Challenges in predicting ΔrxnG in solution: The chelate effect.

Gibbs energies for reactions involving aqueous ions are challenging to predict due to the large solvation energies of such ions. A stringent test would be the ab initio reproduction of the aqueous-phase chelate effect, an entropic effect in reactions of very small enthalpy changes. This paper examines what is required to achieve such a reproduction for the paradigmatic reaction M(NH3)4 2+ + 2 en → M(en)2 2+ + 4 NH3 (en = 1,2-ethylenediamine), for which ΔrxnG* and ΔrxnH* are -2.3 and +1.6 kcal mol-1, respectively, if M = Zn. Explicit solvation via simulation was avoided in order to allow sufficiently accurate electronic structure models; this required the use of continuum solvation models (CSMs), and a great deal of effort was made in attempting to lower the relative errors of ΔsolvG*[M(NH3)4 2+] vs ΔsolvG*[M(en)2 2+] from the CSMs available in Gaussian software. CSMs in ADF and JDFTx software were also tested. A uniform 2.2 kcal mol-1 accuracy in ΔrxnG* for all three metal-atom choices M = {Zn, Cd, Hg} was eventually achieved, but not from any of the known CSMs tested, nor from cavity size reoptimization, nor from semicontinuum modeling: post facto solvation energy corrections [one per solute type, NH3, en, M(NH3)4 2+, M(en)2 2+] were needed. It is hoped that this study will aid (and encourage) further CSM development for coordination-complex ions.

Comment on "Local, solvation pressures and conformational changes in ethylenediamine aqueous solutions probed using Raman spectroscopy" by M. Cáceres, A. Lobato, N. J. Mendoza, L. J. Bonales and V. G. Baonza, Phys. Chem. Chem. Phys., 2016, 18, 26192.

Liquid ethylenediamine contains both trans and gauche conformers, but there are conflicting claims in the literature that the relative abundance of gauche conformers is either completely quenched (J. Mol. Struct., 1999, 482, 639-646) or enhanced (Phys. Chem. Chem. Phys., 2016, 18, 26192-26198) in 1 M aqueous solutions. Density-functional-theory spectra predictions are employed here to resolve the conflict. In both the 1999 and 2016 reports, the effects seen were misinterpreted, and are instead due to loss of direct amine-amine H-bond interaction upon dilution, which appears to be complete at ∼0.66 mole fraction of water. Both trans and gauche conformers of ethylenediamine are concluded to be present, in both liquid and aqueous phases, with as yet no solid evidence for a shift in conformer ratio.

Transcriptomic analysis of Rhizobium leguminosarum biovar viciae in symbiosis with host plants Pisum sativum and Vicia cracca.

Rhizobium leguminosarum bv. viciae forms nitrogen-fixing nodules on several legumes, including pea (Pisum sativum) and vetch (Vicia cracca), and has been widely used as a model to study nodule biochemistry. To understand the complex biochemical and developmental changes undergone by R. leguminosarum bv. viciae during bacteroid development, microarray experiments were first performed with cultured bacteria grown on a variety of carbon substrates (glucose, pyruvate, succinate, inositol, acetate, and acetoacetate) and then compared to bacteroids. Bacteroid metabolism is essentially that of dicarboxylate-grown cells (i.e., induction of dicarboxylate transport, gluconeogenesis and alanine synthesis, and repression of sugar utilization). The decarboxylating arm of the tricarboxylic acid cycle is highly induced, as is gamma-aminobutyrate metabolism, particularly in bacteroids from early (7-day) nodules. To investigate bacteroid development, gene expression in bacteroids was analyzed at 7, 15, and 21 days postinoculation of peas. This revealed that bacterial rRNA isolated from pea, but not vetch, is extensively processed in mature bacteroids. In early development (7 days), there were large changes in the expression of regulators, exported and cell surface molecules, multidrug exporters, and heat and cold shock proteins. fix genes were induced early but continued to increase in mature bacteroids, while nif genes were induced strongly in older bacteroids. Mutation of 37 genes that were strongly upregulated in mature bacteroids revealed that none were essential for nitrogen fixation. However, screening of 3,072 mini-Tn5 mutants on peas revealed previously uncharacterized genes essential for nitrogen fixation. These encoded a potential magnesium transporter, an AAA domain protein, and proteins involved in cytochrome synthesis.

Nitrous oxide dimer: an ab initio coupled-cluster study of isomers, interconversions, and infrared fundamental bands, and experimental observation of a new fundamental for the polar isomer.

Improved quantum chemistry (coupled-cluster) results are presented for spectroscopic parameters and the potential energy surface for the N(2)O dimer. The calculations produce three isomer structures, of which the two lowest energy forms are those observed experimentally: a nonpolar C(2h)-symmetry planar slipped-antiparallel geometry (with inward-located O atoms) and a higher-energy polar C(s)-symmetry planar slipped-parallel geometry. Harmonic vibrational frequencies and infrared intensities for these isomers are calculated. The low-frequency intermolecular vibrational mode predictions should be useful for future spectroscopic searches, and there is good agreement in the one case where an experimental value is available. The frequency shifts for the high-frequency intramolecular stretching vibrations, relative to the monomer, were calculated and used to help locate a new infrared band of the polar isomer, which corresponds to the weaker out-of-phase combination of the nu(1) antisymmetric stretch of the individual monomers. The new band was observed in the region of the monomer nu(1) fundamental for both ((14)N(2)O)(2) and ((15)N(2)O)(2) using a tunable infrared diode laser to probe a pulsed supersonic jet expansion, and results are presented.

Transcriptomic analysis of Rhizobium leguminosarum bacteroids in determinate and indeterminate nodules.

Two common classes of nitrogen-fixing legume root nodules are those that have determinate or indeterminate meristems, as in Phaseolus bean and pea, respectively. In indeterminate nodules, rhizobia terminally differentiate into bacteroids with endoreduplicated genomes, whereas bacteroids from determinate nodules are less differentiated and can regrow. We used RNA sequencing to compare bacteroid gene expression in determinate and indeterminate nodules using two Rhizobium leguminosarum strains whose genomes differ due to replacement of the symbiosis (Sym) plasmid pRP2 (strain Rlp4292) with pRL1 (strain RlvA34), thereby switching symbiosis hosts from Phaseolus bean (determinate nodules) to pea (indeterminate nodules). Both bacteroid types have gene expression patterns typical of a stringent response, a stressful environment and catabolism of dicarboxylates, formate, amino acids and quaternary amines. Gene expression patterns were indicative that bean bacteroids were more limited for phosphate, sulphate and iron than pea bacteroids. Bean bacteroids had higher levels of expression of genes whose products are predicted to be associated with metabolite detoxification or export. Pea bacteroids had increased expression of genes associated with DNA replication, membrane synthesis and the TCA (tricarboxylic acid) cycle. Analysis of bacteroid-specific transporter genes was indicative of distinct differences in sugars and other compounds in the two nodule environments. Cell division genes were down-regulated in pea but not bean bacteroids, while DNA synthesis was increased in pea bacteroids. This is consistent with endoreduplication of pea bacteroids and their failure to regrow once nodules senesce.

Short-range order in liquid aluminum chloride: ab initio molecular dynamics simulations and quantum-chemical calculations.

We use ab initio molecular dynamics simulations based on density-functional theory and quantum-chemistry calculations on molecular clusters to examine the structure of liquid AlCl3. In the past, conflicting descriptions of the short-range-order in molten AlCl3, based on either edge-sharing dimers or corner-sharing oligomers, have been proposed. This liquid also poses a simulation challenge, due to the possibility of ring-like trimers which can be metastable on the order of >10 ps. Simulations which begin with monomers, either random or ordered, appear to be able to produce proper ratios of ring-trimer to dimer-plus-tail molecular structures without the need to achieve long-time scale chemical equilibrium. Single-molecule calculations lend further support to the conclusion that the liquid is composed largely of edge-sharing dimers.

Nucleotide sequence of the gene coding for Clostridium botulinum (Clostridium argentinense) type G neurotoxin: genealogical comparison with other clostridial neurotoxins.

The neurotoxin gene from Clostridium botulinum type G was cloned as a series of overlapping DNA fragments generated using polymerase chain reaction (PCR) technology and primers designed to conserved regions of published botulinal toxin (BoNT) sequences. The 5'-end of the gene was obtained using a primer based on a conserved region of the nontoxic-nonhaemagglutinin gene lying upstream of the toxin gene. Translation of the nucleotide sequence derived from the cloned PCR fragments demonstrated that the gene encodes a protein of 1297 amino acid residues (rmm 149, 147). Comparative alignment of the determined BoNT/G sequence with those of other clostridial neurotoxins revealed highest sequence relatedness (approx. 58% amino acid identity) with BoNT/B of proteolytic and non-proteolytic C. botulinum. Tetanus toxin (TeTx) and other BoNT types revealed lower levels of relatedness with BoNT/G (approximate range 35-42% amino acid identity).

A new bacteriophage vector for cloning in Bacillus subtilis and the use of phi 105 for protein synthesis in maxicells.

Zabarovsky and Allikmets [Gene 42 (1986) 119-123] have described a cloning procedure based on partial filling-in of vector and target DNA cohesive ends, which strongly enriches for recombinant molecules with single insertions. Improved Bacillus subtilis bacteriophage phi 105 vectors containing unique cloning sites for SalI have been constructed to take advantage of the partial fill-in method. The new vectors have been used to construct B. subtilis genomic libraries from which several sporulation loci have been isolated, including five not previously cloned. On inserting a promoterless lacZ gene into the cloning site, beta-galactosidase (beta Gal) was detected at a late stage in lytic phage growth, indicating that phage transcription is directed through the cloning site. When UV-irradiated cells ('maxicells') were infected with the recombinant phage containing the lacZ gene, in the presence of labelled amino acids, a protein of the expected Mr for beta Gal was visualised, in addition to the phage proteins. This system should provide a useful general approach for the identification of the products of cloned genes from B. subtilis and other Gram-positive organisms.

An efficient expression and secretion system based on Bacillus subtilis phage phi 105 and its use for the production of B. cereus beta-lactamase I.

A novel expression system based on the Bacillus subtilis bacteriophage phi 105 has been developed to permit the high-level synthesis and secretion of beta-lactamase I (BlaI) from Bacillus cereus. Shotgun insertion of a promoterless lacZ gene into the phage genome permitted the identification of a clone producing large amounts of beta-galactosidase (beta Gal), indicating the transcription of the reporter gene from a strong phage promoter. The insertion also blocked lysis of the host cell. Although the insertion in the original prophage was complex, plasmid vectors and prophage derivatives have been developed to facilitate the replacement of lacZ with other genes for expression. The new prophages contain two additional mutations: an ind mutation, which greatly enhances the normally poor transformability of phi 105 lysogens, and a cts mutation, which allows thermo-induction of phage development and protein production. Induction of a derivative prophage containing the blaI gene from B. cereus resulted in the production of up to 500 micrograms of secreted BlaI per ml of culture supernatant.

Distribution of a sub-class of bacterial ABC polar amino acid transporter and identification of an N-terminal region involved in solute specificity.

A new sub-class of binding protein-dependent transporter with specificity for a broad range of polar amino acids has been identified by sequence comparison, in Rhizobium leguminosarum, Rhodobacter capsulatus, Escherichia coli and Pseudomonas fluorescens. Southern blotting and PCR analysis has shown that transporters from this new sub-class are widely distributed in Gram-negative bacteria, including, in addition to the above, Citrobacter freundii, Erwinia carotovorum and Rhizobium meliloti. ABC transporters of polar amino acids can be divided into two groups: those with narrow solute specificity and the newly identified sub-class with broad solute specificity. The binding and inner membrane proteins from transporters with a broad solute specificity are larger by approximately 30% than those with a narrow solute specificity. Multiple alignment of the inner membrane proteins from all sequenced polar amino acid transporters indicates there is an N-terminal conserved region that may be involved in solute specificity. A conserved arginine or lysine at residue 30 of this region is changed to glutamate in arginine transporters. Residue 53 also has a strong correlation with the charge on the transported solute, with basic amino acid transporters replacing an aliphatic amino acid at this position with a negatively charged amino acid. The general amino acid permease from R. leguminosarum, which will transport aliphatic as well as basic and acidic amino acids, juxtaposes two prolines at residues 52 and 53 of the N-terminal conserved region.

Sequence of the gene coding for the neurotoxin of Clostridium botulinum type A associated with infant botulism: comparison with other clostridial neurotoxins.

The neurotoxin gene from a strain of Clostridium botulinum type A causing infant botulism was cloned as a series of overlapping polymerase chain reaction (PCR) fragments generated using primers designed to conserved regions of published botulinal toxin (BoNT) sequences. Translation of the nucleotide sequence derived from cloned PCR fragments demonstrated that the toxin gene encodes a protein of 1,296 amino acid residues. Comparative alignment of the derived infant BoNT/A sequence with those of other published neurotoxins revealed highest sequence relatedness with BoNT/A of classical food-borne botulism. The sequence identity between infant and classical BoNT/A was 94.9% for the light chain (corresponding to 23 amino acid changes) and 87.1% for the heavy chain (corresponding to 109 amino acid changes).

Studies on the large subunit ribosomal RNA genes and intergenic spacer regions of non-proteolytic Clostridium botulinum types B, E and F.

The large-subunit ribosomal ribonucleic acid (23S rRNA) genes of non-proteolytic (group II) strains of Clostridium botulinum toxin types B, E and F were amplified using the polymerase chain reaction (PCR), and cloned in Escherichia coli. Sequence determination showed that the 23S rRNA genes were 2910 nucleotides in length, and comparative analysis revealed approximately 99.5% sequence similarity. The 23S rRNA gene sequence of a strain phenotypically resembling non-proteolytic C. botulinum, except in not producing botulinal neurotoxin, was also determined and displayed 99.5% sequence similarity with those from toxigenic strains. A diagnostic sequence within the 23S rRNA characteristic for non-proteolytic C. botulinum was identified and used for the design of an oligonucleotide probe. Molecular hybridizations with PCR-amplified rDNA targets provided a precise and reliable method of identifying non-proteolytic (or Group II) C. botulinum and closely related non-toxigenic strains.

Molecular characterization of DNA encoding 23S rRNA and 16S-23S rRNA intergenic spacer regions of Aeromonas hydrophila.

Amplification of the gene encoding 23S rRNA of Aeromonas hydrophila by polymerase chain reaction, with primers complementary to conserved regions of 16S and the 3'-end of 23S rRNA genes, resulted in a DNA fragment of approximately 3 kb. This fragment was cloned in Escherichia coli, and its nucleotide sequence determined. The region encoding 23S rRNA shows high homology with the published sequences of 23S rRNA from other members of the gamma division of Proteobacteria. The sequence of the intergenic spacer region, between the 16S and 23S rRNA genes, was determined in five clones. Three types of spacer were identified: two clones were identical and encoded tRNA(Ile) and tRNA(Ala) while the remaining three clones contained tRNA(Glu), only two had the same spacer sequences. This variation in sequence indicates that the different clones may be derived from different ribosomal RNA operons.

Analysis of DNA encoding 23S rRNA and 16S-23S rRNA intergenic spacer regions from Plesiomonas shigelloides.

Amplification of the gene encoding 23S rRNA of Plesiomonas shigelloides by polymerase chain reaction (PCR), with primers complementary to conserved regions of 16S and the 3' end of 23S rRNA genes, resulted in a DNA fragment of approximately 3 kb. This fragment was cloned in Escherichia coli and its nucleotide sequence determined. The region encoding 23S rRNA shows high homology with the published sequences of 23S rRNA from other members of the gamma division of Proteobacteria. The sequence of the intergenic spacer region, between the 16S and 23S rRNA genes, was determined in a further two clones. In one the sequence of a single tRNA(Glu) was found which was absent from the other two. This variation in sequence suggests that the different clones may be derived from different ribosomal RNA operons.

Change of a single amino acid in the leader peptide of a staphylococcal beta-lactamase prevents the appearance of the enzyme in the medium.

A plasmid encoded beta-lactamase gene from Staphylococcus aureus (strain 3804) was cloned and sequenced. The nucleotide sequence is very similar to those obtained for other such beta-lactamase genes. The beta-lactamase has an N-terminal region characteristic of an exported protein and assay of activity shows that the enzyme is found extracellularly in S. aureus. A residue in the N-terminal region near to the postulated cleavage site was changed by site-directed mutagenesis from a serine into a proline. Comparison of beta-lactamase activity outside the cell in strains containing the cloned wild-type and mutagenised genes shows that this single amino acid completely prevents the appearance of the enzyme in the medium.

Nucleotide sequence of the gene coding for Clostridium barati type F neurotoxin: comparison with other clostridial neurotoxins.

The neurotoxin gene from Clostridium barati ATCC43756 was cloned as a series of overlapping polymerase chain reaction (PCR) generated fragments using primers designed to conserve toxin sequences previously published. The toxin gene has an open reading frame (ORF) of 1268 amino acids giving a calculated molecular mass of 141,049 Da. The sequence identity between the C. barati ATCC43756 and non-proteolytic C. botulinum 202F neurotoxins is 64.2% for the light chain and 73.6% for the heavy chain. This is much lower than reported identities for the type E neurotoxins from C. botulinum and C. butyricum (96% identity between light chains and 98.8% between the heavy chains). Previously identified conserved regions in other botulinal neurotoxins were also conserved in that of C. barati. An ORF upstream of the toxin coding region was revealed. This shows strong homology to the 3' end of the gene coding for the nontoxic-nonhemagglutinin (NTNH) component of the progenitor toxin from C. botulinum type C neurotoxin.

Sequence of the gene encoding type F neurotoxin of Clostridium botulinum.

Primers designed to conserved regions of botulinum and tetanus clostridial toxins were used to amplify DNA fragments from non-proteolytic Clostridium botulinum type F (202F) DNA using polymerase chain reaction technology. The fragments were cloned and the complete nucleotide sequence of the gene encoding type F toxin determined. Analysis of the nucleotide sequence demonstrated the presence of an open frame encoding a protein of 1274 amino acids, similar to other botulinum neurotoxins. Upstream of the toxin gene is the end of an open reading frame which encodes the C-terminus of a protein with homology to non-toxic-non-hemagglutinin component of type C progenitor toxin.

Combining a hybrid genetic algorithm and a heat transfer model to optimise an insulated box for use in the transport of perishables.

Temperature sensitive products are often transported in non-refrigerated systems, protected from environmental temperatures by thermal insulation and the provision of a source of cold inside the package. This work presents a method for optimising the design of thermally insulated boxes for lowest cost (while ensuring that product temperatures are maintained within set limits) by combining a heat transfer model with genetic algorithm optimisation. An example optimisation problem is presented in which a temperature sensitive product is transported across the US and must be maintained between -1 and 8 degrees C. Independent optimisation operations successfully identified solutions that were very similar, though not identical, thereby providing confidence in the approach to determine optimal solutions to complex problems. The system developed provides a rapid method to optimise the design of an insulated box for minimum cost while maintaining the product in the appropriate temperature range.

Carbocation branching observed in a simulation.

We have observed the branching rearrangement of a straight-chain secondary carbocation (C9H19+) in an ab initio molecular dynamics (AIMD) reverse-annealing (rising-temperature) simulation. The mechanism observed is one involving closed (protonated-cyclopropane) structures, previously observed in traditional geometry optimization calculations. However, the simulations give us a better understanding of the dynamics involved, leading to two advances: a simpler description of carbenium ion structures in general and the discovery of important entropy effects.