Meta-omic profiling reveals ubiquity of genes encoding for the nitrogen-rich biopolymer cyanophycin in activated sludge microbiomes.

Recovering nitrogen (N) from municipal wastewater is a promising approach to prevent nutrient pollution, reduce energy use, and transition toward a circular N bioeconomy, but remains a technologically challenging endeavor. Existing N recovery techniques are optimized for high-strength, low-volume wastewater. Therefore, developing methods to concentrate dilute N from mainstream wastewater will bridge the gap between existing technologies and practical implementation. The N-rich biopolymer cyanophycin is a promising candidate for N bioconcentration due to its pH-tunable solubility characteristics and potential for high levels of accumulation. However, the cyanophycin synthesis pathway is poorly explored in engineered microbiomes. In this study, we analyzed over 3,700 publicly available metagenome assembled genomes (MAGs) and found that the cyanophycin synthesis gene cphA was ubiquitous across common activated sludge bacteria. We found that cphA was present in common phosphorus accumulating organisms (PAO) Ca. 'Accumulibacter' and Tetrasphaera, suggesting potential for simultaneous N and P bioconcentration in the same organisms. Using metatranscriptomic data, we confirmed the expression of cphA in lab-scale bioreactors enriched with PAO. Our findings suggest that cyanophycin synthesis is a ubiquitous metabolic activity in activated sludge microbiomes. The possibility of combined N and P bioconcentration could lower barriers to entry for N recovery, since P concentration by PAO is already a widespread biotechnology in municipal wastewater treatment. We anticipate this work to be a starting point for future evaluations of combined N and P bioaccumulation, with the ultimate goal of advancing widespread adoption of N recovery from municipal wastewater.

Recombinant Multi-l-Arginyl-Poly-l-Aspartate (Cyanophycin) as an Emerging Biomaterial.

Multi-l-arginyl-poly-l-aspartate (MAPA) is a non-ribosomal polypeptide which synthesis is directed by cyanophycin synthetase, and its production can be achieved using recombinant microorganisms carrying the cphA gene. Along its poly-aspartate backbone, arginine or lysine links to each aspartate via an isopeptide bond. MAPA is a zwitterionic polyelectrolyte full of charged carboxylic, amine, and guanidino groups. In aqueous solution, MAPA exhibits dual thermal and pH responses similar to those stimuli-responsive polymers. Being biocompatible, the films containing MAPA can support cell proliferation and elicits minimal immune response in macrophages. Dipeptides from MAPA after enzymatic treatments can provide nutritional benefits. In light of the increasing interest in MAPA, this article focuses on the recent discovery of the function of cyanophycin synthetase and the potentials of MAPA as a biomaterial.

Structure and function of a hexameric cyanophycin synthetase 2.

Cyanophycin is a natural polymer composed of a poly-aspartate backbone with arginine attached to each of the aspartate sidechains. Produced by a wide range of bacteria, which mainly use it as a store of fixed nitrogen, it has many promising industrial applications. Cyanophycin can be synthesized from the amino acids Asp and Arg by the widespread cyanophycin synthetase 1 (CphA1), or from the dipeptide ß-Asp-Arg by the cyanobacterial enzyme cyanophycin synthetase 2 (CphA2). CphA2 enzymes display a range of oligomeric states, from dimers to dodecamers. Recently, the crystal structure of a CphA2 dimer was solved but could not be obtained in complex with substrate. Here, we report cryo-EM structures of the hexameric CphA2 from Stanieria sp. at ~2.8 Å resolution, both with and without ATP analog and cyanophycin. The structures show a two-fold symmetrical, trimer-of-dimers hexameric architecture, and substrate-binding interactions that are similar to those of CphA1. Mutagenesis experiments demonstrate the importance of several conserved substrate-binding residues. We also find that a Q416A/R528G double mutation prevents hexamer formation and use this double mutant to show that hexamerization augments the rate of cyanophycin synthesis. Together, these results increase our mechanistic understanding of how an interesting green polymer is biosynthesized.

A fluorescence method to detect cyanophycin in the symbiotic cyanobacterium, Anabaena azollae.

The cyanophycin content of the heterocystous nitrogen-fixing symbiotic cyanobacterium, Anabaena azollae, which inhabits an ovoid cavity in the dorsal leaf lobes of the fern, Azolla filiculoides, is seldom analyzed. To study the cyanophycin content in vegetative cells and heterocysts of A. azollae, we used three fluorochromes: aluminum trichloride, lead citrate and Wilson citroboric solution and Coomassie brilliant blue. Blue and yellow fluorescence were emitted from the polar nodes and cytoplasm cyanophycin granules of the heterocysts when stained with the three fluorochromes. The cyanophycin observed without staining or with Coomassie brilliant blue staining did not alter the results obtained using the fluorochromes. We found that aluminum trichloride, lead acetate and Wilson citroboric solution could be used to detect cyanophycin.

Discovery of cyanophycin dipeptide hydrolase enzymes suggests widespread utility of the natural biopolymer cyanophycin.

Cyanophycin is a bacterial polymer mainly used for nitrogen storage. It is composed of a peptide backbone of L-aspartate residues with L-arginines attached to their side chains through isopeptide bonds. Cyanophycin is degraded in two steps: Cyanophycinase cleaves the polymer into ß-Asp-Arg dipeptides, which are hydrolyzed into free Asp and Arg by enzymes possessing isoaspartyl dipeptide hydrolase activity. Two unrelated enzymes with this activity, isoaspartyl dipeptidase (IadA) and isoaspartyl aminopeptidase (IaaA) have been shown to degrade ß-Asp-Arg dipeptides, but bacteria which encode cyanophycin-metabolizing genes can lack iaaA and iadA genes. In this study, we investigate a previously uncharacterized enzyme whose gene can cluster with cyanophycin-metabolizing genes. This enzyme, which we name cyanophycin dipeptide hydrolase (CphZ), is specific for dipeptides derived from cyanophycin degradation. Accordingly, a co-complex structure of CphZ and ß-Asp-Arg shows that CphZ, unlike IadA or IaaA, recognizes all portions of its ß-Asp-Arg substrate. Bioinformatic analyses showed that CphZ is found in very many proteobacteria and is homologous to an uncharacterized protein encoded in the "arginine/ornithine transport" (aot) operon of many pseudomonas species, including Pseudomonas aeruginosa. In vitro assays show that AotO is indeed a CphZ, and in cellulo growth experiments show that this enzyme and the aot operon allow P. aeruginosa to take up and use ß-Asp-Arg as a sole carbon and nitrogen source. Together the results establish the novel, highly specific enzyme subfamily of CphZs, suggesting that cyanophycin is potentially used by a much wider range of bacteria than previously appreciated.

Corrigendum: Sustainable production of the cyanophycin biopolymer in tobacco in the greenhouse and field.

[This corrects the article DOI: 10.3389/fbioe.2022.896863.].

The structure of cyanophycinase in complex with a cyanophycin degradation intermediate.

BACKGROUND: Cyanophycinases are serine protease family enzymes which are required for the metabolism of cyanophycin, the natural polymer multi-L-arginyl-poly(L-aspartic acid). Cyanophycinases degrade cyanophycin to ß-Asp-Arg dipeptides, which enables use of this important store of fixed nitrogen. METHODS: We used genetic code expansion to incorporate diaminopropionic acid into cyanophycinase in place of the active site serine, and determined a high-resolution structure of the covalent acyl-enzyme intermediate resulting from attack of cyanophycinase on a short cyanophycin segment. RESULTS: The structure indicates that cyanophycin dipeptide residues P1 and P1' bind shallow pockets adjacent to the catalytic residues. We observe many cyanophycinase - P1 dipeptide interactions in the co-complex structure. Calorimetry measurements show that at least two cyanophycin dipeptides are needed for high affinity binding to cyanophycinase. We also characterized a putative cyanophycinase which we found to be structurally very similar but that shows no activity and could not be activated by mutation of its active site. GENERAL SIGNIFICANCE: Despite its peptidic structure, cyanophycin is resistant to degradation by peptidases and other proteases. Our results help show how cyanophycinase can specifically bind and degrade this important polymer.

Cyanophycin Granule Polypeptide: a Neglected High Value-Added Biopolymer, Synthesized in Activated Sludge on a Large Scale.

Recovery of microbial synthetic polymers with high economic value and market demand in activated sludge has attracted extensive attention. This work analyzed the synthesis of cyanophycin granule peptide (CGP) in activated sludge and its adsorption capacity for heavy metals and dyes. The distribution and expression of synthetic genes for eight biopolymers in two wastewater treatment plants (WWTPs) were analyzed by metagenomics and metatranscriptomics. The results indicate that the abundance and expression level of CGP synthase (cphA) are similar to those of polyhydroxyalkanoate polymerase, implying high synthesis of CGP in activated sludges. CGP in activated sludge is mainly polymerized from aspartic acid and arginine, and its secondary structure is mainly ß-sheet. The crude yields of CGP are as high as 104 ± 26 and 76 ± 13 mg/g dry sludge in winter and in summer, respectively, comparable to those of polyhydroxyalkanoate and alginate. CGP has a stronger adsorption capacity for anionic pollutants (Cr (VI) and methyl orange) than for cationic pollutants because it is rich in guanidine groups. This study highlights prospects for recovery and application of CGP from WWTPs. IMPORTANCE The conversion of organic pollutants into bioresources by activated sludge can reduce the carbon dioxide emission of wastewater treatment plants. Identification of new high value-added biopolymers produced by activated sludge is beneficial to recover bioresources. Cyanophycin granule polypeptide (CGP), first discovered in cyanobacteria, has unique chemical and material properties suitable for industrial food, medicine, cosmetics, water treatment, and agriculture applications. Here, we revealed for the first time that activated sludge has a remarkable ability to produce CGP. These findings could further facilitate the conversion of wastewater treatment plants into resource recycling plants.

Sustainable Production of the Cyanophycin Biopolymer in Tobacco in the Greenhouse and Field.

The production of biodegradable polymers as coproducts of other commercially relevant plant components can be a sustainable strategy to decrease the carbon footprint and increase the commercial value of a plant. The biodegradable polymer cyanophycin granular polypeptide (CGP) was expressed in the leaves of a commercial tobacco variety, whose seeds can serve as a source for biofuel and feed. In T0 generation in the greenhouse, up to 11% of the leaf dry weight corresponded to the CGP. In T1 generation, the maximum content decreased to approximately 4% dw, both in the greenhouse and first field trial. In the field, a maximum harvest of 4 g CGP/plant could be obtained. Independent of the CGP content, most transgenic plants exhibited a slight yield penalty in the leaf biomass, especially under stress conditions in greenhouse and field trials. After the harvest, the leaves were either Sun dried or ensiled. The resulting material was used to evaluate the extraction of CGP compared to that in the laboratory protocol. The farm-level analysis indicates that the extraction of CGP from tobacco plants can provide alternative income opportunities for tobacco farmers. The CGP yield/ha indicates that the CGP production in plants can be economically feasible depending on the cultivation and extraction costs. Moreover, we analyzed the consumer acceptance of potential applications associated with GM tobacco in four European countries (Germany, Finland, Italy and the Netherlands) and found unexpectedly high acceptance.

Cyanophycin accumulated under nitrogen-fluctuating and high-nitrogen conditions facilitates the persistent dominance and blooms of Raphidiopsis raciborskii in tropical waters.

Nutrient storage is considered a critical strategy for algal species to adapt to a fluctuating nutrient supply. Luxury phosphorus (P) uptake into storage of polyphosphate extends the duration of cyanobacterial dominance and their blooms under P deficiency. However, it is unclear whether nitrogen (N) storage in the form of cyanophycin supports persistent cyanobacterial dominance or blooms in the tropics where N deficiency commonly occurs in summer. In this study, we examined genes for cyanophycin synthesis and degradation in Raphidiopsis raciborskii, a widespread and dominant cyanobacterium in tropical waters; and detected the cyanophycin accumulation under fluctuating N concentrations and its ecological role in the population dynamics of the species. The genes for cyanophycin synthesis (cphA) and degradation (cphB) were highly conserved in 21 out of 23 Raphidiopsis strains. This suggested that the synthesis and degradation of cyanophycin are evolutionarily conserved to support the proliferation of R. raciborskii in N-fluctuating and/or deficient conditions. Isotope 15N-NaNO3 labeling experiments showed that R. raciborskii QDH7 always commenced to synthesize and accumulate cyanophycin under fluctuating N conditions, regardless of whether exogenous N was deficient. When the NO3--N concentration exceeded 1.2 mg L-1, R. raciborskii synthesized cyanophycin primarily through uptake of 15N-NaNO3. However, when the NO3--N concentration was below 1.0 mg L-1, cyanophycin-based N was derived from unlabeled N2, as evidenced by increased dinitrogenase activity. Cells grown under NO3--N < 1.0 mg L-1 had lower cyanophycin accumulation rates than cells grown under NO3--N > 1.2 mg L-1. Our field investigation in a large tropical reservoir underscored the association between cyanophycin content and the population dynamics of R. raciborskii. The cyanophycin content was high in N-sufficient (NO3--N > 0.45 mg L-1) periods, and decreased in N-deficient summer. In summer, R. raciborskii sustained a relatively high biomass and produced few heterocysts (< 1%). These findings indicated that cyanophycin-released N, rather than fixed N, supported persistent R. raciborskii blooms in N-deficient seasons. Our study suggests that the highly adaptive strategy in a N2-fixing cyanobacterial species makes mitigating its bloom more difficult than previously assumed.

Cyanophycin Modifications-Widening the Application Potential.

A circular bioeconomy approach is essential to slowing down the fearsome ongoing climate change. Replacing polymers derived from fossil fuels with biodegradable biobased polymers is one crucial part of this strategy. Cyanophycin is a polymer consisting of amino acids produced by cyanobacteria with many potential applications. It consists mainly of aspartic acid and arginine, however, its composition may be changed at the production stage depending on the conditions of the polymerization reaction, as well as the characteristics of the enzyme cyanophycin synthetase, which is the key enzyme of catalysis. Cyanophycin synthetases from many sources were expressed heterologously in bacteria, yeast and plants aiming at high yields of the polymer or at introducing different amino acids into the structure. Furthermore, cyanophycin can be modified at the post-production level by chemical and enzymatic methods. In addition, cyanophycin can be combined with other compounds to yield hybrid materials. Although cyanophycin is an attractive polymer for industry, its usage as a sole material remains so far limited. Finding new variants of cyanophycin may bring this polymer closer to real-world applications. This short review summarizes all modifications of cyanophycin and its variants that have been reported within the literature until now, additionally addressing their potential applications.

Incorporation of alternative amino acids into cyanophycin by different cyanophycin synthetases heterologously expressed in Corynebacterium glutamicum.

Cyanophycin (multi-L-arginyl-poly-L-aspartic acid; also known as cyanophycin grana peptide [CGP]) is a biopolymer that could be used in various fields, for example, as a potential precursor for the synthesis of polyaspartic acid or for the production of CGP-derived dipeptides. To extend the applications of this polymer, it is therefore of interest to synthesize CGP with different compositions. A recent re-evaluation of the CGP synthesis in C. glutamicum has shown that C. glutamicum is a potentially interesting microorganism for CGP synthesis with a high content of alternative amino acids. This study shows that the amount of alternative amino acids can be increased by using mutants of C. glutamicum with altered amino acid biosynthesis. With the DM1729 mutant, the lysine content in the polymer could be increased up to 33.5 mol%. Furthermore, an ornithine content of up to 12.6 mol% was achieved with ORN2(Pgdh4). How much water-soluble or insoluble CGP is synthesized is strongly related to the used cyanophycin synthetase. CphADh synthesizes soluble CGP exclusively. However, soluble CGP could also be isolated from cells expressing CphA6308Δ1 or CphA6308Δ1_C595S in addition to insoluble CGP in all examined strains. The point mutation in CphA6308Δ1_C595S partially resulted in a higher lysine content. In addition, the CGP content could be increased to 36% of the cell dry weight under optimizing growth conditions in C. glutamicum ATCC13032. All known alternative major amino acids for CGP synthesis (lysine, ornithine, citrulline, and glutamic acid) could be incorporated into CGP in C. glutamicum.

Direct Delivery of Health Promoting ß-Asp-Arg Dipeptides via Stable Co-expression of Cyanophycin and the Cyanophycinase CphE241 in Tobacco Plants.

Feed supplementation with ß-arginine-aspartate dipeptides (ß-Asp-Arg DP) shows growth promoting effects in feeding trials with fish and might also be beneficial for pig and poultry farming. Currently, these DPs are generated from purified cyanophycin (CGP), with the help of the CGP-degrading enzyme cyanophycinase (CGPase). As alternative to an in vitro production, the DPs might be directly produced in feed crops. We already demonstrated that CGP can be produced in plastids of tobacco and potato, yielding up to 9.4% of the dry weight (DW). We also transiently co-expressed CGPases in the cytosol without degrading CGP in intact cells, while degradation occurs in the homogenized plant tissue. However, transient co-expression is not feasible for field-grown CGP plants, which is necessary for bulk production. In the present study, we proved that stable co-expression of the CGPase CphE241 in CGP-producing tobacco is sufficient to degrade 2.0% CGP/DW nearly completely within 3 h after homogenization of the leaves. In intact senescing leaves, CGP is partially released to the cytosol and degraded into DPs which limits the overall accumulation of CGP but not the level of the stable DPs. Even after 48 h, 54 µmol ß-Asp-Arg DP/g DW could be detected in the extract, which correspond to 1.5% DP/DW and represents 84% of the expected amount. Thus, we developed a system for the production of ß-Asp-Arg DP in field-grown plants.

Wound Healing Attributes of Polyelectrolyte Multilayers Prepared with Multi-l-arginyl-poly-l-aspartate Pairing with Hyaluronic Acid and γ-Polyglutamic Acid.

Biodegradable multi-l-arginyl-poly-l-aspartate (MAPA), more commonly cyanophycin, prepared with recombinant Escherichia coli contains a polyaspartate backbone with lysine and arginine as side chains. Two assemblies of polyelectrolyte multilayers (PEMs) are fabricated at three different concentration ratios of insoluble MAPA (iMAPA) with hyaluronic acid (iMAPA/HA) and with γ-polyglutamic acid (iMAPA/γ-PGA), respectively, utilizing a layer-by-layer approach. Both films with iMAPA and its counterpart, HA or γ-PGA, as the terminal layer are prepared to assess the effect on film roughness, cell growth, and cell migration. iMAPA incorporation is higher for a higher concentration of the anionic polymer due to better charge interaction. The iMAPA/HA films when compared to iMAPA/γ-PGA multilayers show least roughness. The growth rates of L929 fibroblast cells on the PEMs are similar to those on glass substrate, with no supplementary effect of the terminal layer. However, the migration rates of L929 cells increase for all PEMs. γ-PGA incorporated films impart 50% enhancement to the cell migration after 12 h of culture as compared to the untreated glass, and the smooth films containing HA display a maximum 82% improvement. The results present the use of iMAPA to construct a new layer-by-layer system of polyelectrolyte biopolymers with a potential application in wound dressing.

Characterization of an efficient extracellular cyanophycinase and its encoding cphEStrept. gene from Streptomyces pratensis strain YSM.

Until now, no enzymes were described that hydrolyze cyanophycin granular protein (CGP) from a species of the genus Streptomyces. An isolate able to hydrolyze CGP was identified as Streptomyces pratensis strain YSM. The CGPase from S. pratensis strain YSM had an optimum activity at 42 °C and pH 8.5, and was able to degrade CGP at a rate of 12 ±â€¯0.3 µg/mL min. Additionally, this CGPase hydrolyzes water-soluble CGP significantly faster than water-insoluble CGP. The molecular mass of CGPase subunits from S. pratensis strain YSM as determined by SDS-PAGE was about 43 kDa, and the enzyme was entirely inhibited by serine-protease inhibitors. The CGPase coding gene (cphEStrept.) was amplified from genomic DNA using primers designed form consensus sequence of putative CGPase sequences. The cphEStrept. was 1427 bp encoding a CGPase of 420 amino acids that showed about 44% and 22% similarities to CGPase from Pseudomonas anguilliseptica BI and Synechocystis sp. PCC 6803, respectively. The catalytic triad and serine-protease residues (GXSXG) were identified in the CphEStrept. sequence. Dipeptides and tetrapeptides were identified as hydrolysis products. Biotechnological exploitation of S. pratensis strain YSM for CGPase production might have an advantage due to the reduction of separation costs and its ability to degrade CGP in phosphate buffer saline using actively growing or resting cells.

Microbial production of cyanophycin: From enzymes to biopolymers.

Cyanophycin is an attractive biopolymer with chemical and material properties that are suitable for industrial applications in the fields of food, medicine, cosmetics, nutrition, and agriculture. For efficient production of cyanophycin, considerable efforts have been exerted to characterize cyanophycin synthetases (CphAs) and optimize fermentations and downstream processes. In this paper, we review the characteristics of diverse CphAs from cyanobacteria and non-cyanobacteria. Furthermore, strategies for cyanophycin production in microbial strains, including Escherichia coli, Pseudomonas putida, Ralstonia eutropha, Rhizopus oryzae, and Saccharomyces cerevisiae, heterologously expressing different cphA genes are reviewed. Additionally, chemical and material properties of cyanophycin and its derivatives produced through biological or chemical modifications are reviewed in the context of their industrial applications. Finally, future perspectives on microbial production of cyanophycin are provided to improve its cost-effectiveness.

Re-evaluation of cyanophycin synthesis in Corynebacterium glutamicum and incorporation of glutamic acid and lysine into the polymer.

Corynebacterium glutamicum was only examined in the early 2000s as a possible microorganism for the production of the polyamide cyanophycin (multi-L-arginyl-poly-[L-aspartic acid], CGP). CGP is a potential precursor for the synthesis of polyaspartic acid and CGP-derived dipeptides which may be of use in peptide-based clinical diets, as dietary supplements, or in livestock feeds. In the past, C. glutamicum was disregarded for CGP production due to low CGP contents and difficulties in isolating the polymer. However, considering recent advances in CGP research, the capabilities of this organism were revisited. In this study, several cyanophycin synthetases (CphA) as well as expression vectors and cultivation conditions were evaluated. The ability of C. glutamicum to incorporate additional amino acids such as lysine and glutamic acid was also examined. The strains C. glutamicum pVWEx1::cphAΔ1 and C. glutamicum pVWEx1::cphABP1 accumulated up to 14% of their dry weight CGP, including soluble CGP containing more than 40 mol% of the alternative side-chain amino acid lysine. The soluble, lysine-rich form of the polymer was not detected in C. glutamicum in previous studies. Additionally, an incorporation of up to 6 mol% of glutamic acid into the backbone of CGP synthesized by C. glutamicum pVWEx1::cphADh was detected. The strain accumulated up to 17% of its dry weight in soluble CGP. Although glutamic acid had previously been found to replace arginine in the side chain, this is the first time that glutamic acid was found to substitute aspartic acid in the backbone.

Cyanophycin Synthesis Optimizes Nitrogen Utilization in the Unicellular Cyanobacterium Synechocystis sp. Strain PCC 6803.

Cyanophycin is a carbon/nitrogen storage polymer widely distributed in most cyanobacterial strains and in a few heterotrophic bacteria. It is a nonribosomal polypeptide consisting of equimolar amounts of aspartate and arginine. Here, we focused on the physiological function and cell biology of cyanophycin in the unicellular nondiazotrophic cyanobacterium Synechocystis sp. strain PCC 6803. To study the cellular localization of the cyanophycin-synthesizing enzyme CphA during cyanophycin synthesis and degradation, we fused it to green fluorescent protein. When CphA was inactive, it localized diffusely in the cytoplasm. When cyanophycin synthesis was triggered, CphA first aggregated into foci and later localized on the surface of cyanophycin granules. In the corresponding cell extracts, localization of CphA on the cyanophycin granule surface required Mg2+ During cyanophycin degradation, CphA dissociated from the granule surface and returned to its inactive form in the cytoplasm. To investigate the physiological role of cyanophycin, we compared wild-type cells with a CphA-deficient mutant. Under standard laboratory conditions, the ability to synthesize cyanophycin did not confer a growth advantage. To mimic the situation in natural habitats, cells were cultured with a fluctuating and limiting nitrogen supplementation and/or day/night cycles. Under all of these conditions, cyanophycin provided a fitness advantage to the wild type over the mutant lacking cyanophycin. During resuscitation from nitrogen starvation, wild-type cells accumulated cyanophycin during the night and used it as an internal nitrogen source during the day. This demonstrates that cyanophycin can be used as a temporary nitrogen storage to uncouple nitrogen assimilation from photosynthesis.IMPORTANCE We clarified the elusive biological function of cyanophycin in the nondiazotrophic cyanobacterium Synechocystis sp. PCC 6803. Cyanophycin is a dynamic carbon/nitrogen storage polymer (multi-arginyl-l-polyaspartate) that is conditionally present in most cyanobacteria and a few heterotrophic bacteria as cellular inclusion granules. Here, we show that the cyanophycin-synthesizing enzyme CphA in the nonactive state localizes diffusely in the cytoplasm. When cyanophycin synthesis is triggered, active CphA first aggregates into foci and then covers the surface of mature cyanophycin granules, which in vitro requires Mg2+ as a cofactor. Cyanophycin accumulation enables Synechocystis sp. to optimize nitrogen assimilation under nitrogen-poor conditions, in particular when the nitrogen supply fluctuates and during day/night cycles, by allowing continuous nitrogen assimilation and storage. Therefore, cyanophycin provides the wild-type cyanobacterium with a clear fitness advantage over non-cyanophycin-producing cells in natural environments with fluctuating nitrogen supply.

Influence of the Membrane Dye R18 and of DMSO on Cell Penetration of Guanidinium-Rich Peptides.

A quantitative analysis by confocal fluorescence microscopy of the entry into HEK293 and MCF-7 cells by fluorescein-labeled octaarginine (1) and by three octa-Adp derivatives (2 - 4, octamers of the ß-Asp-Arg-dipeptide, derived from the biopolymer cyanophycin) is described, including the effects of the membrane dye R18 and of DMSO on cell penetration.

Ruegeria denitrificans sp. nov., a marine bacterium in the family Rhodobacteraceae with the potential ability for cyanophycin synthesis.

Strain CECT 5091T, an aerobic, marine, Gram-reaction- and Gram-stain-negative, chemoheterotrophic bacterium was isolated from oysters harvested off the Spanish Mediterranean coast. Analysis of the 16S rRNA gene sequence placed the strain within the genus Ruegeria, in the family Rhodobacteraceae, with 16S rRNA gene similarities of 98.7, 98.7 and 98.4 % to Ruegeria conchae, Ruegeria atlanticaand Ruegeria arenilitoris, respectively. Average nucleotide identities (ANI) and in silico DNA-DNA hybridization (DDH) were determined, comparing the genome sequence of CECT 5091T with those of the type strains of 12 species of the genus Ruegeria: the values obtained were always below the thresholds (95-96 % ANI, 70 % in silico DDH) used to define genomic species, proving that CECT 5091T represents a novel species of the genus Ruegeria. The strain was slightly halophilic and mesophilic, with optimum growth at 26 °C, pH 7.0 and 3 % salinity, it required sodium and magnesium ions for growth and was able to reduce nitrate to dinitrogen. Carbon sources for growth include some carbohydrates (d-ribose, d-glucose, l-rhamnose, N-acetyl-d-glucosamine) and multiple organic acids and amino acids. The major cellular fatty acid was summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), representing 70 % of the total fatty acids. Carbon monoxide oxidation, cyanophycin synthetic ability and phosphatidylglycerol, diphosphatidylglycerol and phosphatidylcholine production are predicted from genome annotation, while bacteriochlorophyll a production was absent. The DNA G+C content of the genome was 56.7 mol%. We propose the name Ruegeriadenitrificans sp. nov. and strain CECT 5091T (=5OM10T=LMG 29896T) as the type strain for the novel species.