Investigations on the solubility behavior of cyanophycin. Solubility of cyanophycin in solutions of simple inorganic salts

On the basis of a previous report on the occurrence of water-soluble cyanophycin (CGP, cyanophycin granule polypeptide) in a recombinant strain of Escherichia coli expressing the cyanophycin synthetase (CphA) of Desulfitobacterium hafniense published by others, the conditions of its production were investigated in this study. Although the incubation temperature, aeration level, and NaCl concentration during cultivation had effects on the in vivo production of water-soluble CGP, it could be isolated as a major variant irrespective of the cultivation conditions. The occurrence of the soluble variant was also not dependent on the E. coli host or on the origin of cphA. Furthermore, it was shown that water-insoluble CGP can be in vitro solubilized to extents of up to about 80% (w/w) in solutions of different inorganic salts such as LiCl, NaCl, KCl, RbCl, KBr, MgCl(2), or CaCl(2). Evidence was obtained that the salt ions bind tightly to CGP. If the ions were not removed from the salt solution by dialysis or dilution, the CGP remained stable in solution. This method to solubilize water-insoluble CGP could also be applied to high concentrations of the polymer. CGP that remained insoluble after the first treatment could only marginally be solubilized in following treatments. The polydisperse CGP molecules were solubilized to the same extent over the whole molecular weight range with no preference of a particular molecular weight.     

Tuber-specific cphA expression to enhance cyanophycin production in potatoes

The production of biodegradable polymers that can be used to substitute petrochemical compounds in commercial products in transgenic plants is an important challenge for plant biotechnology. Nevertheless, it is often accompanied by reduced plant fitness. To decrease the phenotypic abnormalities of the sprout and to increase polymer production, we restricted cyanophycin accumulation to the potato tubers by using the cyanophycin synthetase gene ( cph A_Te) from Thermosynechococcus elongatus BP-1, which is under the control of the tuber-specific class 1 promoter (B33). Tuber-specific cytosolic (pB33- cph A_Te) as well as tuber-specific plastidic (pB33-PsbY- cph A_Te) expression resulted in significant polymer accumulation solely in the tubers. In plants transformed with pB33- cph A_Te, both cyanophycin synthetase and cyanophycin were detected in the cytoplasm leading to an increase up to 2.3% cyanophycin of dry weight and resulting in small and deformed tubers. In B33-PsbY- cph A_Te tubers, cyanophycin synthetase and cyanophycin were exclusively found in amyloplasts leading to a cyanophycin accumulation up to 7.5% of dry weight. These tubers were normal in size, some clones showed reduced tuber yield and sometimes exhibited brown sunken staining starting at tubers navel. During a storage period over of 32 weeks of one selected clone, the cyanophycin content was stable in B33-PsbY- cph A_Te tubers but the stress symptoms increased. However, all tubers were able to germinate. Nitrogen fertilization in the greenhouse led not to an increased cyanophycin yield, slightly reduced protein content, decreased starch content, and changes in the amounts of bound and free arginine and aspartate, as compared with control tubers were observed.     

Occurrence of several genes encoding putative reductive dehalogenases in Desulfitobacterium hafniense/frappieri and Dehalococcoides ethenogenes

Desulfitobacterium frappieri PCP-1 has the capacity to dehalogenate several halogenated aromatic compounds by reductive dehalogenation, however, the genes encoding the enzymes involved in such processes have not yet been identified. Using a degenerate oligonucleotide corresponding to a conserved sequence of CprA/PceA reductive dehalogenases, a cprA-like gene fragment was amplified by PCR from this bacterial strain. A Desulfitobacterium frappieri PCP-1 cosmid library was screened with the PCR product, allowing the cloning and sequencing of a 1.9-kb fragment. This fragment contains a nucleic acid sequence identical to one genomic contig of Desulfitobacterium hafniense, a bacterium closely related to Desulfitobacterium frappieri that is also involved in reductive dehalogenation. Other genes related to the Desulfitobacterium dehalogenans cpr locus were identified in this contig. Interestingly, the gene arrangement shows the presence of two copies of cprA-, cprB-, cprC-, cprD-, cprK-, and cprT-related genes, suggesting that gene duplication occurred within this chromosomic region. The screening of Delfitobacterium hafniense genomic contigs with a CprA-deduced amino acid sequence revealed two other cprA-like genes. Microbial genomes available in gene databases were also analyzed for sequences related to CprA/PceA. Two open reading frames encoding other putative reductive dehalogenases in Desulfitobacterium hafniense contigs were detected, along with 17 in the Dehalococcoides ethenogenes genome, a bacterium involved in the reductive dehalogenation of tetrachloroethene to ethene. The fact that several gene encoding putative reductive dehalogenases exist in Delfitobacterium hafniense, probably in other members of the genus Desulfitobacterium, and in Dehalococcoides ethenogenes suggests that these bacteria use distinct but related enzymes to achieve the dehalogenation of several chlorinated compounds [corrected].     

Sulfonates as terminal electron acceptors for growth of sulfite-reducing bacteria (Desulfitobacterium spp.) and sulfate-reducing bacteria: effects of inhibitors of sulfidogenesis.

This study demonstrates the ability of Desulfitobacterium spp. to utilize aliphatic sulfonates as terminal electron acceptors (TEA) for growth. Isethionate (2-hydroxyethanesulfonate) reduction by Desulfitobacterium hafniense resulted in acetate as well as sulfide accumulation in accordance with the expectation that the carbon portion of isethionate was oxidized to acetate and the sulfur was reduced to sulfide. The presence of a polypeptide, approximately 97 kDa, was evident in isethionate-grown cells of Desulfitobacterium hafniense, Desulfitobacterium sp. strain PCE 1, and the two sulfate-reducing bacteria (SRB)-Desulfovibrio desulfuricans IC1 (T. J. Lie, J. R. Leadbetter, and E. R. Leadbetter, Geomicrobiol. J. 15:135-149, 1998) and Desulfomicrobium norvegicum; this polypeptide was not detected when these bacteria were grown on TEA other than isethionate, suggesting involvement in its metabolism. The sulfate analogs molybdate and tungstate, effective in inhibiting sulfate reduction by SRB, were examined for their effects on sulfonate reduction. Molybdate effectively inhibited sulfonate reduction by strain IC1 and selectively inhibited isethionate (but not cysteate) reduction by Desulfitobacterium dehalogenans and Desulfitobacterium sp. strain PCE 1. Desulfitobacterium hafniense, however, grew with both isethionate and cysteate in the presence of molybdate. In contrast, tungstate only partially inhibited sulfonate reduction by both SRB and Desulfitobacterium spp. Similarly, another inhibitor of sulfate reduction, 1,8-dihydroxyanthraquinone, effectively inhibited sulfate reduction by SRB but only partially inhibited sulfonate reduction by both SRB and Desulfitobacterium hafniense.     

Production of cyanophycin, a suitable source for the biodegradable polymer polyaspartate, in transgenic plants

The production of biodegradable polymers in transgenic plants in order to replace petrochemical compounds is an important challenge for plant biotechnology. Polyaspartate, a biodegradable substitute for polycarboxylates, is the backbone of the cyanobacterial storage material cyanophycin. Cyanophycin, a copolymer of l-aspartic acid and l-arginine, is produced via non-ribosomal polypeptide biosynthesis by the enzyme cyanophycin synthetase. A gene from Thermosynechococcus elongatus BP-1 encoding cyanophycin synthetase has been expressed constitutively in tobacco and potato. The presence of the transgene-encoded messenger RNA (mRNA) correlated with changes in leaf morphology and decelerated growth. Such transgenic plants were found to produce up to 1.1% dry weight of a polymer with cyanophycin-like properties. Aggregated material, able to bind a specific cyanophycin antibody, was detected in the cytoplasm and the nucleus of the transgenic plants.     

Biosynthesis of the cyanobacterial reserve polymer multi-L-arginyl-poly-L-aspartic acid (cyanophycin): mechanism of the cyanophycin synthetase reaction studied with synthetic primers.

Biosynthesis of the cyanobacterial nitrogen reserve cyanophycin (multi-L-arginyl-poly-L-aspartic acid) is catalysed by cyanophycin synthetase, an enzyme that consists of a single kind of polypeptide. Efficient synthesis of the polymer requires ATP, the constituent amino acids aspartic acid and arginine, and a primer like cyanophycin. Using synthetic peptide primers, the course of the biosynthetic reaction was studied. The following results were obtained: (a) sequence analysis suggests that cyanophycin synthetase has two ATP-binding sites and hence probably two active sites; (b) the enzyme catalyses the formation of cyanophycin-like polymers of 25-30 kDa apparent molecular mass in vitro; (c) primers are elongated at their C-terminus; (d) the constituent amino acids are incorporated stepwise, in the order aspartic acid followed by arginine, into the growing polymer. A mechanism for the cyanophycin synthetase reaction is proposed; (e) the specificity of the enzyme for its amino-acid substrates was also studied. Glutamic acid cannot replace aspartic acid as the acidic amino acid, whereas lysine can replace arginine but is incorporated into cyanophycin at a much lower rate.     

Molecular characterization of the cyanophycin synthetase from Synechocystis sp. strain PCC6308

A 3878-bp genomic region from the cyanobacterium Synechocystis sp. strain PCC6308, amplified by inverse PCR, harbored the structural genes cphA (2625 bp) and cphB (819 bp) encoding cyanophycin synthetase and cyanophycinase, respectively. Both primary structures exhibited a high degree of similarity to the corresponding translational products from other cyanobacteria. Five regions were localized in the cyanophycin synthetase consensus sequence by their resemblance to conserved sites of ATP-dependent carboxylate-amine/thiol ligases and three substrate ligases. The functionality of cphA was proven by heterologous expression of active enzyme and synthesis of cyanophycin in Escherichia coli, which led to a maximum cyanophycin content of 26.6% (w/w) of cell dry mass. Furthermore, a modified radiometric enzyme assay for a more reliable and feasible measurement of cyanophycin synthetase activity was developed and applied to reveal the substrate specificity of the enzyme.     

Recognition of pyrrolysine tRNA by the Desulfitobacterium hafniense pyrrolysyl-tRNA synthetase

Pyrrolysine (Pyl), the 22nd co-translationally inserted amino acid, is incorporated in response to a UAG amber stop codon. Pyrrolysyl-tRNA synthetase (PylRS) attaches Pyl to its cognate tRNA, the special amber suppressor tRNA(Pyl). The genes for tRNA(Pyl) (pylT) and PylRS (pylS) are found in all members of the archaeal family Methanosarcinaceae, and in Desulfitobacterium hafniense. The activation and aminoacylation properties of D. hafniense PylRS and the nature of the tRNA(Pyl) identity elements were determined by measuring the ability of 24 mutant tRNA(Pyl) species to be aminoacylated with the pyrrolysine analog N-epsilon-cyclopentyloxycarbonyl-l-lysine. The discriminator base G73 and the first base pair (G1.C72) in the acceptor stem were found to be major identity elements. Footprinting analysis showed that PylRS binds tRNA(Pyl) predominantly along the phosphate backbone of the T-loop, the D-stem and the acceptor stem. Significant contacts with the anticodon arm were not observed. The tRNA(Pyl) structure contains the highly conserved T-loop contact U54.A58 and position 57 is conserved as a purine, but the canonical T- to D-loop contact between positions 18 and 56 was not present. Unlike most tRNAs, the tRNA(Pyl) anticodon was shown not to be important for recognition by bacterial PylRS.     

PII-regulated arginine synthesis controls accumulation of cyanophycin in Synechocystis sp. strain PCC 6803

Cyanophycin (multi-L-arginyl-poly-L-aspartic acid) is a nitrogen storage polymer found in most cyanobacteria and some heterotrophic bacteria. The cyanobacterium Synechocystis sp. strain PCC 6803 accumulates cyanophycin following a transition from nitrogen-limited to nitrogen-excess conditions. Here we show that the accumulation of cyanophycin depends on the activation of the key enzyme of arginine biosynthesis, N-acetyl-L-glutamate kinase, by signal transduction protein PII.     

Resolution of culture Clostridium bifermentans DPH-1 into two populations, a Clostridium sp. and tetrachloroethene-dechlorinating Desulfitobacterium hafniense strain JH1

Clostridium bifermentans strain DPH-1 reportedly dechlorinates tetrachloroethene (PCE) to cis-1,2-dichloroethene. Cultivation-based approaches resolved the DPH-1 culture into two populations: a nondechlorinating Clostridium sp. and PCE-dechlorinating Desulfitobacterium hafniense strain JH1. Strain JH1 carries pceA, encoding a PCE reductive dehalogenase, and shares other characteristics with Desulfitobacterium hafniense strain Y51.     

The amino-terminal domain of pyrrolysyl-tRNA synthetase is dispensable in vitro but required for in vivo activity

Pyrrolysine (Pyl) is co-translationally inserted into a subset of proteins in the Methanosarcinaceae and in Desulfitobacterium hafniense programmed by an in-frame UAG stop codon. Suppression of this UAG codon is mediated by the Pyl amber suppressor tRNA, tRNA(Pyl), which is aminoacylated with Pyl by pyrrolysyl-tRNA synthetase (PylRS). We compared the behavior of several archaeal and bacterial PylRS enzymes towards tRNA(Pyl). Equilibrium binding analysis revealed that archaeal PylRS proteins bind tRNA(Pyl) with higher affinity (K(D)=0.1-1.0 microM) than D. hafniense PylRS (K(D)=5.3-6.9 microM). In aminoacylation the archaeal PylRS enzymes did not distinguish between archaeal and bacterial tRNA(Pyl) species, while the bacterial PylRS displays a clear preference for the homologous cognate tRNA. We also show that the amino-terminal extension present in archaeal PylRSs is dispensable for in vitro activity, but required for PylRS function in vivo.     

Molecular characterization of a thermostable cyanophycin synthetase from the thermophilic cyanobacterium Synechococcus sp. strain MA19 and in vitro synthesis of cyanophycin and related polyamides.

The thermophilic cyanobacterium Synechococcus sp. strain MA19 contained the structural genes for cyanophycin synthetase (cphA) and cyanophycinase (cphB), which were identified, cloned, and sequenced in this study. The translation products of cphA and cphB exhibited high levels of similarity to corresponding proteins of other cyanobacteria, such as Anabaena variabilis and Synechocystis sp. Recombinant cells of Escherichia coli harboring cphA colinear with lacPO accumulated cyanophycin that accounted for up to 25% (wt/wt) of the dry cell matter in the presence of isopropyl-beta-D-thiogalactopyranoside (IPTG). The cyanophycin synthetase was enriched 123-fold to electrophoretic homogeneity from the soluble fraction of the recombinant cells by anion-exchange chromatography, affinity chromatography, and gel filtration chromatography. The purified cyanophycin synthetase maintained the parental thermophilic character and was active even after prolonged incubation at 50 degrees C; in the presence of ectoine the enzyme retained 90% of its activity even after 2 h of incubation. The in vitro activity of the enzyme depended on ATP, primers, and both substrates, L-arginine and L-aspartic acid. In addition to native cyanophycin, the purified enzyme accepted a modified cyanophycin containing less arginine, alpha-arginyl aspartic acid dipeptide, and poly-alpha,beta-DL-aspartic acid as primers and also incorporated beta-hydroxyaspartic acid instead of L-aspartic acid or L-canavanine instead of L-arginine at a significant rate. The lack of specificity of this thermostable enzyme with respect to primers and substrates, the thermal stability of the enzyme, and the finding that the enzyme is suitable for in vitro production of cyanophycin make it an interesting candidate for biotechnological processes.     

Features of the biotechnologically relevant polyamide family “cyanophycins” and their biosynthesis in prokaryotes and eukaryotes

Cyanophycin, inclusions in cyanobacteria discovered by the Italian scientist Borzi in 1887, were characterized as a polyamide consisting of aspartic acid and arginine. Its synthesis in cyanobacteria was analyzed regarding growth conditions, responsible gene product, requirements, polymer structure and properties. Heterologous expression of diverse cyanophycin synthetases (CphA) in Escherichia coli enabled further enzyme characterization. Cyanophycin is a polyamide with variable composition and physiochemical properties dependent on host and cultivation conditions in contrast to the extracellular polyamides poly-γ-glutamic acid and poly-ε-l-lysine. Furthermore, recombinant prokaryotes and transgenic eukaryotes, including plants expressing different cphA genes, were characterized as suitable for production of insoluble cyanophycin regarding higher yields and modified composition for other requirements and applications. In addition, cyanophycin was characterized as a source for the synthesis of polyaspartic acid or N-containing bulk chemicals and dipeptides upon chemical treatment or degradation by cyanophycinases, respectively. Moreover, water-soluble cyanophycin derivatives with altered amino acid composition were isolated from transgenic plants, yeasts and recombinant bacteria. Thereby, the range of dipeptides could be extended by biological processes and by chemical modification, thus increasing the range of applications for cyanophycin and its dipeptides, including agriculture, food supplementations, medical and cosmetic purposes, synthesis of the polyacrylate substitute poly(aspartic acid) and other applications.     

Isolation and characterization of a novel As(V)-reducing bacterium: implications for arsenic mobilization and the genus Desulfitobacterium.

Dissimilatory arsenate-reducing bacteria have been implicated in the mobilization of arsenic from arsenic-enriched sediments. An As(V)-reducing bacterium, designated strain GBFH, was isolated from arsenic-contaminated sediments of Lake Coeur d'Alene, Idaho. Strain GBFH couples the oxidation of formate to the reduction of As(V) when formate is supplied as the sole carbon source and electron donor. Additionally, strain GBFH is capable of reducing As(V), Fe(III), Se(VI), Mn(IV) and a variety of oxidized sulfur species. 16S ribosomal DNA sequence comparisons reveal that strain GBFH is closely related to Desulfitobacterium hafniense DCB-2(T) and Desulfitobacterium frappieri PCP-1(T). Comparative physiology demonstrates that D. hafniense and D. frappieri, known for reductively dechlorinating chlorophenols, are also capable of toxic metal or metalloid respiration. DNA-DNA hybridization and comparative physiological studies suggest that D. hafniense, D. frappieri, and strain GBFH should be united into one species. The isolation of an Fe(III)- and As(V)-reducing bacterium from Lake Coeur d'Alene suggests a mechanism for arsenic mobilization in these contaminated sediments while the discovery of metal or metalloid respiration in the genus Desulfitobacterium has implications for environments cocontaminated with arsenious and chlorophenolic compounds.     

The Desulfitobacterium genus

Desulfitobacterium spp. are strictly anaerobic bacteria that were first isolated from environments contaminated by halogenated organic compounds. They are very versatile microorganisms that can use a wide variety of electron acceptors, such as nitrate, sulfite, metals, humic acids, and man-made or naturally occurring halogenated organic compounds. Most of the Desulfitobacterium strains can dehalogenate halogenated organic compounds by mechanisms of reductive dehalogenation, although the substrate spectrum of halogenated organic compounds varies substantially from one strain to another, even with strains belonging to the same species. A number of reductive dehalogenases and their corresponding gene loci have been isolated from these strains. Some of these loci are flanked by transposition sequences, suggesting that they can be transmitted by horizontal transfer via a catabolic transposon. Desulfitobacterium spp. can use H2 as electron donor below the threshold concentration that would allow sulfate reduction and methanogenesis. Furthermore, there is some evidence that syntrophic relationships occur between Desulfitobacterium spp. and sulfate-reducing bacteria, from which the Desulfitobacterium cells acquire their electrons by interspecies hydrogen transfer, and it is believed that this relationship also occurs in a methanogenic consortium. Because of their versatility, desulfitobacteria can be excellent candidates for the development of anaerobic bioremediation processes. The release of the complete genome of Desulfitobacterium hafniense strain Y51 and information from the partial genome sequence of D. hafniense strain DCB-2 will certainly help in predicting how desulfitobacteria interact with their environments and other microorganisms, and the mechanisms of actions related to reductive dehalogenation.     

Production of cyanophycin in <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> through the expression of a cyanophycin synthetase encoding gene

Cyanophycin or cyanophycin granule peptide is a protein that results from non-ribosomal protein synthesis in microorganisms such as cyanobacteria. The amino acids in cyanophycin can be used as a feedstock in the production of a wide range of chemicals such as acrylonitrile, polyacrylic acid, 1,4-butanediamine, and urea. In this study, an auxotrophic mutant (Rhizopus oryzae M16) of the filamentous fungus R. oryzae 99-880 was selected to express cyanophycin synthetase encoding genes. These genes originated from Synechocystis sp. strain PCC6803, Anabaena sp. strain PCC7120, and a codon optimized version of latter gene. The genes were under control of the pyruvate decarboxylase promoter and terminator elements of R. oryzae. Transformants were generated by the biolistic transformation method. In only two transformants both expressing the cyanophycin synthetase encoding gene from Synechocystis sp. strain PCC6803 was a specific enzyme activity detected of 1.5 mU/mg protein. In one of these transformants was both water-soluble and insoluble cyanophycin detected. The water-soluble fraction formed the major fraction and accounted for 0.5% of the dry weight. The water-insoluble CGP was produced in trace amounts. The amino acid composition of the water-soluble form was determined and constitutes of equimolar amounts of arginine and aspartic acid.     

Cyanophycin production in a phycoerythrin-containing marine synechococcus strain of unusual phylogenetic affinity

Thirty-two strains of phycoerythrin-containing marine picocyanobacteria were screened for the capacity to produce cyanophycin, a nitrogen storage compound synthesized by some, but not all, cyanobacteria. We found that one of these strains, Synechococcus sp. strain G2.1 from the Arabian Sea, was able to synthesize cyanophycin. The cyanophycin extracted from the cells was composed of roughly equimolar amounts of arginine and aspartate (29 and 35 mol%, respectively), as well as a small amount of glutamate (15 mol%). Phylogenetic analysis, based on partial 16S ribosomal DNA (rDNA) sequence data, showed that Synechococcus sp. strain G2.1 formed a well-supported clade with several strains of filamentous cyanobacteria. It was not closely related to several other well-studied marine picocyanobacteria, including Synechococcus strains PCC7002, WH7805, and WH8018 and Prochlorococcus sp. strain MIT9312. This is the first report of cyanophycin production in a phycoerythrin-containing strain of marine or halotolerant Synechococcus, and its discovery highlights the diversity of this ecologically important functional group.