Organic solute accumulation in osmotically stressed cyanobacteria

Abstract Three groups of cyanobacteria are recognized on the basis of their organic osmotica and upper salinity limit for growth. In general, the least halotolerant forms accumulate disaccharides, while cyanobacteria of intermediate halotolerance synthesize the heteroside glucosylglycerol and the most halotolerant isolates accumulate betaines in response to salt stress. However, certain strains also accumulate additional organic solutes, depending upon the growth temperature, the ambient salinity and the duration of salt stress.     

Efficient phage-mediated pigment biosynthesis in oceanic cyanobacteria

Although the oceanic cyanobacterium Prochlorococcus harvests light with a chlorophyll antenna [1-3] rather than with the phycobilisomes that are typical of cyanobacteria, some strains express genes that are remnants of the ancestral Synechococcus phycobilisomes [4]. Similarly, some Prochlorococcus cyanophages, which often harbor photosynthesis-related genes [5], also carry homologs of phycobilisome pigment biosynthesis genes [6, 7]. Here, we investigate four such genes in two cyanophages that both infect abundant Prochlorococcus strains [8]: homologs of heme oxygenase (ho1), 15,16-dihydrobiliverdin:ferredoxin oxidoreductase (pebA), ferredoxin (petF) in the myovirus P-SSM2, and a phycocyanobilin:ferredoxin oxidoreductase (pcyA) homolog in the myovirus P-SSM4. We demonstrate that the phage homologs mimic the respective host activities, with the exception of the divergent phage PebA homolog. In this case, the phage PebA single-handedly catalyzes a reaction for which uninfected host cells require two consecutive enzymes, PebA and PebB. We thus renamed the phage enzyme phycoerythrobilin synthase (PebS). This gene, and other pigment biosynthesis genes encoded by P-SSM2 (petF and ho1), are transcribed during infection, suggesting that they can improve phage fitness. Analyses of global ocean metagenomes show that PcyA and Ho1 occur in both cyanobacteria and their phages, whereas the novel PebS-encoding gene is exclusive to phages.     

Comparative genomics of NAD biosynthesis in cyanobacteria

Biosynthesis of NAD(P) cofactors is of special importance for cyanobacteria due to their role in photosynthesis and respiration. Despite significant progress in understanding NAD(P) biosynthetic machinery in some model organisms, relatively little is known about its implementation in cyanobacteria. We addressed this problem by a combination of comparative genome analysis with verification experiments in the model system of Synechocystis sp. strain PCC 6803. A detailed reconstruction of the NAD(P) metabolic subsystem using the SEED genomic platform (http://theseed.uchicago.edu/FIG/index.cgi) helped us accurately annotate respective genes in the entire set of 13 cyanobacterial species with completely sequenced genomes available at the time. Comparative analysis of operational variants implemented in this divergent group allowed us to elucidate both conserved (de novo and universal pathways) and variable (recycling and salvage pathways) aspects of this subsystem. Focused genetic and biochemical experiments confirmed several conjectures about the key aspects of this subsystem. (i) The product of the slr1691 gene, a homolog of Escherichia coli gene nadE containing an additional nitrilase-like N-terminal domain, is a NAD synthetase capable of utilizing glutamine as an amide donor in vitro. (ii) The product of the sll1916 gene, a homolog of E. coli gene nadD, is a nicotinic acid mononucleotide-preferring adenylyltransferase. This gene is essential for survival and cannot be compensated for by an alternative nicotinamide mononucleotide (NMN)-preferring adenylyltransferase (slr0787 gene). (iii) The product of the slr0788 gene is a nicotinamide-preferring phosphoribosyltransferase involved in the first step of the two-step non-deamidating utilization of nicotinamide (NMN shunt). (iv) The physiological role of this pathway encoded by a conserved gene cluster, slr0787-slr0788, is likely in the recycling of endogenously generated nicotinamide, as supported by the inability of this organism to utilize exogenously provided niacin. Positional clustering and the co-occurrence profile of the respective genes across a diverse collection of cellular organisms provide evidence of horizontal transfer events in the evolutionary history of this pathway.     

Molecular biology of peptide and polyketide biosynthesis in cyanobacteria

Cyanobacteria produce numerous and structurally diverse secondary metabolites, in particular nonribosomal peptide and polyketide structures. Various bioactivities could be assigned to these compounds, and some may prove useful either for development into commercial drugs or as biochemical research tools. Microcystin, a worldwide common cyanobacterial hepatotoxin, was the first metabolite whose nonribosomal biosynthesis could be confirmed by knock-out mutagenesis. The microcystin synthetase complex consists of peptide synthetases, polyketide synthases, and hybrid enzymes, and reveals a number of novel enzymatic features, signifying the potential of cyanobacterial biosynthetic systems for combinatorial biochemistry. Recent studies have shown the presence of peptide synthetase genes and polyketide synthase genes within a number of cyanobacterial genomes. This knowledge may be very valuable for future screening projects aimed at the detection of new bioactive compounds.     

Compatible solute accumulation and stress-mitigating effects in barley genotypes contrasting in their salt tolerance

The accumulation of compatible solutes is often regarded as a basic strategy for the protection and survival of plants under abiotic stress conditions, including both salinity and oxidative stress. In this work, a possible causal link between the ability of contrasting barley genotypes to accumulate/synthesize compatible solutes and their salinity stress tolerance was investigated. The impact of H(2)O(2) (one of the components of salt stress) on K(+) flux (a measure of stress 'severity') and the mitigating effects of glycine betaine and proline on NaCl-induced K(+) efflux were found to be significantly higher in salt-sensitive barley genotypes. At the same time, a 2-fold higher accumulation of leaf and root proline and leaf glycine betaine was found in salt-sensitive cultivars. The total amino acid content was also less affected by salinity in salt-tolerant cultivars. In these, potassium was found to be the main contributor to cytoplasmic osmolality, while in salt-sensitive genotypes, glycine betaine and proline contributed substantially to cell osmolality, compensating for reduced cytosolic K(+). Significant negative correlations (r= -0.89 and -0.94) were observed between Na(+)-induced K(+) efflux (an indicator of salt tolerance) and leaf glycine betaine and proline. These results indicate that hyperaccumulation of known major compatible solutes in barley does not appear to play a major role in salt-tolerance, but rather, may be a symptom of salt-susceptibility.     

Making proteins green; biosynthesis of chlorophyll-binding proteins in cyanobacteria

Chlorophyll (Chl) is an essential component of the photosynthetic apparatus. Embedded into Chl-binding proteins, Chl molecules play a central role in light harvesting and charge separation within the photosystems. It is critical for the photosynthetic cell to not only ensure the synthesis of a sufficient amount of new Chl-binding proteins but also avoids any misbalance between apoprotein synthesis and the formation of potentially phototoxic Chl molecules. According to the available data, Chl-binding proteins are translated on membrane bound ribosomes and their integration into the membrane is provided by the SecYEG/Alb3 translocon machinery. It appears that the insertion of Chl molecules into growing polypeptide is a prerequisite for the correct folding and finishing of Chl-binding protein synthesis. Although the Chl biosynthetic pathway is fairly well-described on the level of enzymatic steps, a link between Chl biosynthesis and the synthesis of apoproteins remains elusive. In this review, I summarize the current knowledge about this issue putting emphasis on protein–protein interactions. I present a model of the Chl biosynthetic pathway organized into a multi-enzymatic complex and physically attached to the SecYEG/Alb3 translocon. Localization of this hypothetical large biosynthetic centre in the cyanobacterial cell is also discussed as well as regulatory mechanisms coordinating the rate of Chl and apoprotein synthesis.     

Glucosylglycerate: a secondary compatible solute common to marine cyanobacteria from nitrogen-poor environments

The synthesis and accumulation of compatible solutes represent an essential part of the salt acclimation strategy of microorganisms. Glucosylglycerol is considered to be the typical compatible solute among marine cyanobacteria. However, genes that encode enzymes for the synthesis of glucosylglycerol were not detected in the genome sequences of marine picoplanktonic Prochlorococcus strains. Instead, we noticed the presence of genes that putatively encode for glucosylglycerate (GGA) synthesis among Prochlorococcus and most other closely related marine picocyanobacteria. Recombinant proteins from Prochlorococcus marinus SS120 and Synechococcus sp. PCC 7002 exhibited glucosyl-phosphoglycerate synthase (GpgS) activity, and GpgS is a key enzyme of GGA synthesis. GGA accumulation was found to be salt- as well as nitrogen-regulated in the coastal strain Synechococcus sp. PCC 7002. Moreover, GGA was also detected in all picoplanktonic Prochlorococcus and Synechococcus strains harbouring gpgS genes, especially under N-limiting conditions. These results suggest that marine picocyanobacteria acquired the capacity to synthesize the negatively charged compound GGA during their evolution. Our results establish GGA as the fifth most widespread compatible solute among cyanobacteria. Additionally, GGA appears to replace glutamate as an anion to counter monovalent cations in marine picocyanobacteria from N-poor environments.     

Phylogenetic distribution of compatible solute synthesis genes support a freshwater origin for cyanobacteria

Previous work using ancestral state reconstruction of habitat salinity preference proposed that the early cyanobacteria likely lived in a freshwater environment. The aim of this study was to test that hypothesis by performing phylogenetic analyses of the genes underlying salinity preferences in the cyanobacteria. Phylogenetic analysis of compatible solute genes shows that sucrose synthesis genes were likely ancestral in the cyanobacteria, and were also likely inherited during the cyanobacterial endosymbiosis and into the photosynthetic algae and land plants. In addition, the genes for the synthesis of compatible solutes that are necessary for survival in marine and hypersaline environments (such as glucosylglycerol, glucosylglycerate, and glycine betaine) were likely acquired independently high up (i.e., more recently) in the cyanobacterial tree. Because sucrose synthesis is strongly associated with growth in a low salinity environment, this independently supports a freshwater origin for the cyanobacteria. It is also consistent with geologic evidence showing that the early oceans were much warmer and saltier than modern oceans—sucrose synthesis alone would have been insufficient for early cyanobacteria to have colonized early Precambrian oceans that had a higher ionic strength. Indeed, the acquisition of an expanded set of new compatible solute genes may have enabled the historical colonization of marine and hypersaline environments by cyanobacteria, midway through their evolutionary history.     

Compatible solute effects on thermostability of glutamine synthetase and aspartate transcarbamoylase from Methanococcus jannaschii

Methanococcus jannaschii accumulates alpha- and beta-glutamate as osmolytes. The effect of these and other solutes on the thermostability of two multisubunit metabolic enzymes from M. jannaschii, aspartate transcarbamoylase catalytic trimer (ATCase C3) and glutamine synthetase (GS), has been measured and compared to solute effects on bacterial mesophilic counterparts in order to explore if osmolytes accumulated by each organism can preferentially stabilize the proteins to thermal unfolding. For both ATCase enzymes and for the B. subtilis GS, the solutes normally accumulated by the organism were very effective in protecting the enzyme from losing activity at high temperatures, although solute effects on loss of secondary structure did not necessarily correlate with this thermoprotection of activity. The recombinant M. jannaschii GS exhibited quite different behavior. The pure enzyme had a thermal unfolding transition with a midpoint temperature (Tm) less than 60 degrees C, well under the growth temperature of the organism (85 degrees C). None of the small molecule solutes tested (including the K+-glutamate isomers accumulated by M. jannaschii) significantly stabilized the protein to incubation at 85 degrees C. Instead, protein-protein interactions, as illustrated by E. coli GroEL or ribosomal protein L2 stabilization of GS, appeared to be the dominant factor in stabilizing this archaeal enzyme at the growth temperature.     

Post-translational control of tetrapyrrole biosynthesis in plants, algae, and cyanobacteria

The tetrapyrrole biosynthetic pathway provides the vital cofactors and pigments for photoautotrophic growth (chlorophyll), several essential redox reactions in electron transport chains (haem), N- and S-assimilation (sirohaem), and photomorphogenic processes (phytochromobilin). While the biochemistry of the pathway is well understood and almost all genes encoding enzymes of tetrapyrrole biosynthesis have been identified in plants, the post-translational control and organization of the pathway remains to be clarified. Post-translational mechanisms controlling metabolic activities are of particular interest since tetrapyrrole biosynthesis needs adaptation to environmental challenges. This review surveys post-translational mechanisms that have been reported to modulate metabolic activities and organization of the tetrapyrrole biosynthesis pathway.     

Unsaturated fatty acid composition and biosynthesis in Oscillatoria limnetica and other cyanobacteria

A number of cyanobacteria showing a high degree of adaptation to life under reduced oxygen tensions as witnessed by their potency of facultative anoxygenic CO₂ photoassimilation with sulfide as electron donor were found to lack polyunsaturated fatty acids in their lipids. Lack of polyunsaturated fatty acids was found in representatives of different taxonomic groups. One of the strains lacking polyenoic acids was Oscillatoria limnetica, which can alternatively grow acrobically or anaerobically with sulfide as electron donor. This organism was found to synthesize monounsaturated fatty acids by desaturation of their saturated counterparts, in the presence as well as in the absence of molecular oxygen.Abbreviations ACP Acyl carrier protein - DCMU 3(3,4-dichlorophenyl)-1,1-dimethylurea     

Structure and biosynthesis of toxins from blue-green algae (cyanobacteria)

Microcystis andNodularia species produce cyclic hepta- and pentapeptides, microcystins and nodularin, respectively, both containing the same unusual C₂₀ amino acid, abbreviated Adda. Biosynthesis of nodularin fromNodularia and especially of Adda employs a pathway similar to that employed byMicrocystis for producting microcystins. Nearly 30 new microcystins have been isolated in our laboratory from cyanobacteria species and their structures assigned, largely employing tandem FAB mass spectrometry (FABMS/CID/MS). Acyclic peptides, some of them presumed precursors of nodularin and microcystins, have now been isolated and characterized. The numerous analogs identified or synthesized allow the identification of important parameters in a structure-activity relationship study.     

The AtProT family. Compatible solute transporters with similar substrate specificity but differential expression patterns

Proline transporters (ProTs) mediate transport of the compatible solutes Pro, glycine betaine, and the stress-induced compound gamma-aminobutyric acid. A new member of this gene family, AtProT3, was isolated from Arabidopsis (Arabidopsis thaliana), and its properties were compared to AtProT1 and AtProT2. Transient expression of fusions of AtProT and the green fluorescent protein in tobacco (Nicotiana tabacum) protoplasts revealed that all three AtProTs were localized at the plasma membrane. Expression in a yeast (Saccharomyces cerevisiae) mutant demonstrated that the affinity of all three AtProTs was highest for glycine betaine (K(m) = 0.1-0.3 mM), lower for Pro (K(m) = 0.4-1 mM), and lowest for gamma-aminobutyric acid (K(m) = 4-5 mM). Relative quantification of the mRNA level using real-time PCR and analyses of transgenic plants expressing the beta-glucuronidase (uidA) gene under control of individual AtProT promoters showed that the expression pattern of AtProTs are complementary. AtProT1 expression was found in the phloem or phloem parenchyma cells throughout the whole plant, indicative of a role in long-distance transport of compatible solutes. beta-Glucuronidase activity under the control of the AtProT2 promoter was restricted to the epidermis and the cortex cells in roots, whereas in leaves, staining could be demonstrated only after wounding. In contrast, AtProT3 expression was restricted to the above-ground parts of the plant and could be localized to the epidermal cells in leaves. These results showed that, although intracellular localization, substrate specificity, and affinity are very similar, the transporters fulfill different roles in planta.     

Carotenoid biosynthesis in cyanobacteria: structural and evolutionary scenarios based on comparative genomics

Carotenoids are widely distributed pigments in nature and their biosynthetic pathway has been extensively studied in various organisms. The recent access to the overwhelming amount genomic data of cyanobacteria has given birth to a novel approach called comparative genomics. The putative enzymes involved in the carotenoid biosynthesis among the cyanobacteria were determined by similarity-based tools. The reconstruction of biosynthetic pathway was based on the related enzymes. It is interesting to find that nearly all the cyanobacteria share quite similar pathway to synthesize beta-carotene except for Gloeobacter violaceus PCC 7421. The enzymes, crtE-B-P-Qb-L, involved in the upstream pathway are more conserved than the subsequent ones (crtW-R). In addition, many carotenoid synthesis enzymes exhibit diversity in structure and function. Such examples in the families of zeta -carotene desaturase, lycopene cylases and carotene ketolases were described in this article. When we mapped these crt genes to the cyanobacterial genomes, the crt genes showed great structural variation among species. All of them are dispersed on the whole chromosome in contrast to the linear adjacent distribution of the crt gene cluster in other eubacteria. Moreover, in unicellular cyanobacteria, each step of the carotenogenic pathway is usually catalyzed by one gene product, whereas multiple ketolase genes are found in filamentous cyanobacteria. Such increased numbers of crt genes and their correlation to the ecological adaptation were carefully discussed.     

Biochemical diversity for biosynthesis of aromatic amino acids among the cyanobacteria.

We examined the enzymology and regulatory patterns of the aromatic amino acid pathway in 48 strains of cyanobacteria including representatives from each of the five major grouping. Extensive diversity was found in allosteric inhibition patterns of 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase, not only between the major groupings but also within several of the generic groupings. Unimetabolite inhibition by phenylalanine occurred in approximately half of the strains examined; in the other strains unimetabolite inhibition by tyrosine and cumulative, concerted, and additive patterns were found. The additive patterns suggest the presence of regulatory isozymes. Even though both arogenate and prephenate dehydrogenase activities were found in some strains, it seems clear that the arogenate pathway to tyrosine is a common trait that has been highly conserved among cyanobacteria. No arogenate dehydratase activities were found. In general, prephenate dehydratase activities were activated by tyrosine and inhibited by phenylalanine. Chorismate mutase, arogenate dehydrogenase, and shikimate dehydrogenase were nearly always unregulated. Most strains preferred NADP as the cofactor for the dehydrogenase activities. The diversity in the allosteric inhibition patterns for 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase, cofactor specificities, and the presence or absence of prephenate dehydrogenase activity allowed the separation of subgroupings within several of the form genera, namely, Synechococcus, Synechocystis, Anabaena, Nostoc, and Calothrix.     

Plastoquinone-9 biosynthesis in cyanobacteria differs from that in plants and involves a novel 4-hydroxybenzoate solanesyltransferase

PQ-9 (plastoquinone-9) has a central role in energy transformation processes in cyanobacteria by mediating electron transfer in both the photosynthetic as well as the respiratory electron transport chain. The present study provides evidence that the PQ-9 biosynthetic pathway in cyanobacteria differs substantially from that in plants. We identified 4-hydroxybenzoate as being the aromatic precursor for PQ-9 in Synechocystis sp. PCC6803, and in the present paper we report on the role of the membrane-bound 4-hydroxybenzoate solanesyltransferase, Slr0926, in PQ-9 biosynthesis and on the properties of the enzyme. The catalytic activity of Slr0926 was demonstrated by in vivo labelling experiments in Synechocystis sp., complementation studies in an Escherichia coli mutant with a defect in ubiquinone biosynthesis, and in vitro assays using the recombinant as well as the native enzyme. Although Slr0926 was highly specific for the prenyl acceptor substrate 4-hydroxybenzoate, it displayed a broad specificity with regard to the prenyl donor substrate and used not only solanesyl diphosphate, but also a number of shorter-chain prenyl diphosphates. In combination with in silico data, our results indicate that Slr0926 evolved from bacterial 4-hydroxybenzoate prenyltransferases catalysing prenylation in the course of ubiquinone biosynthesis.