Phosphorus deprivation affects composition and spatial distribution of membrane lipids in legume nodules.

In legumes, symbiotic nitrogen (N) fixation (SNF) occurs in specialized organs called nodules after successful interactions between legume hosts and rhizobia. In a nodule, N-fixing rhizobia are surrounded by symbiosome membranes, through which the exchange of nutrients and ammonium occurs between bacteria and the host legume. Phosphorus (P) is an essential macronutrient, and N2-fixing legumes have a higher requirement for P than legumes grown on mineral N. As in the previous studies, in P deficiency, barrel medic (Medicago truncatula) plants had impaired SNF activity, reduced growth, and accumulated less phosphate in leaves, roots, and nodules compared with the plants grown in P sufficient conditions. Membrane lipids in M. truncatula tissues were assessed using electrospray ionization-mass spectrometry. Galactolipids were found to increase in P deficiency, with declines in phospholipids (PL), especially in leaves. Lower PL losses were found in roots and nodules. Subsequently, matrix-assisted laser desorption/ionization-mass spectrometry imaging was used to spatially map the distribution of the positively charged phosphatidylcholine (PC) species in nodules in both P-replete and P-deficient conditions. Our results reveal heterogeneous distribution of several PC species in nodules, with homogeneous distribution of other PC classes. In P poor conditions, some PC species distributions were observed to change. The results suggest that specific PC species may be differentially important in diverse nodule zones and cell types, and that membrane lipid remodeling during P stress is not uniform across the nodule.

Characterization of the Nit6803 nitrilase homolog from the cyanotroph Pseudomonas fluorescens NCIMB 11764.

We report the purification and characterization of a nitrilase (E.C. 3.5.5.1) (Nit11764) essential for the assimilation of cyanide as the sole nitrogen source by the cyanotroph, Pseudomonas fluorescens NCIMB 11764. Nit11764, is a member of a family of homologous proteins (nitrile_sll0784) for which the genes typically reside in a conserved seven-gene cluster known as Nit1C. The physical properties and substrate specificity of Nit11764 resemble those of Nit6803, the current reference protein for the family, and the only true nitrilase that has been crystallized. The substrate binding pocket of the two enzymes places the substrate in direct proximity to the active site nucleophile (C160) and conserved catalytic triad (Glu44, Lys126). The two enzymes exhibit a similar substrate profile, however, for Nit11764, cinnamonitrile, was found to be an even better substrate than fumaronitrile the best substrate previously identified for Nit6803. A higher affinity for cinnamonitrile (Km 1.27 mM) compared to fumaronitrile (Km 8.57 mM) is consistent with docking studies predicting a more favorable interaction with hydrophobic residues lining the binding pocket. By comparison, 3,4-dimethoxycinnamonitrile was a poorer substrate the substituted methoxyl groups apparently hindering entry into the binding pocket. in situ 1H NMR studies revealed that only one of the two nitrile substituents in the dinitrile, fumaronitrile, was attacked yielding trans-3-cyanoacrylate (plus ammonia) as a product. The essentiality of Nit11764 for cyanotrophy remains uncertain given that cyanide itself is a poor substrate and the catalytic efficiencies for even the best of nitrile substrates (~5 × 103 M-1 s-1) is less than stellar.

Draft Genome Sequence of the Cyanotroph Pseudomonas monteilii BCN3.

We report here the first draft genome of Pseudomonas monteilii BCN3, a cyanotroph isolated from sewage sludge. The genome consists of approximately 6,029,517 bp with a G+C content of 61.89% and 5,369 annotated protein-coding genes.

Linkage of the Nit1C gene cluster to bacterial cyanide assimilation as a nitrogen source.

A genetic linkage between a conserved gene cluster (Nit1C) and the ability of bacteria to utilize cyanide as the sole nitrogen source was demonstrated for nine different bacterial species. These included three strains whose cyanide nutritional ability has formerly been documented (Pseudomonas fluorescens Pf11764, Pseudomonas putida BCN3 and Klebsiella pneumoniae BCN33), and six not previously known to have this ability [Burkholderia (Paraburkholderia) xenovorans LB400, Paraburkholderia phymatum STM815, Paraburkholderia phytofirmans PsJN, Cupriavidus (Ralstonia) eutropha H16, Gluconoacetobacter diazotrophicus PA1 5 and Methylobacterium extorquens AM1]. For all bacteria, growth on or exposure to cyanide led to the induction of the canonical nitrilase (NitC) linked to the gene cluster, and in the case of Pf11764 in particular, transcript levels of cluster genes (nitBCDEFGH) were raised, and a nitC knock-out mutant failed to grow. Further studies demonstrated that the highly conserved nitB gene product was also significantly elevated. Collectively, these findings provide strong evidence for a genetic linkage between Nit1C and bacterial growth on cyanide, supporting use of the term cyanotrophy in describing what may represent a new nutritional paradigm in microbiology. A broader search of Nit1C genes in presently available genomes revealed its presence in 270 different bacteria, all contained within the domain Bacteria, including Gram-positive Firmicutes and Actinobacteria, and Gram-negative Proteobacteria and Cyanobacteria. Absence of the cluster in the Archaea is congruent with events that may have led to the inception of Nit1C occurring coincidentally with the first appearance of cyanogenic species on Earth, dating back 400-500 million years.

Complete Genome Sequence of a Cyanotroph, Pseudomonas fluorescens NCIMB 11764, Employing Single-Molecule Real-Time Technology.

We report here the application of single-molecule real-time sequencing for determining the entire genome structure of the cyanotroph Pseudomonas fluorescens NCIMB 11764.

Quasi-epitaxial growth of [Ru(bpy)3 ](2+) by confinement in clay nanoplatelets yields polarized emission.

A nano confinement strategy is presented to control the spatial orientation and emission polarization of phosphorescent metal complexes. Through nano-confinement of the phosphorescent metal complex [Ru(bpy)3 ](2+) by attaching it to anionic clay nanoplatelets, it is possible to simultaneously lock the spatial orientation of the complex and fix its emission polarization. This quasi-epitaxial approach may provide a future work strategy directed at light emitting diodes and lasers.

In-plane modulus of singular 2:1 clay lamellae applying a simple wrinkling technique.

Knowledge of the mechanical properties of singular clay lamellae is of crucial importance for the optimization of clay-polymer nanocomposites. On the basis of controlled stress release, singular 2:1 clay lamellae show regular wrinkles on a deformable substrate. A subsequent two-dimensional Fourier transformation gives an in-plane modulus of the clay lamella of approximately 150 GPa. Only readily-available topographical atomic force microscopy is required for analysis rendering that fast and facile procedure generally applicable for nanoplatelet characterization.

Clay-based nanocomposite coating for flexible optoelectronics applying commercial polymers.

Transparency, flexibility, and especially ultralow oxygen (OTR) and water vapor (WVTR) transmission rates are the key issues to be addressed for packaging of flexible organic photovoltaics and organic light-emitting diodes. Concomitant optimization of all essential features is still a big challenge. Here we present a thin (1.5 µm), highly transparent, and at the same time flexible nanocomposite coating with an exceptionally low OTR and WVTR (1.0 × 10(-2) cm(3) m(-2) day(-1) bar(-1) and <0.05 g m(-2) day(-1) at 50% RH, respectively). A commercially available polyurethane (Desmodur N 3600 and Desmophen 670 BA, Bayer MaterialScience AG) was filled with a delaminated synthetic layered silicate exhibiting huge aspect ratios of about 25,000. Functional films were prepared by simple doctor-blading a suspension of the matrix and the organophilized clay. This preparation procedure is technically benign, is easy to scale up, and may readily be applied for encapsulation of sensitive flexible electronics.

Nanoplatelets of sodium hectorite showing aspect ratios of ≈20,000 and superior purity.

Applying a combination of melt synthesis followed by long-term annealing a fluorohectorite is obtained which is unique with respect to homogeneity, purity, and particle size. Counterintuitively, the hectorite undergoes a disorder-to-order transition upon swelling to the level of the bilayer hydrate. Alkylammonium-exchanged samples show at any chain length only a single basal spacing corroborating a nicely homogeneous layer charge density. Its intracrystalline reactivity improves greatly upon annealing, making it capable to spontaneously and completely disintegrate into single clay lamellae of 1 nm thickness. Realizing exceptional aspect ratios of around 20,000 upon delamination, this synthetic clay will offer unprecedented potential as functional filler in highly transparent nanocomposites with superior gas barrier and mechanical properties.

Draft genome sequence of the cyanide-utilizing bacterium Pseudomonas fluorescens strain NCIMB 11764.

We report here the 6.97-Mb draft genome sequence of Pseudomonas fluorescens strain NCIMB 11764, which is capable of growth on cyanide as the sole nitrogen source. The draft genome sequence allowed the discovery of several genes implicated in enzymatic cyanide turnover and provided additional information contributing to a better understanding of this organism's unique cyanotrophic ability. This is the first sequenced genome of a cyanide-assimilating bacterium.

How to maximize the aspect ratio of clay nanoplatelets.

Melt-synthesis yielded lithium-fluorohectorites (Li-hect(x)) with variable layer charge (x = 0.4, 0.6, 0.8, 1.0). Counterintuitively, both tactoid diameter and intracrystalline reactivity increased concomitantly with increasing layer charge. This way hectorites with very large diameters were obtained (d(50%) = 48 µm) that nevertheless still spontaneously delaminate when immersed into water and nano-platelets with huge aspect ratios (>10 000) are formed. Melt-synthesis of Li-hect(x) has been performed in an open glassy carbon crucible allowing for easy scaling to batches of 500 g. These unprecedented huge aspect ratio fillers promise great potential for flame retardants and barrier applications.

UV-cured, flexible, and transparent nanocomposite coating with remarkable oxygen barrier.

A polymer-layered silicate nanocomposite coating is prepared by combining a novel synthetic lithium-hectorite and an UV-curable, cationic polyurethane. Oxygen transmission measurements clearly indicate the supremacy of the lithium-hectorite as compared to a standard montmorillonite. In addition, a very high degree of optical transparency of the nanocomposite coating is achieved, rendering this material highly interesting for flexible packaging and encapsulation applications.

Homogeneous precipitation by formamide hydrolysis: synthesis, reversible hydration, and aqueous exfoliation of the layered double hydroxide (LDH) of Ni and Al.

Homogenous precipitation by formamide hydrolysis results in the formation of a formate-intercalated layered double hydroxide (LDH) of Ni(II) and Al(III). The formate-LDH is sensitive to the atmospheric humidity and reversibly exchanges its intercalated water with atmospheric moisture. The hydration/dehydration cycle is complete within a narrow range of 0-30% relative humidity with significant hysteresis and involves a randomly interstratified intermediate phase. When immersed in water, the formate ion grows its hydration sphere (osmotic swelling), eventually leading to the exfoliation of the metal hydroxide layers into lamellar particles having in-plane dimensions of 100-200 nm and a thickness of 9-12 nm. These nanoplatelets restack to thicker tactoids again upon evaporation of the dispersion. The intercalated formate ion can be exchanged with nitrate ions in solution but not with iodide ions. These observations have implications for many applications of LDHs in the area of carbon dioxide sorption and catalysis.

Tailoring shear-stiff, mica-like nanoplatelets.

This work introduces a novel facile method to produce shear-stiff, mica-like nanoplatelets by efficient exfoliation. The essence of this procedure is the nonreversible alteration of the interlamellar reactivity of a synthetic fluorohectorite by simple cation exchange. The possibility of switching from highly hydrated to collapsed interlayers permits a highly efficient exfoliation in the swollen state while providing shear-stiffness in the collapsed state. This method restricts cation exchange in the mica-like nanoplatelets to the outer surfaces, which represents a significant advantage for use in nanocomposites as compared to conventional organoclays which contain up to 40%/wt of organocations. It is expected that this new type of rigid, shear-stiff, clay-based nanoplatelets will be superior for reinforcement when used in composite materials like polymer layered silicate nanocomposites or artificial nacre.

Bacterial cyanide oxygenase is a suite of enzymes catalyzing the scavenging and adventitious utilization of cyanide as a nitrogenous growth substrate.

Cyanide oxygenase (CNO) from Pseudomonas fluorescens NCIMB 11764 catalyzes the pterin-dependent oxygenolytic cleavage of cyanide (CN) to formic acid and ammonia. CNO was resolved into four protein components (P1 to P4), each of which along with a source of pterin cofactor was obligately required for CNO activity. Component P1 was characterized as a multimeric 230-kDa flavoprotein exhibiting the properties of a peroxide-forming NADH oxidase (oxidoreductase) (Nox). P2 consisted of a 49.7-kDa homodimer that showed 100% amino acid identity at its N terminus to NADH peroxidase (Npx) from Enterococcus faecalis. Enzyme assays further confirmed the identities of both Nox and Npx enzymes (specific activity, 1 U/mg). P3 was characterized as a large oligomeric protein (approximately 300 kDa) that exhibited cyanide dihydratase (CynD) activity (specific activity, 100 U/mg). Two polypeptides of 38 kDa and 43 kDa were each detected in the isolated enzyme, the former believed to confer catalytic activity based on its similar size to other CynD enzymes. The amino acid sequence of an internal peptide of the 43-kDa protein was 100% identical to bacterial elongation factor Tu, suggesting a role as a possible chaperone in the assembly of CynD or a multienzyme CNO complex. The remaining P4 component consisted of a 28.9-kDa homodimer and was identified as carbonic anhydrase (specific activity, 2,000 U/mg). While the function of participating pterin and the roles of Nox, Npx, CynD, and CA in the CNO-catalyzed scavenging of CN remain to be determined, this is the first report describing the collective involvement of these four enzymes in the metabolic detoxification and utilization of CN as a bacterial nitrogenous growth substrate.

The transcriptional regulator AlgR controls cyanide production in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic lung infections in cystic fibrosis (CF) patients. One characteristic of P. aeruginosa CF isolates is the overproduction of the exopolysaccharide alginate, controlled by AlgR. Transcriptional profiling analyses comparing mucoid P. aeruginosa strains to their isogenic algR deletion strains showed that the transcription of cyanide-synthesizing genes (hcnAB) was approximately 3-fold lower in the algR mutants. S1 nuclease protection assays corroborated these findings, indicating that AlgR activates hcnA transcription in mucoid P. aeruginosa. Quantification of hydrogen cyanide (HCN) production from laboratory isolates revealed that mucoid laboratory strains made sevenfold more HCN than their nonmucoid parental strains. In addition, comparison of laboratory and clinically derived nonmucoid strains revealed that HCN was fivefold higher in the nonmucoid CF isolates. Moreover, the average amount of cyanide produced by mucoid clinical isolates was 4.7 +/- 0.85 micromol of HCN/mg of protein versus 2.4 +/- 0.40 micromol of HCN/mg of protein for nonmucoid strains from a survey conducted with 41 P. aeruginosa CF isolates from 24 patients. Our data indicate that (i) mucoid P. aeruginosa regardless of their origin (laboratory or clinically derived) produce more cyanide than their nonmucoid counterparts, (ii) AlgR regulates HCN production in P. aeruginosa, and (iii) P. aeruginosa CF isolates are more hypercyanogenic than nonmucoid laboratory strains. Taken together, cyanide production may be a relevant virulence factor in CF lung disease, the production of which is regulated, in part, by AlgR.

Enzymatic assimilation of cyanide via pterin-dependent oxygenolytic cleavage to ammonia and formate in Pseudomonas fluorescens NCIMB 11764.

Utilization of cyanide as a nitrogen source by Pseudomonas fluorescens NCIMB 11764 occurs via oxidative conversion to carbon dioxide and ammonia, with the latter compound satisfying the nitrogen requirement. Substrate attack is initiated by cyanide oxygenase (CNO), which has been shown previously to have properties of a pterin-dependent hydroxylase. CNO was purified 71-fold and catalyzed the quantitative conversion of cyanide supplied at micromolar concentrations (10 to 50 micro M) to formate and ammonia. The specific activity of the partially purified enzyme was approximately 500 mU/mg of protein. The pterin requirement for activity could be satisfied by supplying either the fully (tetrahydro) or partially (dihydro) reduced forms of various pterin compounds at catalytic concentrations (0.5 micro M). These compounds included, for example, biopterin, monapterin, and neopterin, all of which were also identified in cell extracts. Substrate conversion was accompanied by the consumption of 1 and 2 molar equivalents of molecular oxygen and NADH, respectively. When coupled with formate dehydrogenase, the complete enzymatic system for cyanide oxidation to carbon dioxide and ammonia was reconstituted and displayed an overall reaction stoichiometry of 1:1:1 for cyanide, O(2), and NADH consumed. Cyanide was also attacked by CNO at a higher concentration (1 mM), but in this case formamide accumulated as the major reaction product (formamide/formate ratio, 0.6:0.3) and was not further degraded. A complex reaction mechanism involving the production of isocyanate as a potential CNO monooxygenation product is proposed. Subsequent reduction of isocyanate to formamide, whose hydrolysis occurs as a CNO-bound intermediate, is further envisioned. To our knowledge, this is the first report of enzymatic conversion of cyanide to formate and ammonia by a pterin-dependent oxygenative mechanism.