Hypersaline environments with salt concentrations up to NaCl saturation are inhabited by a great diversity of microorganisms belonging to the three domains of life. They all must cope with the low water activity of their environment, but different strategies exist to provide osmotic balance of the cells' cytoplasm with the salinity of the medium. One option used by many halophilic Archaea and a few representatives of the Bacteria is to accumulate salts, mainly KCl and to adapt the entire intracellular machinery to function in the presence of molar concentrations of salts. A more widespread option is the synthesis or accumulation of organic osmotic, so-called compatible solutes. Here, we review the mechanisms of osmotic adaptation in a number of model organisms, including the KCl accumulating Halobacterium salinarum (Archaea) and Salinibacter ruber (Bacteria), Halomonas elongata as a representative of the Bacteria that synthesize organic osmotic solutes, eukaryotic microorganisms including the unicellular green alga Dunaliella salina and the black yeasts Hortaea werneckii and the basidiomycetous Wallemia ichthyophaga, which use glycerol and other compatible solutes. The strategies used by these model organisms and by additional halophilic microorganisms presented are then compared to obtain an integrative picture of the adaptations to life at high salt concentrations in the microbial world.

Strategies of adaptation of microorganisms of the three domains of life to high salt concentrations.

RNA-seq and especially differential RNA-seq-type transcriptomic analyses (dRNA-seq) are powerful analytical tools, as they not only provide insights into gene expression changes but also provide detailed information about all promoters active at a given moment, effectively giving a deep insight into the transcriptional landscape. Synechocystis sp. PCC 6803 (Synechocystis 6803) is a unicellular model cyanobacterium that is widely used in research fields from ecology, photophysiology to systems biology, modelling and biotechnology. Here, we analysed the response of the Synechocystis 6803 primary transcriptome to different, environmentally relevant stimuli. We established genome-wide maps of the transcriptional start sites active under 10 different conditions relevant for photosynthetic growth and identified 4,091 transcriptional units, which provide information about operons, 5' and 3' untranslated regions (UTRs). Based on a unique expression factor, we describe regulons and relevant promoter sequences at single-nucleotide resolution. Finally, we report several sRNAs with an intriguing expression pattern and therefore likely function, specific for carbon depletion (CsiR1), nitrogen depletion (NsiR4), phosphate depletion (PsiR1), iron stress (IsaR1) or photosynthesis (PsrR1). This dataset is accompanied by comprehensive information providing extensive visualization and data access to allow an easy-to-use approach for the design of experiments, the incorporation into modelling studies of the regulatory system and for comparative analyses.

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Comparative Analysis of the Primary Transcriptome of Synechocystis sp. PCC 6803

Porphyra umbilicalis (laver) belongs to an ancient group of red algae (Bangiophyceae), is harvested for human food, and thrives in the harsh conditions of the upper intertidal zone. Here we present the 87.7-Mbp haploid Porphyra genome (65.8% G + C content, 13,125 gene loci) and elucidate traits that inform our understanding of the biology of red algae as one of the few multicellular eukaryotic lineages. Novel features of the Porphyra genome shared by other red algae relate to the cytoskeleton, calcium signaling, the cell cycle, and stress-tolerance mechanisms including photoprotection. Cytoskeletal motor proteins in Porphyra are restricted to a small set of kinesins that appear to be the only universal cytoskeletal motors within the red algae. Dynein motors are absent, and most red algae, including Porphyra, lack myosin. This surprisingly minimal cytoskeleton offers a potential explanation for why red algal cells and multicellular structures are more limited in size than in most multicellular lineages. Additional discoveries further relating to the stress tolerance of bangiophytes include ancestral enzymes for sulfation of the hydrophilic galactan-rich cell wall, evidence for mannan synthesis that originated before the divergence of green and red algae, and a high capacity for nutrient uptake. Our analyses provide a comprehensive understanding of the red algae, which are both commercially important and have played a major role in the evolution of other algal groups through secondary endosymbioses.

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Insights into the red algae and eukaryotic evolution from the genome of Porphyra umbilicalis (Bangiophyceae, Rhodophyta)

Water is vital for many biological processes and is essential for all living organisms. However, numerous macroorganisms and microorganisms have adapted to survive in environments in which water is scarce; such organisms are collectively termed xerotolerant. With increasing global desertification due to climate change and human-driven desertification processes, it is becoming ever more important to understand how xerotolerant organisms cope with a lack of water. In this Review, we discuss the environmental, physiological and molecular adaptations that enable xerotolerant bacteria to survive in environments in which water is scarce and highlight insights from modern 'omics' technologies. Understanding xerotolerance will inform and hopefully aid efforts to regulate and even reverse desertification.

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Xerotolerant bacteria: surviving through a dry spell

Cyanobacteria: Promising biocatalysts for sustainable chemical production

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.

Salt acclimation of cyanobacteria and their application in biotechnology.

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. 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. 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.

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

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. 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. 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.

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

Synechocystis sp. PCC 6803 isthe most popularcyanobacterial model for prokaryotic photosynthesis and for metabolic engineering to produce biofuels. Genomic and transcriptomic comparisons between closely relatedbacteriaarepowerfulapproachestoinferinsightsintotheirmetabolicpotentials andregulatorynetworks. To enable acomparative approach, we generated the draft genome sequenceofSynechocystissp. PCC 6714, aclosely 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 uniquegenes inSynechocystissp. PCC 6803 and 895 genes in Synechocystis sp. PCC 6714. The genetic differences include a prophage in the genome of strain 6714, a different compositionofthe pooloftransposable elements, anda ∼40 kb genomic island encodingseveral glycosyltransferases and transportproteins.Weverifiedseveralphysiological differencesthat were predicted on the basis of the respective genome sequence. Strain 6714 exhibited a lower tolerance to Zn 21 ions, associated with the lackof acorresponding 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).

Comparative Genome Analysis of the Closely Related Synechocystis Strains PCC 6714 and PCC 6803

Summary

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.

Nadin Pade

Papers

Salt acclimation of cyanobacteria and their application in biotechnology.

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

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

Comparative Genome Analysis of the Closely Related Synechocystis Strains PCC 6714 and PCC 6803

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