The glucosylglycerol-degrading enzyme GghA is involved in acclimation to fluctuating salinities by the cyanobacterium Synechocystis sp. strain PCC 6803.

The ggpS gene, which encodes the key enzyme for the synthesis of the compatible solute glucosylglycerol (GG), has a promoter region that overlaps with the upstream-located gene slr1670 in the cyanobacterium Synechocystissp. PCC 6803. Like ggpS, the slr1670 gene is salt-induced and encodes a putative glucosylhydrolase. A mutant strain with a slr1670 deletion was generated and found to be unable to adapt the internal GG concentrations in response to changes in external salinities. Whereas cells of the wild-type reduced the internal pool of GG when exposed to gradual and abrupt hypo-osmotic treatments, or when the compatible solute trehalose was added to the growth medium, the internal GG pool of ∆slr1670 mutant cells remained unchanged. These findings indicated that the protein Slr1670 is involved in GG breakdown. The biochemical activity of this GG-hydrolase enzyme was verified using recombinant Slr1670 protein, which split GG into glucose and glycerol. These results validate that Slr1670, which was named GghA, acts as a GG hydrolase. GghA is involved in GG turnover in fluctuating salinities, and similar proteins are found in the genomes of other GG-synthesizing cyanobacteria.

Systems analysis of ethanol production in the genetically engineered cyanobacterium Synechococcus sp. PCC 7002.

BACKGROUND: Future sustainable energy production can be achieved using mass cultures of photoautotrophic microorganisms, which are engineered to synthesize valuable products directly from CO2 and sunlight. As cyanobacteria can be cultivated in large scale on non-arable land, these phototrophic bacteria have become attractive organisms for production of biofuels. Synechococcus sp. PCC 7002, one of the cyanobacterial model organisms, provides many attractive properties for biofuel production such as tolerance of seawater and high light intensities. RESULTS: Here, we performed a systems analysis of an engineered ethanol-producing strain of the cyanobacterium Synechococcus sp. PCC 7002, which was grown in artificial seawater medium over 30 days applying a 12:12 h day-night cycle. Biosynthesis of ethanol resulted in a final accumulation of 0.25% (v/v) ethanol, including ethanol lost due to evaporation. The cultivation experiment revealed three production phases. The highest production rate was observed in the initial phase when cells were actively growing. In phase II growth of the producer strain stopped, but ethanol production rate was still high. Phase III was characterized by a decrease of both ethanol production and optical density of the culture. Metabolomics revealed that the carbon drain due to ethanol diffusion from the cell resulted in the expected reduction of pyruvate-based intermediates. Carbon-saving strategies successfully compensated the decrease of central intermediates of carbon metabolism during the first phase of fermentation. However, during long-term ethanol production the producer strain showed clear indications of intracellular carbon limitation. Despite the decreased levels of glycolytic and tricarboxylic acid cycle intermediates, soluble sugars and even glycogen accumulated in the producer strain. The changes in carbon assimilation patterns are partly supported by proteome analysis, which detected decreased levels of many enzymes and also revealed the stress phenotype of ethanol-producing cells. Strategies towards improved ethanol production are discussed. CONCLUSIONS: Systems analysis of ethanol production in Synechococcus sp. PCC 7002 revealed initial compensation followed by increasing metabolic limitation due to excessive carbon drain from primary metabolism.

Ethanol, glycogen and glucosylglycerol represent competing carbon pools in ethanol-producing cells of Synechocystis sp. PCC 6803 under high-salt conditions.

Cyanobacteria are photoautotrophic micro-organisms, which are increasingly being used as microbial cell factories to produce, for example, ethanol directly from solar energy and CO2. Here, we analysed the effects of different salt concentrations on an ethanol-producing strain of Synechocystis sp. PCC 6803 that overexpresses the pyruvate decarboxylase (pdc) from Zymomonas mobilis and the native alcohol dehydrogenase (adhA). Moderate salinities of 2 % NaCl had no negative impact on ethanol production, whereas the addition of 4 % NaCl resulted in significantly decreased ethanol yields compared to low-salt conditions. Proteomic analysis identified a defined set of proteins with increased abundances in ethanol-producing cells. Among them, we found strong up-regulation of α-1,4 glucan phosphorylase (GlgP, Slr1367) in the producer strain, which consistently resulted in a massive depletion of glycogen pools in these cells regardless of the salinity. The salt-induced accumulation of the compatible solute glucosylglycerol was not affected by the ethanol production. Glycogen and probably compatible solutes could present competing pools with respect to organic carbon, explaining the decreased ethanol production at the highest salinity.

Cyanobacterial populations in biological soil crusts of the northwest Negev Desert, Israel-effects of local conditions and disturbance.

Biological soil crusts (BSCs) fulfil numerous ecological functions in arid and semiarid areas. Cyanobacteria are important BSC organisms, which are responsible for carbon fixation, N2 fixation and binding of soil via extracellular polysaccharides. The cyanobacterial populations were characterised in different sampling plots established in three experimental stations along a rainfall gradient within NW Negev Desert, Israel. Cyanobacterial crust thickness and osmolyte accumulation therein decreased in plots with lower moisture. The cyanobacterial population structure also changed in different plots. We observed an increase of subsection III cyanobacteria such as Microcoleus spp. and Leptolyngbya spp. and a decreasing proportion of strains belonging to subsections I and IV in drier areas on the rainfall gradient. This population shift was also observed in the sampling plots, which were situated at various relief positions within the sand dune experimental sites. We also characterised the cyanobacterial populations within mechanically disturbed plots. After 4 years, they reached between 80% and 50% of the control populations in the northernmost and southern stations, respectively. Our results suggest that the cyanobacterial population is sensitive not only to macroscale factors but may also be subject to local climate variations and that 4 years was insufficient for complete recovery of the cyanobacterial population.

What distinguishes cyanobacteria able to revive after desiccation from those that cannot: the genome aspect.

Filamentous cyanobacteria are the main founders and primary producers in biological desert soil crusts (BSCs) and are likely equipped to cope with one of the harshest environmental conditions on earth including daily hydration/dehydration cycles, high irradiance and extreme temperatures. Here, we resolved and report on the genome sequence of Leptolyngbya ohadii, an important constituent of the BSC. Comparative genomics identified a set of genes present in desiccation-tolerant but not in dehydration-sensitive cyanobacteria. RT qPCR analyses showed that the transcript abundance of many of them is upregulated during desiccation in L. ohadii. In addition, we identified genes where the orthologs detected in desiccation-tolerant cyanobacteria differs substantially from that found in desiccation-sensitive cells. We present two examples, treS and fbpA (encoding trehalose synthase and fructose 1,6-bisphosphate aldolase respectively) where, in addition to the orthologs present in the desiccation-sensitive strains, the resistant cyanobacteria also possess genes with different predicted structures. We show that in both cases the two orthologs are transcribed during controlled dehydration of L. ohadii and discuss the genetic basis for the acclimation of cyanobacteria to the desiccation conditions in desert BSC.

Trimethylated homoserine functions as the major compatible solute in the globally significant oceanic cyanobacterium Trichodesmium.

The oceanic N2-fixing cyanobacterium Trichodesmium spp. form extensive surface blooms and contribute significantly to marine carbon and nitrogen cycles in the oligotrophic subtropical and tropical oceans. Trichodesmium grows in salinities from 27 to 43 parts per thousand (ppt), yet its salt acclimation strategy remains enigmatic because the genome of Trichodesmium erythraeum strain IMS101 lacks all genes for the biosynthesis of any known compatible solute. Using NMR and liquid chromatography coupled to mass spectroscopy, we identified the main compatible solute in T. erythraeum strain IMS101 as the quaternary ammonium compound N,N,N-trimethyl homoserine (or homoserine betaine) and elucidated its biosynthetic pathway. The identification of this compatible solute explains how Trichodesmium spp. can thrive in the marine system at varying salinities and provides further insight into the diversity of microbial salt acclimation.

Insights into isoprene production using the cyanobacterium Synechocystis sp. PCC 6803.

BACKGROUND: Cyanobacteria are phototrophic prokaryotes that convert inorganic carbon as CO2 into organic compounds at the expense of light energy. They need only inorganic nutrients and can be cultivated to high densities using non-arable land and seawater. This has made cyanobacteria attractive organisms for the production of biofuels and chemical feedstock. Synechocystis sp. PCC 6803 is one of the most widely used cyanobacterial model strains. Based on its available genome sequence and genetic tools, Synechocystis has been genetically modified to produce different biotechnological products. Efficient isoprene production is an attractive goal because this compound is widely used as chemical feedstock. RESULTS: Here, we report on our attempts to generate isoprene-producing strains of Synechocystis using a plasmid-based strategy. As previously reported, a codon-optimized plant isoprene synthase (IspS) was expressed under the control of different Synechocystis promoters that ensure strong constitutive or light-regulated ispS expression. The expression of the ispS gene was quantified by qPCR and Western blotting, while the amount of isoprene was quantified using GC-MS. In addition to isoprene measurements in the headspace of closed culture vessels, single photon ionization time-of-flight mass spectrometry (SPI-MS) was applied, which allowed online measurements of isoprene production in open-cultivation systems under various conditions. Under standard conditions, a good correlation existed between ispS expression and isoprene production rate. The cultivation of isoprene production strains under NaCl-supplemented conditions decreased isoprene production despite enhanced ispS mRNA levels. The characterization of the metabolome of isoprene-producing strains indicated that isoprene production might be limited by insufficient precursor levels. Transcriptomic analysis revealed the upregulation of mRNA and regulatory RNAs characteristic of acclimation to metabolic stress. CONCLUSIONS: Our best production strains produced twofold higher isoprene amounts in the presence of low NaCl concentrations than previously reported strains. These results will guide future attempts to establish isoprene production in cyanobacterial hosts.

Simulated soil crust conditions in a chamber system provide new insights on cyanobacterial acclimation to desiccation.

Environmental research often faces two major hurdles: (i) fluctuating spatial and temporal conditions and consequently large variability in the organisms' abundance and performance, and (ii) complex, costly logistics involved in field experiments. Measurements of physiological parameters or molecular analyses often represent single shot experiments. To study desiccation acclimation of filamentous cyanobacteria, the founders and main primary producers in desert biological soil crusts (BSC), we constructed an environmental chamber that can reproducibly and accurately simulate ambient conditions and measure microorganism performance. We show that recovery from desiccation of BSC cyanobacteria and Leptolyngbya ohadii isolated thereof are strongly affected by dehydration rate following morning dew. This effect is most pronounced in cells exposed to high light and temperature in the dry phase. Simultaneous measurements of water content, gas exchange and fluorescence were performed during dehydration. Photosynthetic performance measured by fluorescence begins declining when light intensity reaches values above 100 µmol photons m(-2) s(-1), even in fully hydrated cells. In contrast, photosynthetic rates measured using O2 evolution and CO2 uptake increased during rising irradiance to the point where the water content declined below ∼ 50%. Thus, fluorescence cannot serve as a reliable measure of photosynthesis in desert cyanobacteria. The effects of drying on gas exchange are discussed.

Floridoside and isofloridoside are synthesized by trehalose 6-phosphate synthase-like enzymes in the red alga Galdieria sulphuraria.

Compatible solutes are small molecules that are involved in acclimation to various abiotic stresses, especially high salinity. Among the red algae, the main photosynthetic products floridoside and isofloridoside (galactosylglycerols) are known also to contribute to the osmotic acclimation of cells. However, the genes encoding (iso)floridoside biosynthetic enzymes are still unknown. To identify candidate genes, we examined the genome of the floridoside- and isofloridoside-accumulating extremophilic red alga Galdieria sulphuraria belonging to the Cyanidiales. We hypothesized that two candidate genes, Gasu_10960 and Gasu_26940, code for enzymes involved in floridoside and isofloridoside biosynthesis. These proteins comprise a sugar phosphate synthase and a sugar phosphate phosphatase domain. To verify their biochemical activity, both genes were in vitro translated into the entire proteins. The protein translation mixture containing Gasu_10960 synthesized small amounts of isofloridoside, whereas the Gasu_26940 translation mix also produced small amounts of floridoside. Moreover, the expression of Gasu_10960 in a salt-sensitive mutant of the cyanobacterium Synechocystis sp. PCC 6803 resulted in increased salt tolerance as a consequence of the presence of isofloridoside in the complemented cells. Thus, our experiments suggest that the Gasu_26940 and Gasu_10960 genes of G. sulphuraria encode the enzymatically active floridoside and isofloridoside phosphate synthase/phosphatase fusion proteins, respectively, crucial for salt acclimation.

Comparative genome analysis of the closely related Synechocystis strains PCC 6714 and PCC 6803.

Synechocystis sp. PCC 6803 is the most popular cyanobacterial model for prokaryotic photosynthesis and for metabolic engineering to produce biofuels. Genomic and transcriptomic comparisons between closely related bacteria are powerful approaches to infer insights into their metabolic potentials and regulatory networks. To enable a comparative approach, we generated the draft genome sequence of Synechocystis sp. PCC 6714, a closely related strain of 6803 (16S rDNA identity 99.4%) that also is amenable to genetic manipulation. Both strains share 2838 protein-coding genes, leaving 845 unique genes in Synechocystis sp. PCC 6803 and 895 genes in Synechocystis sp. PCC 6714. The genetic differences include a prophage in the genome of strain 6714, a different composition of the pool of transposable elements, and a ∼ 40 kb genomic island encoding several glycosyltransferases and transport proteins. We verified several physiological differences that were predicted on the basis of the respective genome sequence. Strain 6714 exhibited a lower tolerance to Zn(2+) ions, associated with the lack of a corresponding export system and a lowered potential of salt acclimation due to the absence of a transport system for the re-uptake of the compatible solute glucosylglycerol. These new data will support the detailed comparative analyses of this important cyanobacterial group than has been possible thus far. Genome information for Synechocystis sp. PCC 6714 has been deposited in Genbank (accession no AMZV01000000).

Salt acclimation of cyanobacteria and their application in biotechnology.

The long evolutionary history and photo-autotrophic lifestyle of cyanobacteria has allowed them to colonize almost all photic habitats on Earth, including environments with high or fluctuating salinity. Their basal salt acclimation strategy includes two principal reactions, the active export of ions and the accumulation of compatible solutes. Cyanobacterial salt acclimation has been characterized in much detail using selected model cyanobacteria, but their salt sensing and regulatory mechanisms are less well understood. Here, we briefly review recent advances in the identification of salt acclimation processes and the essential genes/proteins involved in acclimation to high salt. This knowledge is of increasing importance because the necessary mass cultivation of cyanobacteria for future use in biotechnology will be performed in sea water. In addition, cyanobacterial salt resistance genes also can be applied to improve the salt tolerance of salt sensitive organisms, such as crop plants.

Bioenergetics of the moderately halophilic bacterium Halobacillus halophilus: composition and regulation of the respiratory chain.

In their natural environments, moderately halophilic bacteria are confronted not only with high salinities but also with low oxygen tensions due to the high salinities. The growth of H. halophilus is strictly aerobic. To analyze the dependence of respiration on the NaCl concentration and oxygen availability of the medium, resting cell experiments were performed. The respiration rates were dependent on the NaCl concentration of the growth medium, as well as on the NaCl concentration of the assay buffer, indicating regulation on the transcriptional and the activity level. Respiration was accompanied by the generation of an electrochemical proton potential (Δµ(H+)) across the cytoplasmic membrane whose magnitude was dependent on the external pH. Genes encoding proteins involved in respiration and Δµ(H+) generation, such as a noncoupled NADH dehydrogenase (NDH-2), complex II, and complex III, were identified in the genome. In addition, genes encoding five different terminal oxidases are present. Inhibitor profiling revealed the presence of NDH-2 and complex III, but the nature of the oxidases could not be resolved using this approach. Expression analysis demonstrated that all the different terminal oxidases were indeed expressed, but by far the most prominent was cta, encoding cytochrome caa3 oxidase. The expression of all of the different oxidase genes increased at high NaCl concentrations, and the transcript levels of cta and qox (encoding cytochrome aa3 oxidase) also increased at low oxygen concentrations. These data culminate in a model of the composition and variation of the respiratory chain of H. halophilus.

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.