Novel cyanobacterial bioreporters of phosphorus bioavailability based on alkaline phosphatase and phosphate transporter genes of <Emphasis Type="Italic">Anabaena</Emphasis> sp. PCC 7120

There is heterogeneity in the way cyanobacteria respond to P starvation and subsequently how they adapt to environments with low or fluctuating P concentrations. In this study, we have fused the promoterless lux operon luxCDABE to the promoter regions of Anabaena sp. PCC 7120 phoA genes putatively encoding alkaline phosphatases, phoA (all2843) and phoA-like (alr5291) and to the promoter region of one operon putatively encoding a high affinity phosphate transporter pst1 (all4575-4572). The self-bioluminescent strains constructed in this way, Anabaena AP (phoA promoter), Anabaena AP-L (phoA-like promoter), and Anabaena PST (pst1 promoter) have been used to study the expression of these genes in response to P starvation and P re-feeding with inorganic and organic phosphate sources. Our data showed that the pst1 promoter was activated at much higher level than the phoA-like promoter following P starvation; however, we did not observe activation of the phoA promoter. The P re-feeding experiments revealed that both strains, Anabaena (A.) PST and A. AP-L could be used as novel bioreporters of P availability in environmental samples. Both strains were used to estimate bioavailable P in environmental samples (fresh- and wastewaters) with a wide range of soluble P concentrations. The results indicated that most of the P in the water samples was in chemical forms available to the cyanobacterium; however there were some differences in the estimates given by both strains as A. PST appeared to be more adequate for the samples with the lowest P load while A. AP-L gave similar or even higher values of P concentrations than those chemically measured in samples with higher P load.     

o‐ and m‐Benzenedicarbaldehyde

o‐Benzene­dicarb­aldehyde (systematic name: benzene‐1,2‐dicarb­aldehyde), C₈H₆O₂, exhibits a weak intramolecular hydrogen bond between an aldehyde H atom and the O atom of the adjacent aldehyde group, with a C?O distance of 2.852 (2) Å. m‐Benzene­dicarb­aldehyde (systematic name: benzene‐1,3‐dicarb­aldehyde), C₈H₆O₂, occurs as two different isomorphs. In all three crystals, there are intermolecular C—­H?O contacts involving both aldehyde and ring H atoms.     

THE CHEMISTRY AND BIOLOGY OF BACTERIAL LIGHT EMISSION

Abstract—Bioluminescent bacteria may be isolated from sea water, and grown on a medium containing fish (or meat or yeast) extract. Cells harvested at the peak of luminescence can be lysed osmotically, releasing into the medium the soluble enzyme bacterial luciferase, which catalyzes the bioluminescent oxidation of reduced riboflavin 5′-phosphate and long chain aldehyde by molecular oxygen. Luciferase is the simplest possible heterpolymeric protein, with an α (catalytic, 42,000 daltons)-β (regulatory, 37,000 daltons) dimeric structure. Luciferase is not constitutive; it is induced by a substance produced by the bacteria themselves and excreted into the medium. Control also involves repression (glucose) and cyclic nucleotides. Recent work has resulted in the characterization of an intermediate in the light emitting reaction postulated to be luciferase-bound 4a-peroxy-dihydro FMN. The final steps in the in vitro light-emitting reaction involve reaction of this peroxy intermediate with aldehyde in a mixed function oxidase-type reaction, yielding an excited luciferase-flavin and long chain acid. The excited state is postulated to be the luciferase-bound 4a-hydroxy-dihydro-FMN. Although the identity of the in vivo aldehyde, its localization and its metabolism is unknown, studies with mutants which fail to synthesize aldehyde suggest that the 14 carbon fatty acid is a precursor. Moreover, although bacterial luciferase is highly soluble (200 mg ml^-1 in aqueous solution) there is recent evidence from our laboratory and others that its function may involve the cytoplasmic membrane. The function of light emission is of particular interest since a considerable amount of energy is involved; assuming a quantum yield of 10%, the cell foregoes the production of about 60 ATP molecules per photon. A fully induced cell emits about 10⁴ quanta/s and about 20% (!) of the oxygen consumption of the cell has been estimated to go via the light emitting pathway. One function is in light organs of higher organisms, where they occur as symbionts. The inducible (and repressible) nature of the luminescent system may be appreciated in terms of ecological options; the bacteria may be biologically very versatile. Induction by an inducer produced by the bacteria themselves would occur only under conditions where it accumulates, as in a luminous organ of a host. In the open ocean such an accumulation does not occur; the luminous system would thus not be synthesized and energy loss via luminescence is averted, allowing the bacteria to compete in an alternate “life style”.     

Vectors for Glucose-Dependent Protein Expression in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Based on the p426 series of expression vectors developed by Mumberg et al. (Gene 156, 119–122, 1995), we have generated a set of plasmids that allow the glucose-dependent expression of target genes in the yeast, Saccharomyces cerevisiae. The ADH1 promoter in plasmid p426-ADH1 was replaced by the 1-kb 5′-region from either of the following genes: HXK1, YGR243, HXT4 and HXT7. Expression mediated by the respective 5′-regions was monitored with EGFP, yEGFP3-CLN2pest and TurboGFP as marker genes. Fluorescence is induced 2.7-fold using the HXK1, 2.3-fold using the YGR243-, 5-fold using the HXT7- and 12.6-fold using the HXT4 5′-regions upon depletion of glucose to a concentration of <0.5 g/l.     

Dibromo(pyridoxal semicarbazone‐κ3N1,O3,O3′)copper(II)

The title compound, di­bromo(3‐hydroxy‐5‐hydroxy­methyl‐2‐methyl‐4‐pyridine­carbox­aldehyde semicarbazone‐κ³N¹,O³,O^3′)copper(II), [CuBr₂(C₉H₁₂N₄O₃)], consists of discrete complex units with the tridentate pyridoxal semicarbazone ligand as a zwitterion in an almost planar configuration. The Cu^II ions are in a distorted square‐pyramidal coordination, with the equatorial Br atom at a distance of 2.4017 (6) Å and the apical Br atom at a distance of 2.6860 (6) Å.     

Ionic Liquid Promoted One-Pot Synthesis of Furo[2,3-<Emphasis Type="Italic">d</Emphasis>]pyrimidine-2,4(1<Emphasis Type="Italic">H</Emphasis>,3<Emphasis Type="Italic">H</Emphasis>)-diones

Summary. The three-component condensation of aldehyde, N,N′-dimethylbarbituric acid and alkyl or aryl isocyanide afforded the corresponding furo[2,3-d]pyrimidine-2,4(1H,3H)-diones in 1-butyl-3-methylimidazolium bromide as an ionic liquid in high yields at room temperature within several minutes.     

A multi-channel bioluminescent bacterial biosensor for the on-line detection of metals and toxicity. Part II: technical development and proof of concept of the biosensor

This research study deals with the on-line detection of heavy metals and toxicity within the context of environmental pollution monitoring. It describes the construction and the proof of concept of a multi-channel bioluminescent bacterial biosensor in immobilized phase: Lumisens3. This new versatile device, designed for the non-stop analysis of water pollution, enables the insertion of any bioluminescent strains (inducible or constitutive), immobilized in a multi-well removable card. The technical design of Lumisens3 has benefited from both a classical and a robust approach and includes four main parts: (1) a dedicated removable card contains 64 wells, 3 mm in depth, arranged in eight grooves within which bacteria are immobilized, (2) this card is incubated on a Pelletier block with a CCD cooled camera on top for bioluminescence monitoring, (3) a fluidic network feeds the card with the sample to be analyzed and finally (4) a dedicated computer interface, BIOLUX 1.0, controls all the elements of the biosensor, allowing it to operate autonomously. The proof of concept of this biosensor was performed using a set of four bioluminescent bacteria (Escherichia coli DH1 pBzntlux, pBarslux, pBcoplux, and E. coli XL1 pBfiluxCDABE) in the on-line detection of CdCl₂ 0.5 μM and As₂O₃ 5 μM from an influent. When considering metals individually, the “fingerprints” from the biosensor were as expected. However, when metals were mixed together, cross reaction and synergistic effects were detected. This biosensor allowed us to demonstrate the simultaneous on-line cross detection of one or several heavy metals as well as the measurement of the overall toxicity of the sample.     

Bacteriophage-amplified bioluminescent sensing of <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7

Escherichia coli O157:H7 remains a continuous public health threat, appearing in meats, water, fruit juices, milk, cheese, and vegetables, where its ingestion at concentrations of perhaps as low as 10 to 100 organisms can result in potent toxin exposure and severe damage to the lining of the intestine. Abdominal pain and diarrhea develop, which in the very young or elderly can progress towards hemolytic uremic syndrome and kidney failure. To assist in the detection of E. coli O157:H7, a recombinant bacteriophage reporter was developed that uses quorum sensing (luxI/luxR) signaling and luxCDABE-based bioluminescent bioreporter sensing to specifically and autonomously respond to O157:H7 serotype E. coli. The bacteriophage reporter, derived from phage PP01, was tested in artificially contaminated foodstuffs including apple juice, tap water, ground beef, and spinach leaf rinsates. In apple juice, detection of E. coli O157:H7 at original inoculums of 1 CFU mL⁻¹ occurred within approximately 16 h after a 6-h pre-incubation, detection of 1 CFU mL⁻¹ in tap water occurred within approximately 6.5 h after a 6-h pre-incubation, and detection in spinach leaf rinsates using a real-time Xenogen IVIS imaging system resulted in detection of 1 CFU mL⁻¹ within approximately 4 h after a 2-h pre-incubation. Detection in ground beef was not successful, however, presumably due to the natural occurrence of quorum sensing autoinducer (N-3-(oxohexanoyl)-l-homoserine lactone; OHHL), which generated false-positive bioreporter signals in the ground beef samples.     

2‐Hydroxy‐4,6‐dimethoxy‐3‐(3‐methylbutanoyl)benzaldehyde

The structure of the title compound, C₁₄H₁₈O₅, has two independent molecules related by a local noncrystallographic a‐glide plane perpendicular to the b axis. The pseudo‐glide plane shows a discontinuity at z = 0. Both molecules have an intramolecular hydrogen bond between the hydroxy and aldehyde groups. There are stacks of molecules along the a‐axis direction. Neighboring molecules in the stack have an interplanar angle of 1.6 (1)°, interplanar distances ranging between 3.399 (3) and 3.417 (3) Å, and a ring offset of 1.38 (1) Å.     

Are luminescent bacteria suitable for online detection and monitoring of toxic compounds in drinking water and its sources?

Biosensors based on luminescent bacteria may be valuable tools to monitor the chemical quality and safety of surface and drinking water. In this review, an overview is presented of the recombinant strains available that harbour the bacterial luciferase genes luxCDABE, and which may be used in an online biosensor for water quality monitoring. Many bacterial strains have been described for the detection of a broad range of toxicity parameters, including DNA damage, protein damage, membrane damage, oxidative stress, organic pollutants, and heavy metals. Most lux strains have sensitivities with detection limits ranging from milligrams per litre to micrograms per litre, usually with higher sensitivities in compound-specific strains. Although the sensitivity of lux strains can be enhanced by various molecular manipulations, most reported detection thresholds are still too high to detect levels of individual contaminants as they occur nowadays in European drinking waters. However, lux strains sensing specific toxic effects have the advantage of being able to respond to mixtures of contaminants inducing the same effect, and thus could be used as a sensor for the sum effect, including the effect of compounds that are as yet not identified by chemical analysis. An evaluation of the suitability of lux strains for monitoring surface and drinking water is therefore provided.     

3‐Hydro­xy­benz­aldehyde

The title compound, C₇H₆O₂, forms infinite chains where the mol­ecules are hydrogen bonded via the hydroxyl and aldehyde groups, with an O?O distance of 2.719 (3) Å. Interchain interactions are weak. The geometry of the ring differs from the ideal form due to the effect of the substituents. Abinitio (Hartree–Fock self‐consistent field–molecular orbital and density functional theory) calculations for the free mol­ecule reproduce well the observed small distortions of the ring. In the crystal, the geometry deviates from the ideal C_s symmetry of the free mol­ecule, as given by the ab initio calculations. The aldehyde and hydroxyl groups are twisted around the single bonds which join them to the ring as a result of the intermolecular hydrogen‐bond interactions. These are also responsible for an elongation of the hydroxy C—OH bond compared with that calculated for the free mol­ecule.     

Uranium–Carbene–Imido Metalla‐Allenes: Ancillary‐Ligand‐Controlled cis‐/trans‐Isomerisation and Assessment of trans Influence in the R2C=UIV=NR′ Unit (R=Ph2PNSiMe3; R′=CPh3)

Uranium(IV)–carbene–imido complexes [U(BIPM^TMS)(NCPh₃)(κ²‐N,N′‐BIPY)] ( 2 ; BIPM^TMS=C(PPh₂NSiMe₃)₂; BIPY=2,2‐bipyridine) and [U(BIPM^TMS)(NCPh₃)(DMAP)₂] ( 3 ; DMAP=4‐dimethylamino‐pyridine) that contain unprecedented, discrete R₂C=U=NR′ units are reported. These complexes complete the family of E=U=E (E=CR₂, NR, O) metalla‐allenes with feasible first‐row hetero‐element combinations. Intriguingly, 2 and 3 contain cis‐ and trans‐C=U=N units, respectively, representing rare examples of controllable cis/trans isomerisation in f‐block chemistry. This work reveals a clear‐cut example of the trans influence in a mid‐valent uranium system, and thus a strong preference for the cis isomer, which is computed in a co‐ligand‐free truncated model—to isolate the electronic trans influence from steric contributions—to be more stable than the trans isomer by approximately 12 kJ mol^?1 with an isomerisation barrier of approximately 14 kJ mol^?1.     

Living ring‐opening polymerization of ϵ‐caprolactone with Ti alkoxides derived from the Cp2TiCl‐catalyzed SET reduction of aldehydes

A series of substituted benzaldehydes were investigated as initiators for the living ring‐opening polymerization (LROP) of ε‐caprolactone (CL) mediated by titanium alkoxides obtained from the Cp₂TiCl‐catalyzed single electron transfer (SET) reduction of the carbonyl group following the in situ reduction of Cp₂TiCl₂ with Zn. The aldehyde initiation was demonstrated (NMR) by the presence of the initiator derived fragment on the polycaprolactone (PCL) chain end. The effect of the nature of the aldehyde functionality (R‐Ph‐CHO, R = H, Cl, PhCH₂O, NMe₂, CH₃O, NO₂, and CHO), reagent ratios ([CL]/[aldehyde] = 50/1 to 400/1, [aldehyde]/[Cp₂TiCl₂] = 1/1 to 1/4, and [Cp₂TiCl₂]/[Zn] = 1/0.5 to 1/2), and temperature (T = 75–120 °C) was investigated over a wide range of values to reveal a living polymerization in all cases with an optimum observed at 90 °C with typical stoichiometric ratios of [CL]/[aldehyde]/[Cp₂TiCl₂]/[Zn] = 100/1/1/2. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 2869–2877, 2008     

Properties and engineering of a mutantSTA promoter ofSaccharomyces diastaticus

A new allelic variant of theSTA2 gene ofS. diastaticus, designated asSTA2 ^K, was cloned and characterized (1; accompanying paper). An application-oriented analysis of the promoter region ofSTA2 ^K is described, with an emphasis on its peculiar structural feature: A 1.1-kb natural deletion located 189 nucleotides upstream of the translation start codon. The strength of theSTA2 ^K promoter was found comparable to that of known strong constitutive yeast promoters(ADH1, GAPDH). Regulated glucoamylase expression was demonstrated by chimeric promoters, which were constructed by placing theSTA2 ^K promoter under the control of either thePH05 orCYC1 upstream regulatory sequences. On high-copy-number vectors, induction of the UASpho5-STA2^K chimeric promoter by phosphate depletion resulted in a destructive overexpression of the secreted glucoamylase, which completely halted cell growth, and promoted cell decay. In contrast, UAScyc1 was shown to mediate a fine-tuned regulation both by glucose concentration and, indirectly, by starch, the substrate for the glucoamylase to produce glucose.     

Unusual Reactions of (+)‐Car‐2‐ene and (+)‐Car‐3‐ene with Aldehydes on K10 Clay

The reactions of (+)‐car‐2‐ene ( 1 ) and (+)‐car‐3‐ene ( 2 ) with aldehydes in the presence of montmorillonite clay were studied for the first time (Schemes 3 and 5). The major products of these reactions are optically active, substituted hexahydroisobenzofurans, probably formed as a result of an attack of the protonated aldehyde at the cyclopropane ring. Quite unexpectedly, the products are cis‐configured at the ring‐fusion site; the fact was established by means of quantum‐chemical calculations and NMR data. It appeared that the behavior of the 2 : 3 mixture 1 / 2 in reactions with aldehydes in the presence of K10 clay differed substantially from the reactivities of the corresponding individual monoterpenes.     

5,5′‐Di‐tert‐butyl‐2,2′‐di­hydroxy‐3,3′‐methyl­enedibenz­aldehyde and 6,6′‐di‐tert‐butyl‐8,8′‐methyl­ene­bis­(spiro­[4H‐1,3‐benzodioxin‐2,1′‐cyclo­hexane])

Two related compounds containing p‐tert‐butyl‐o‐methyl­ene‐linked phenol or phenol‐derived subunits are described, namely 5,5′‐di‐tert‐butyl‐2,2′‐di­hydroxy‐3,3′‐methyl­ene­di­benz­aldehyde, C₂₃H₂₈O₄, (I), and 6,6′‐di‐tert‐butyl‐8,8′‐methyl­ene­bis­(spiro­[4H‐1,3‐benzo­di­oxin‐2,1′‐cyclo­hexane]), C₃₅H₄₈O₄, (II). Both compounds adopt a `butterfly' shape, with the two phenol or phenol‐derived O atoms in distal positions. Phenol and aldehyde groups in (I) are involved in intramolecular hydrogen bonds and the two dioxin rings in (II) are in distorted half‐chair conformations.     

Molecular properties of the copolymers of <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-diallyl-<Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-dimethylammonium chloride and maleic acid

The hydrodynamic and conformational properties of molecules of poly(N,N-diallyl-N,N-dimethylammonium chloride) and N,N-diallyl-N,N-dimethylammonium chloride-maleic acid copolymers of different compositions in solutions with various ionic-strength and pH values, as well as of the polyelectrolyte complex based on the copolymer with dodecyl sulfate anions in chloroform, are studied. For poly(N,N-diallyl-N,N-dimethylammonium chloride) molecules in a 1 M NaCl solution, the Kuhn segment length and the hydrodynamic diameter of the chain are estimated as A = 3.9 nm and d = 0.48 nm, respectively. In acidic solutions with pH 3.5, the copolymers demonstrate behavior typical for polyelectrolytes. In an alkaline solution with pH 13, when 1 M NaCl is added to the solution of the copolymer containing 29 mol % maleic acid units, there is an antipolyelectrolyte effect that manifests itself as an increase in the intrinsic viscosity of the copolymer and in the hydrodynamic radius of its molecules. It is found that an increase in the fraction of maleic acid units in the copolymer from 12 to 42 mol % brings about a reduction in the equilibrium rigidity of its macromolecules from 4.1 to 2.2 nm. The equilibrium rigidity of polyelectrolyte-complex molecules is higher than that of initial copolymer molecules owing to steric interactions arising between the aliphatic chains of dodecyl sulfate anions. In an electric field, the molecules of the complex are oriented owing to the induced dipole moment resulting from the displacement of dodecyl sulfate anions along the chain contour.     

One‐Pot Synthesis of 5,7,8,9,9a,10‐Hexahydro‐8‐thioxotetrahydropyrido[2,3‐d : 6,5‐d′]dipyrimidine‐2,4,6(1H,3H,5aH)‐triones via a Four‐Component Coupling Reaction of Aldehydes,Amines, Barbituric Acids,and Thiouracil

An efficient one‐pot approach to the synthesis of 5,7,8,9,9a,10‐hexahydro‐8‐thioxopyrido[2,3‐d : 6,5‐d′]dipyrimidine‐2,4,6(1H,3H,5aH)‐triones 5 via a four‐component reaction of an aldehyde 1 , an amine 2 , a barbituric acid 3 , and thiouracil ( 4 ) is reported for the first time. This new multicomponent reaction is accomplished in refluxing EtOH in the presence of tungstophosphoric acid (H₃PW₁₂O₄₀) as a catalyst. A variety of hexahydropyrido[2,3‐d : 6,5‐d′]dipyrimidinetrione derivatives were successfully synthesized in excellent yields with this protocol (Table 2).     

О,О‐Dialkyl‐S‐(1,1‐dimethyl‐2‐oxoethyl)dithiophosphates: Synthesis and Reactions with n‐Nucleophiles

Hydrolysis of the new types of iminium salts was used to synthesize О,О‐dialkyl‐S‐(1,1‐dimethyl‐2‐oxoethyl)dithiophosphates or 2‐dialkoxythiophosphorylthio‐substituted aldehydes with carbon isochain. Reactions of aldehydes with N‐, N,N‐, and O,N‐nucleophiles gave new phosphorylated imines containing an acetal group at different positions, perhydro‐1,3‐diazol and oxazol with the diisopropoxythiophosphorylthio group in a side chain, and hydrazone of this aldehyde and diphenylphosphinylacetic acid hydrazide.