Transcriptomics-guided identification of an algicidal protease of the marine bacterium Kordia algicida OT-1.

In recent years, interest in algicidal bacteria has risen due to their ecological importance and their potential as biotic regulators of harmful algal blooms. Algicidal bacteria shape the plankton communities of the oceans by inhibiting or lysing microalgae and by consuming the released nutrients. Kordia algicida strain OT-1 is a model marine algicidal bacterium that was isolated from a bloom of the diatom Skeletonema costatum. Previous work has suggested that algicidal activity is mediated by secreted proteases. Here, we utilize a transcriptomics-guided approach to identify the serine protease gene KAOT1_RS09515, hereby named alpA1 as a key element in the algicidal activity of K. algicida. The protease AlpA1 was expressed and purified from a heterologous host and used in in vitro bioassays to validate its activity. We also show that K. algicida is the only algicidal species within a group of four members of the Kordia genus. The identification of this algicidal protease opens the possibility of real-time monitoring of the ecological impact of algicidal bacteria in natural phytoplankton blooms.

The Algicidal Bacterium Kordia algicida Shapes a Natural Plankton Community.

Plankton communities consist of complex microbial consortia that change over time. These fluctuations can be only partially explained by limiting resources. Biotic factors such as herbivores and pathogens also contribute to the control of algal blooms. Here we address the effects of algicidal bacteria on a natural plankton community in an indoor enclosure experiment. The algicidal bacteria, introduced into plankton taken directly from the North Sea during a diatom bloom, caused the rapid decline of the bloom-forming Chaetoceros socialis within only 1 day. The haptophyte Phaeocystis, in contrast, is resistant to the lytic bacteria and could benefit from the removal of the competitor, as indicated by an onset of a bloom in the treated enclosures. This cascading effect caused by the bacterial pathogen accelerated the succession of Phaeocystis, which bloomed with a delay of only several weeks in the in situ waters at Helgoland Roads in the North Sea. The algicidal bacteria can thus modulate the community within the limits of the abiotic and biotic conditions of the local environment. Implications of our findings for plankton ecosystem functioning are discussed.IMPORTANCE Plankton communities change on a seasonal basis in temperate systems, with distinct succession patterns; this is mainly due to algal species that have their optimal timing relative to environmental conditions. We know that bacterial populations are also instrumental in the decay and termination of phytoplankton blooms. Here, we describe algicidal bacteria as modulators of this important species succession. Upon treatment of a natural plankton consortium with an algicidal bacterium, we observed a strong shift in the phytoplankton community structure, compared to controls, resulting in formation of a succeeding Phaeocystis bloom. Blooms of this alga have a substantial impact on global biogeochemical and ecological cycles, as they are responsible for a substantial proportion of primary production during spring in the North Sea. We propose that one of the key factors influencing such community shifts may be algicidal bacteria.

Algal Oxylipins Mediate the Resistance of Diatoms against Algicidal Bacteria.

Algicidal bacteria can lyse microalgal blooms and trigger shifts within plankton communities. Resistant algal species can escape lysis, and have the opportunity to dominate the phytoplankton after a bacterial infection. Despite their important function in ecosystem regulation, little is known about mechanisms of resistance. Here, we show that the diatom Chaetoceros didymus releases eicosanoid oxylipins into the medium, and that the lytic algicidal bacterium, Kordia algicida, induces the production of several wound-activated oxylipins in this resistant diatom. Neither releases nor an induction occurs in the susceptible diatom Skeletonema costatum that is lysed by the bacterium within a few days. Among the upregulated oxylipins, hydroxylated eicosapentaenoic acids (HEPEs) dominate. However, also, resolvins, known lipid mediators in mammals, increase upon exposure of the algae to the algicidal bacteria. The prevailing hydroxylated fatty acid, 15-HEPE, significantly inhibits growth of K. algicida at a concentration of approximately 1 µM. The oxylipin production may represent an independent line of defense of the resistant alga, acting in addition to the previously reported upregulation of proteases.

The metabolite dimethylsulfoxonium propionate extends the marine organosulfur cycle.

Algae produce massive amounts of dimethylsulfoniopropionate (DMSP), which fuel the organosulfur cycle1,2. On a global scale, several petagrams of this sulfur species are produced annually, thereby driving fundamental processes and the marine food web1. An important DMSP transformation product is dimethylsulfide, which can be either emitted to the atmosphere3,4 or oxidized to dimethylsulfoxide (DMSO) and other products5. Here we report the discovery of a structurally unusual metabolite, dimethylsulfoxonium propionate (DMSOP), that is synthesized by several DMSP-producing microalgae and marine bacteria. As with DMSP, DMSOP is a low-molecular-weight zwitterionic metabolite that carries both a positively and a negatively charged functional group. Isotope labelling studies demonstrate that DMSOP is produced from DMSP, and is readily metabolized to DMSO by marine bacteria. DMSOP was found in near nanomolar amounts in field samples and in algal culture media, and thus represents-to our knowledge-a previously undescribed biogenic source for DMSO in the marine environment. The estimated annual oceanic production of oxidized sulfur from this pathway is in the teragram range, similar to the calculated dimethylsulfide flux to the atmosphere3. This sulfoxonium metabolite is therefore a key metabolite of a previously undescribed pathway in the marine sulfur cycle. These findings highlight the importance of DMSOP in the marine organosulfur cycle.

Kinetic studies on strand displacement in de novo designed parallel heterodimeric coiled coils.

Among the protein folding motifs, which are accessible by de novo design, the parallel heterodimeric coiled coil is most frequently used in bioinspired applications and chemical biology in general. This is due to the straightforward sequence-to-structure relationships, which it has in common with all coiled-coil motifs, and the heterospecificity, which allows control of association. Whereas much focus was laid on designing orthogonal coiled coils, systematic studies on controlling association, for instance by strand displacement, are rare. As a contribution to the design of dynamic coiled-coil-based systems, we studied the strand-displacement mechanism in obligate heterodimeric coiled coils to investigate the suitability of the dissociation constants (KD) as parameters for the prediction of the outcome of strand-displacement reactions. We use two sets of heterodimeric coiled coils, the previously reported N-A x B y and the newly characterized C-A x B y . Both comprise KD values in the µM to sub-nM regime. Strand displacement is explored by CD titration and a FRET-based kinetic assay and is proved to be an equilibrium reaction with half-lifes from a few seconds up to minutes. We could fit the displacement data by a competitive binding model, giving rate constants and overall affinities of the underlying association and dissociation reactions. The overall affinities correlate well with the ratios of KD values determined by CD-thermal denaturation experiments and, hence, support the dissociative mechanism of strand displacement in heterodimeric coiled coils. From the results of more than 100 different displacement reactions we are able to classify three categories of overall affinities, which allow for easy prediction of the equilibrium of strand displacement in two competing heterodimeric coiled coils.

Carbon Nanotubes Encapsulated in Coiled-Coil Peptide Barrels.

Specific functionalization of 1D nanomaterials such as near infrared (nIR) fluorescent single-walled carbon nanotubes (SWCNTs) is essential for colloidal stability and tailoring of their interactions with the environment. Here, we show that de novo designed alpha-helical coiled-coil peptide barrels (αHBs) with appropriate pores encapsulate and solubilize SWCNTs. In contrast, barrels without or with narrow pores showed a much smaller efficiency. Absorption/fluorescence spectroscopy and atomic force microscopy indicate that the SWCNTs are incorporated into the αHB's pore. The resulting hybrid SWCNT@αHBs display periodic surface coverage with a 40 nm pitch and remain fluorescent in the nIR. This approach presents a novel concept to encapsulate, discriminate and functionalize SWCNTs non-covalently with peptides and holds great promise for future applications in bioimaging or drug delivery.

Strategies and ecological roles of algicidal bacteria.

In both freshwater and marine ecosystems, phytoplankton are the most dominant primary producers, contributing substantially to aquatic food webs. Algicidal bacteria that can associate to microalgae from the phytoplankton have the capability to control the proliferation and even to lyse them. These bacteria thus play an important role in shaping species composition in pelagic environments. In this review, we discuss and categorise strategies used by algicidal bacteria for the attack on microalgae. We highlight the complex regulation of algicidal activity and defence responses that govern alga-bacteria interactions. We also discuss how algicidal bacteria impact algal physiology and metabolism and survey the existing algicidal metabolites and enzymes. The review illustrates that the ecological role of algicidal bacteria is not yet fully understood and critically discusses the challenges in obtaining ecologically relevant data.

Development of New Supramolecular Lyotropic Liquid Crystals and Their Application as Alignment Media for Organic Compounds.

Most alignment media for the residual dipolar coupling (RDC) based molecular structure determination of small organic compounds consist of rod-like polymers dissolved in organic solvents or of swollen cross-linked polymer gels. Thus far, the synthesis of polymer-based alignment media has been a challenging process, which is often followed by a time-consuming sample preparation. We herein propose the use of non-polymeric alignment media based on benzenetricarboxamides (BTAs), which self-assemble into rod-like supramolecules. Our newly found supramolecular lyotropic liquid crystals (LLCs) are studied in terms of their LLC properties and their suitability as alignment media in NMR spectroscopy. Scalable enantiodifferentiating properties are introduced through a sergeant-and-soldier principle by blending achiral with chiral substituted BTAs.

Arsenite stimulates glutathione export and glycolytic flux in viable primary rat brain astrocytes.

Intoxication with inorganic arsenicals leads to neuropathies and impaired cognitive functions. However, little is known so far on the cellular targets that are involved in the adverse effects of arsenite to brain cells. To test whether arsenite may affect neural glucose and glutathione (GSH) metabolism, primary astrocyte cultures from rat brain were used as a model system. Exposure of cultured astrocytes to arsenite in concentrations of up to 0.3mM did not compromise cell viability during incubations for up to 6h, while 1mM arsenite damaged the cells already within 2h after application. Determination of cellular arsenic contents of astrocytes that had been incubated for 2h with arsenite revealed an almost linear concentration-dependent increase in the specific cellular arsenic content. Exposure of astrocytes to arsenite stimulated the export of GSH and accelerated the cellular glucose consumption and lactate production in a time- and concentration-dependent manner. Half-maximal stimulation of GSH export and glycolytic flux were observed for arsenite in concentrations of 0.1mM and 0.3mM, respectively. The arsenite-induced stimulation of both processes was abolished upon removal of extracellular arsenite. The strong stimulation of GSH export by arsenite was prevented by MK571, an inhibitor of the multidrug resistance protein 1, suggesting that this transporter mediates the accelerated GSH export. In addition, presence of MK571 significantly increased the specific cellular arsenic content, suggesting that Mrp1 may also be involved in arsenic export from astrocytes. The data observed suggest that alterations in glucose and GSH metabolism may contribute to the reported adverse neural consequences of intoxication with arsenite.

Arsenate accumulation and arsenate-induced glutathione export in astrocyte-rich primary cultures.

Arsenate is a toxic compound that has been connected with neuropathies and impaired cognitive functions. To test whether arsenate affects the viability and the GSH metabolism of brain astrocytes, we have used primary astrocyte cultures as model system. Incubation of astrocytes for 2h with arsenate in concentrations of up to 10mM caused an almost linear increase in the cellular arsenic content, but did not acutely compromise cell viability. The presence of moderate concentrations of arsenate caused a time- and concentration-dependent loss of GSH from viable astrocytes which was accompanied by a matching increase in the extracellular GSH content. Half-maximal effects were observed for arsenate in a concentration of about 0.3 mM. The arsenate-induced stimulated GSH export from astrocytes was prevented by MK571, an inhibitor of the multidrug resistance protein 1. Exposure of astrocytes to arsenite increased the specific cellular arsenic content and stimulated GSH export to values that were similar to those observed for arsenate-treated cells, while dimethylarsinic acid was less efficiently accumulated by the cells and did not modulate cellular and extracellular GSH levels. The observed strong stimulation of GSH export from astrocytes by arsenate suggests that disturbances of the astrocytic GSH metabolism may contribute to the observed arsenic-induced neurotoxicity.

Borane and borohydride complexes of the rare-earth elements: synthesis, structures, and butadiene polymerization catalysis.

The reaction of potassium 2,5-bis[N-(2,6-diisopropylphenyl)iminomethyl]pyrrolyl [(dip(2)-pyr)K] with the borohydrides of the larger rare-earth metals, [Ln(BH(4))(3)(thf)(3)] (Ln=La, Nd), afforded the expected products [Ln(BH(4))(2)(dip(2)-pyr)(thf)(2)]. As usual, the trisborohydrides reacted like pseudohalide compounds forming KBH(4) as a by-product. To compare the reactivity with the analogous halides, the dimeric neodymium complex [NdCl(2)(dip(2)-pyr)(thf)](2) was prepared by reaction of [(dip(2)-pyr)K] with anhydrous NdCl(3). Reaction of [(dip(2)-pyr)K] with the borohydrides of the smaller rare-earth metals, [Sc(BH(4))(3)(thf)(2)] and [Lu(BH(4))(3)(thf)(3)], resulted in a redox reaction of the BH(4) (-) group with one of the Schiff base functions of the ligand. In the resulting products, [Ln(BH(4)){(dip)(dip-BH(3))-pyr}(thf)(2)] (Ln=Sc, Lu), a dinegatively charged ligand with a new amido function, a Schiff base, and the pyrrolyl function is bound to the metal atom. The by-product of the reaction of the BH(4) (-) anion with the Schiff base function (a BH(3) molecule) is trapped in a unique reaction mode in the coordination sphere of the metal complex. The BH(3) molecule coordinates in an eta(2) fashion to the metal atom. The rare-earth-metal atoms are surrounded by the eta(2)-coordinated BH(3) molecule, the eta(3)-coordinated BH(4) (-) anion, two THF molecules, and the nitrogen atoms from the Schiff base and the pyrrolyl function. All new compounds were characterized by single-crystal X-ray diffraction. Low-temperature X-ray diffraction data at 6 K were collected to locate the hydrogen atoms of [Lu(BH(4)){(dip)(dip-BH(3))-pyr}(thf)(2)]. The (DIP(2)-pyr)(-) borohydride and chloride complexes of neodymium, [Nd(BH(4))(2)(dip(2)-pyr)(thf)(2)] and [NdCl(2)(dip(2)-pyr)(thf)](2), were also used as Ziegler-Natta catalysts for the polymerization of 1,3-butadiene to yield poly(cis-1,4-butadiene). Very high activities and good cis selectivities were observed by using each of these complexes as a catalyst in the presence of various cocatalyst mixtures.

Unusual reactivity of lanthanide borohydride complexes leading to a borane complex.

Depending on the ionic radius of the central metal atom the BH(4)(-) group, which usually reacts in lanthanide chemistry like a pseudo halide, can be involved in redox chemistry and the resulting product contains an N-BH(3) unit, which binds in an unusual eta(2)-fashion onto the metal atom.

Iron(II) complexes for the efficient catalytic asymmetric transfer hydrogenation of ketones.

Iron(II) carbonyl compounds of the type trans-[Fe(NCMe)(CO)(P-N-N-P)][BF(4)](2) bearing the ethylenediamine-derived diiminodiphosphine ligands (R,R)- or (S,S)-1,2-diphenyl-1,2-diaminoethane were synthesized and characterized, including by their crystal structures. The new complexes are suitable precatalysts for the transfer hydrogenation of ketones at room temperature, giving turnover frequencies of up to 2600 h(-1) with low catalyst loadings (0.025-0.17%). Screening experiments showed that the precatalysts are able to produce alcohols from a wide range of simple ketones. For sterically demanding prochiral ketones, excellent enantioselectivities were obtained (up to 96% ee).

Synthesis and characterization of iron(II) complexes with tetradentate diiminodiphosphine or diaminodiphosphine ligands as precatalysts for the hydrogenation of acetophenone.

Six complexes of the type trans-[Fe(NCMe)2(P-N-N-P)]X2 (X = BF4(-), B{Ar(f)}4(-)) (Ar(f) = 3,5-(CF3)2C6H3) containing diiminodiphosphine ligands and the complexes trans-[Fe(NCMe)2(P-NH-NH-P)][BF4]2 with a diaminodiphosphine ligand were obtained by the reaction of Fe(II) salts with achiral and chiral P-N-N-P or P-NH-NH-P ligands, respectively, in acetonitrile at ambient temperature. The P-N-N-P ligands are derived from reaction of ortho-diphenylphosphinobenzaldehyde with the diamines 1,2-ethylenediamine, 1,3-propylenediamine, (S,S)-1,2-disopropyl-1,2-diaminoethane, and (R,R)-1,2-diphenyl-1,2-diaminoethane. Some complexes could also be obtained for the first time in a one-pot template synthesis under mild reaction conditions. Single crystal X-ray diffraction studies of the complexes revealed a trans distorted octahedral structure around the iron. The iPr or Ph substituents on the diamine were found to be axial in the five-membered Fe-N-CHR-CHR-N- ring of the chiral P-N-N-P ligands. A steric clash between the imine hydrogen and the substituent probably determines this stereochemistry. The diaminodiphosphine complex has longer Fe-N and Fe-P bonds than the analogous diiminodiphosphine complex. The new iron compounds were used as precatalysts for the hydrogenation of acetophenone. The complexes without axial substituents on the diamine had moderate catalytic activity while that with axial Ph substituents had low activity but fair (61%) enantioselectivity for the asymmetric hydrogenation of acetophenone. The fact that the diaminodiphosphine complex has a slightly higher activity than the corresponding diiminodiphosphine complex suggests that hydrogenation of the imine groups in the P-N-N-P ligand may be important for catalyst activation. Evidence is provided, including the first density-functional theory calculations on iron-catalyzed outer-sphere ketone hydrogenation, that the mechanism is similar to that of ruthenium analogues.

Chiral bridged aminotroponiminate complexes of the lanthanides.

The enantiomerically pure bridged aminotroponimines, S,S- and R,R-H2{(iPrATI)2diph}, in which two amino-isopropyl-troponimine moieties are linked by 1,2-diamino-1,2-diphenylethane, were deprotonated with nBuLi to give the corresponding dilithium salts [{Li(THF)}2{(S,S)-(iPrATI)2diph)}] (1a) and [{Li(THF)}2{(R,R)-(iPrATI)2diph)}] (1b). The potassium salts [{K(THF)2}2{(S,S)-(iPrATI)2diph}] (2a) and [{K(THF)2}2{(R,R)-(iPrATI)2diph}] (2b) were obtained by a deprotonation reaction with KH. Transmetallation of 2a and 2b with anhydrous lanthanide trichlorides led to [(S,S)-{(iPrATI)2diph}LnCl(THF)] (Ln = Ho (3a), Er (4a)) and [(R,R)-{(iPrATI)2diph}LnCl(THF)] (Ln = Ho (3b), Er (4b), Yb (5b)), respectively. The corresponding Yb complex [(S,S)-{(iPrATI)2diph}YbCl(THF)] (5a) was obtained by treatment of 1a with YbCl3 at elevated temperature. Performing the same reaction at room temperature results in the metallate complex [(S,S)-{(iPrATI)2diph}YbCl2][Li(THF)4] (6). Reaction of NaC5H5 with afforded [(S,S)-{(iPrATI)2diph}Yb(eta5-C5H5)(THF)] (7). The structures of 1a, 3a, 4a, 5a, 5b, 6, and 7 were confirmed by single crystal X-ray diffraction in the solid-state.

Intramolecular hydroamination with homogeneous zinc catalysts: evaluation of substituent effects in N,N'-disubstituted aminotroponiminate zinc complexes.

A series of symmetrical and unsymmetrical N,N'-disubstituted aminotroponimines (ATIHs) have been prepared. Substituents ranging from linear to cyclic alkyl groups, chelating ethers, and aryl groups were employed. The corresponding aminotroponiminate zinc complexes were then synthesized and characterized by a number of techniques, including by X-ray crystallography. Herein we report on the investigations into their activity in the intramolecular hydroamination of nonactivated alkenes. We also demonstrate that complexes bearing ligands with cyclic alkyl groups show superior activity in a number of selected reactions with functionalized aminoalkenes.