide, suggesting that the latter property determines the upper limit of the salt tolerance of H. werneckii. A refinement of the model of adaptation of H. werneckii to high-salinity environments is proposed.     

An alkaline phosphatase/phosphodiesterase, PhoD, induced by salt stress and secreted out of the cells of Aphanothece halophytica, a halotolerant cyanobacterium

Alkaline phosphatases (APases) are important enzymes in organophosphate utilization. Three prokaryotic APase gene families, PhoA, PhoX, and PhoD, are known; however, their functional characterization in cyanobacteria largely remains to be clarified. In this study, we cloned the phoD gene from a halotolerant cyanobacterium, Aphanothece halophytica (phoD(Ap)). The deduced protein, PhoD(Ap), contains Tat consensus motifs and a peptidase cleavage site at the N terminus. The PhoD(Ap) enzyme was activated by Ca(2+) and exhibited APase and phosphodiesterase (APDase) activities. Subcellular localization experiments revealed the secretion and processing of PhoD(Ap) in a transformed cyanobacterium. Expression of the phoD(Ap) gene in A. halophytica cells was upregulated not only by phosphorus (P) starvation but also under salt stress conditions. Our results suggest that A. halophytica cells possess a PhoD that participates in the assimilation of P under salinity stress.     

Anabaena sp. PCC7120 transformed with glycine methylation genes from Aphanothece halophytica synthesized glycine betaine showing increased tolerance to salt

Photosynthetic, nitrogen-fixing Anabaena strains play an important role in the carbon and nitrogen cycles in tropical paddy fields although they are salt sensitive. Improvement in salt tolerance of Anabaena cells by expressing glycine betaine–synthesizing genes is an interesting subject. Due to the absence of choline in cyanobacteria, choline-oxidizing enzyme could not be used for the synthesis of glycine betaine. Here, the genes encoding glycine-sarcosine and dimethylglycine methyltransferases (ApGSMT-DMT) from a halotolerant cyanobacterium Aphanothece halophytica were expressed in Anabaena sp. strain PCC7120. The ApGSMT-DMT-expressing Anabaena cells were capable of synthesizing glycine betaine without the addition of any substance. The accumulation level of glycine betaine in Anabaena increased with rise of salt concentration. The transformed cells exhibited an improved growth and more tolerance to salinity than the control cells. The present work provides a prospect to engineer a nitrogen-fixing cyanobacterium having enhanced tolerance to stress by manipulating de novo synthesis of glycine betaine.     

Enhanced photosynthesis and redox energy production contribute to salinity tolerance in Dunaliella as revealed by homology-based proteomics

Salinity is a major limiting factor for the proliferation of plants and inhibits central metabolic activities such as photosynthesis. The halotolerant green alga Dunaliella can adapt to hypersaline environments and is considered a model photosynthetic organism for salinity tolerance. To clarify the molecular basis for salinity tolerance, a proteomic approach has been applied for identification of salt-induced proteins in Dunaliella. Seventy-six salt-induced proteins were selected from two-dimensional gel separations of different subcellular fractions and analyzed by mass spectrometry (MS). Application of nanoelectrospray mass spectrometry, combined with sequence-similarity database-searching algorithms, MS BLAST and MultiTag, enabled identification of 80% of the salt-induced proteins. Salinity stress up-regulated key enzymes in the Calvin cycle, starch mobilization, and redox energy production; regulatory factors in protein biosynthesis and degradation; and a homolog of a bacterial Na(+)-redox transporters. The results indicate that Dunaliella responds to high salinity by enhancement of photosynthetic CO(2) assimilation and by diversion of carbon and energy resources for synthesis of glycerol, the osmotic element in Dunaliella. The ability of Dunaliella to enhance photosynthetic activity at high salinity is remarkable because, in most plants and cyanobacteria, salt stress inhibits photosynthesis. The results demonstrated the power of MS BLAST searches for the identification of proteins in organisms whose genomes are not known and paved the way for dissecting molecular mechanisms of salinity tolerance in algae and higher plants.     

Halophily and halotolerance in cyanophytes

The survival, growth and distribution of organisms in hypersaline environments is discussed using cyanophytes (cyanobacteria) as examples. The distinction between halophilic (Na⁺-requiring) and halotolerant organisms is not adequate to describe the entire spectrum of adaptations to salt. The classical division into stenohaline (narrow) and euryhaline (wide) adaptational types, with optima identified as oligo-, meso- and polyhaline, better reflects both organismal adaptations and the environmental conditions to which these are adjusted and is therefore recommended as a conceptual model.Two independent properties of organisms are growth and survival. Organisms requiring narrow ranges of salt concentration are considered specialists and are restricted to environments with relatively constant salinities at any particular concentration. Organisms which tolerate wide ranges of fluctuation in salinity are considered generalists. The existence of separate and distinct microbial assemblages in these two types of environments is demonstrated in marine intertidal zones and seasonal salt works, representative of fluctuating salinity, and in the open ocean. The hypersaline ponds of Yallahs, Jamaica, and Solar Lake, Sinai represent different but relatively constant salinities. It is concluded that cyanophytes speciate along the salinity gradient, and that separate halophilic taxa occupy environments with relatively constant salinities.Proceedings of the fourth College Park Colloquium on Chemical EvolutionLimits of Life, University of Maryland, College Park, 18–20 October 1978.     

Lipid modulation by environmental stresses in two models of extremophiles isolated from Antarctica

Thermoacidophilic and halotolerant microorganisms from the Antarctic continent were studied for their lipid modulation under stress growth conditions. Temperature-induced changes in complex lipids and fatty acids of four strains belonging to the genus Alicyclobacillus involved the relative proportions of different polar lipids and the synthesis of ω-cyclohexyl-acyl chains, which were favoured by high temperatures. Studies were carried out on the lipid composition of four strains of extremely halotolerant bacteria belonging to the genus Micrococcus grown at different salt concentrations from 0 up to 4.5 M NaCl. The main lipids found were two unidentified glycolipids and two phospholipids: 1,2 diacylglycero-3-phosphoryl-glycerol (PG) and cardiolipin (DPG). Among the strains analysed, the lipids of the Micrococcus strain Erebus were shown to be strongly influenced by salt concentrations, in that DPG and one glycolipid were absent at a low salt molarity while, under these conditions, PG was the main lipid found. The predominant fatty acids in all halotolerant strains were of the anteiso type; growth under increasing salinity gave rise to an increase in long chain fatty acids and of straight chain fatty acids, while a decrease in iso fatty acids occurred. Accepted: 20 May 2000     

Regulation of Ethylene Biosynthesis Under Salt Stress in Red Pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) by 1-Aminocyclopropane-1-Carboxylic Acid (ACC) Deaminase-producing Halotolerant Bacteria

The present study was carried out to understand the mechanism of salt stress amelioration in red pepper plants by inoculation of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-producing halotolerant bacteria. In general, ethylene production, ACC concentration, ACC synthase (ACS), and ACC oxidase (ACO) enzyme activities increased with increasing levels of salt stress. Treatment with halotolerant bacteria reduced ethylene production by 47–64%, ACC concentration by 47–55% and ACO activity by 18–19% in salt-stressed (150 mmol NaCl) red pepper seedlings compared to uninoculated controls. ACS activity was lower in red pepper seedlings treated with Bacillus aryabhattai RS341 but higher in seedlings treated with Brevibacterium epidermidis RS15 (44%) and Micrococcus yunnanensis RS222 (23%) under salt-stressed conditions as compared to uninoculated controls. A significant increase was recorded in red pepper plant growth under salt stress when treated with ACC deaminase-producing halotolerant bacteria as compared to uninoculated controls. The results of this study collectively suggest that salt stress enhanced ethylene production by increasing enzyme activities of the ethylene biosynthetic pathway. Inoculation with ACC deaminase-producing halotolerant bacteria plays an important role in ethylene metabolism, particularly by reducing the ACC concentration, although a direct effect on reducing ACO activity was also observed. It is suggested that growth promotion in inoculated red pepper plants under inhibitory levels of salt stress is due to ACC deaminase activity present in the halotolerant bacteria.     

Microbial degradation of pollutants at high salt concentrations

Though our knowledge on microbial degradation of organic pollutants at high salt concentrations is still limited, the list of toxic compounds shown to be degraded or transformed in media of high salinity is growing. Compounds transformed aerobically include saturated and aromatic hydrocarbons (by certain archaeobacteria), certain aromatic compounds, organophosphorus compounds, and formaldehyde (by halotolerant eubacteria). Anaerobic microbial transformations of toxic compounds occurring at high salt concentrations include reduction of nitroaromatic compounds, and possibly transformation of chlorinated aromatic compounds.     

Glycinebetaine in saline conditions: an assessment of the current state of knowledge

Salt stress is one of the environmental threats that have devastating impacts on plant distribution, growth and production. Different plants are believed to have salt tolerance mechanisms that occur at the cellular level. One facet of the cellular mechanisms of adaptation to salinity stress is to accumulate either inorganic and/or organic solutes. Glycinebetaine (GB), as well as other organic solutes, has been referred to as compatible solutes, for the reason that they are innocent with essential biochemical reactions even at high concentrations. GB has been assumed to be involved in osmotic adjustment and/or osmoprotection of cellular functional macromolecules and, hence, can improve tolerance to saline conditions. However, the exact mechanism and direct evidences for such correlative data are still lacking despite many attempts to improve growth under saline conditions by exogenous application as well as genetic engineering of metabolic pathways involved in metabolism of GB. Despite the enormous amount of information accumulated in this regard, the exact function of GB in the adaptation to saline environments is not fully clear to this point, and even GB functions have been argued. Because of that, inconsistencies exist in the published data regarding GB accumulation and functions under salt stress. In this review, we provide an update on evidence supporting each of these arguments to reassess how GB affects plant growth and physiological traits under salt imposition, and whether its effects correlate with salt tolerance.     

Accumulation of LEA proteins in salt (NaCl) stressed young seedlings of rice (Oryza sativa L.) cultivar Bura Rata and their degradation during recovery from salinity stress

Germination and subsequent hydroponic growth under salt stress (100 mmol/L NaCl) triggered an accumulation of six major stress proteins and resulted in a growth arrest of young seedlings of rice (Oryza sativa L.) cv. Bura Rata. Based on two-dimensional electrophoretic resolution, partial amino acid sequencing and immunodetection techniques, four of the salt stress-induced polypeptides were identified as LEA proteins. Under all experimental conditions wherein seedlings exhibited superior halotolerance, salt stress-induced LEA proteins were expressed at low levels. In contrast, accumulation of LEA proteins was found associated with growth arrest. When returned to non-saline media, seedlings stressed with salt for four days recovered immediately. Longer exposure to 100 mmol/L NaCl, however, progressively delayed recovery and reduced the number of seedlings which could recover from salt stress. Recovery from salt stress was consistently accompanied by degradation of the salt stress-induced LEA proteins. The results of this study show that LEA proteins accumulate during the salinity-triggered growth arrest of young Bura Rata seedlings and are mobilised during the recovery of seedlings from salinity stress.     

Genetic control and mechanisms of salt and hyperosmotic shock resistance in cyanobacteria

Exposure to high concentrations of environmental NaCl exerts two stress effects on living cells, increasing the osmotic pressure and the concentration of inorganic ions. Salt stress dramatically suppresses the photosynthetic activity in cells of phototrophic organisms, such as cyanobacteria. During salt adaptation, cyanobacterial cells accumulate osmoprotectors, export excessive Na+ with the help of Na+/H+ antiporters, and actively absorb K+ with the help of K+-transporting systems. These physiological processes are accompanied by induction or suppression of several genes involved in salt adaptation. The review considers the main mechanisms responsible for the resistance of cyanobacterial cells to salt and hyperosmotic stresses. Special emphasis is placed on recent achievements in studying the genetic control of salt resistance and regulation of gene expression during adaptation of cyanobacteria to salt and hyperosmotic stresses.     

Mechanisms of response to salinity in halotolerant microalgae

Summary A limited number of organic solutes are used by microalgae to adjust their internal osmotic pressure in response to changing external salinities. Glycerol and proline are used by the most extremely halotolerant algae. Only glycerol allows growth at salinities approaching saturation. In addition to organic osmoregulatory solutes, inorganic ions also play an important role in osmoregulation. The ability of microalgae to maintain intracellular ions at levels compatible with metabolic functions may set upper limits for their salt tolerance. Requirements for NaCl in the external medium for nutrient transport may define the lower salinity limits for growth observed for some euryhaline algae.Osmotic upshocks generally cause severe temporary inhibition of photosynthesis in euryhaline microalgae. Extensive osmotic downshocks have little effect on photosynthesis in microalgae with strong cell walls, while wall-less species appear to be more sensitive. Rapid glycerol synthesis takes place in response to increased external salinity inChlamydomonas pulsatilla both in light and dark. Starch supplies carbon for glycerol synthesis in the dark and also during the initial periods of inhibition of photosynthesis in the light. Turnover of osmoregulatory solutes such as glycerol and isofloridoside may be an important aspect of the osmoregulatory mechanism.At salinities beyond the growth limit for the green flagellateChlamydomonas pulsatilla, resting spores are formed that enable this alga to survive extreme salinities.     

Matching molecular diversity and ecophysiology of benthic cyanobacteria and diatoms in communities along a salinity gradient

The phylogenetic diversity of oxygenic phototrophic microorganisms in hypersaline microbial mats and their distribution along a salinity gradient were investigated and compared with the halotolerances of closely related cultivated strains. Segments of 16S rRNA genes from cyanobacteria and diatom plastids were retrieved from mat samples by DNA extraction and polymerase chain reaction (PCR), and subsequently analysed by denaturing gradient gel electrophoresis (DGGE). Sequence analyses of DNA from individual DGGE bands suggested that the majority of these organisms was related to cultivated strains at levels that had previously been demonstrated to correlate with characteristic salinity responses. Proportional abundances of amplified 16S rRNA gene segments from phylogenetic groupings of cyanobacteria and diatoms were estimated by image analysis of DGGE gels and were generally found to correspond to abundances of the respective morphotypes determined by microscopic analyses. The results indicated that diatoms accounted for low proportions of cells throughout, that the cyanobacterium Microcoleus chthonoplastes and close relatives dominated the communities up to a salinity of 11% and that, at a salinity of 14%, the most abundant cyanobacteria were related to highly halotolerant cultivated cyanobacteria, such as the recently established phylogenetic clusters of Euhalothece and Halospirulina . Although these organisms in cultures had previously demonstrated their ability to grow with close to optimal rates over a wide range of salinities, their occurrence in the field was restricted to the highest salinities investigated.     

Salinity effects on growth,photosynthetic parameters,and nitrogenase activity in estuarine planktonic cyanobacteria

Salinity has been suggested as being a controlling factor for blooms of N2-fixing cyanobacteria in estuaries. We tested the effect of salinity on the growth, N2 fixation, and photosynthetic activities of estuarine and freshwater isolates of heterocystous bloom-forming cyanobacteria. Anabaena aphanizomenoides and Anabaenopsis sp. were isolated from the Neuse River Estuary, North Carolina, and Cylindrospermopsis raciborskii from Lakes Dora and Griffin, central Florida. Salinity tolerance of these cyanobacteria was compared with that of two Nodularia strains from the Baltic Sea. We measured growth rates, N2 fixation (nitrogenase activity), and CO2 fixation at salinities between 0 and 20 g L(-1) NaCl. We also examined photosynthesis-irradiance relation-ships in response to salinity. Anabaenopsis maintained similar growth rates in the full range of salinities from 2 to 20 g L(-1) NaCl. Anabaena grew at up to 15 g L-', but the maximum salinity 20 g L(-1) NaCl was inhibitory. The upper limit for salinity tolerance of Cylindrospermopsis was 4 g L(-1) NaCl. Nodularia spp. maintained similar growth rates in the full range of salinities from 0 to 20 g L(-1) . Between 0 and 10 g L(-1), the growth rate of Nodularia spumigena was slower than that of the Neuse Estuary strains. In most strains, the sensitivity of nitrogenase activity and CO2 fixation to salinity appeared similar. Anabaenopsis, Anabaena, and the two Nodularia strains rapidly responded to NaCl by increasing their maximum photosynthetic rates (Pmn). Overall, both Neuse River Estuary and Baltic Sea strains showed an ability to acclimate to salt stress over short-(24 h) and long-term (several days to weeks) exposures. The study suggested that direct effect of salinity (as NaCl in these experiments) on cyanobacterial physiology does not alone explain the low frequency and magnitude of blooms of N2-fixing cyanobacteria in estuaries.