Phage-lambda-mediated transduction of non-conjugative plasmids is promoted by transposons

Transduction experiments using phage λ as a vector have shown that non-conjugative plasmids can be transduced from one cell to the other, provided the phage or the plasmid DNA carries a copy of a Tn3-like transposon. The transduction is a result of replicon fusion between the phage and the plasmid DNA occurring during the transposition event.     

Chromosomal insertion of TOL transposons in Pseudomonas aeruginosa PAO.

Insertions of the TOL plasmid transposons Tn4651 and Tn4653 into the Pseudomonas aeruginosa PAO chromosome were isolated by a temperature selection technique. The locations and orientations of 16 insertions were determined by pulsed field gel electrophoresis and Southern hybridization with genomic and TOL DNA probes. All insertions occurred within a 334 kb region of the chromosome (representing less than 6% of the genome) with nine of the inserts clustered within a 10 kb area. Each transposon was able to insert in either orientation. An internal duplication of the 39 kb excisable region of pWW0 was seen in two independent insertions.     

Mini-Mu transposition of bacterial genes on the transmissible plasmid

Using the pRM30 plasmid, an Ap^s deletion derivative of broad host range plasmid RP4 with integrated new miniMu 5 (11 kb), we followed the transfer ofEscherichia coli chromosomal genes to the recipient strain. The miniMu 5-mediated transposition of chromosomal genes occurs onto the plasmid with integrated miniMu5 rather than onto the “recipient” plasmid pNH602. The plasmid DNA in recipient cells was detected by electrophoresis. One of the acquired hybrid plasmids pTB2 was analyzed genetically and by restriction endodeoxyribonuclease digestion. A structure consisting of miniMu-chromosomal segment-miniMu as a product of Mu-mediated transposition was detected.     

Transfer of "artificial transposons" constructed on the basis of insertion element IS1

Terminal inverted repeats of the insertion element IS1 were synthesized chemically and plasmids containing these sequences flanking kanamycin-resistance gene in different combinations were constructed. Further incorporation of a whole-sized copy of the IS1 into such plasmids caused in some cases the autonomous transfer of Km-resistance from plasmid to bacteriophage lambda DNA. The transposition of the Km-resistance gene was only observed in those cases when the gene was enclosed between IS1 copy and one of the terminal repeats. The data obtained are discussed with regard to the evolution of bacterial transposons.     

Notch Signalling: Receptor cis-Inhibition To Achieve Directionality

Lateral inhibition, by which single cells become distinct from their neighbours, can be mediated by Notch signalling during animal development. Signalling directionality is presumably achieved by downregulation of the Notch ligand in signal-receiving cells. New evidence suggests that cis-inhibition of the receptor in the ligand-sending cell might also provide directionality.     

Fed-batch bioconversion of indene to cis-indandiol

The bioconversion of indene to cis-(1S,2R) indandiol, a key intermediate in the synthesis of Merck’s HIV protease inhibitor, CRIXIVAN™ can be achieved during the growth of a Rhodococcus strain. In a previous study, we reported on the application of multi-parameter flow cytometry for the measurement of indene toxicity to the strain, and found that concentrations up to 0.25 g/l of indene (0.037 g indene/g dry cell weight) in batch bioconversions did not influence cell physiology. Using this information, this study reports on the implementation of a single phase indene fed-batch bioconversion. Cytoplasmic membrane (membrane) integrity and membrane polarisation of a large number of cells were measured during such bioconversions using multi-parameter flow cytometry and compared to a control in order to assess any toxic effects of indene feeding. The results indicate that indene supply at a rate of 0.1 g/l/h is feasible without any deleterious effects on cell physiology. The delay in indene metabolism was significantly shorter, with lower concentrations of by-product formation, when it was added to the culture in the stationary phase than when it was added at the beginning of the exponential phase of the fermentation. cis-Indandiol production rates could be enhanced from 20 mg/l/h, in a previously reported silicone oil two-liquid phase system, up to 200 mg/l/h by a combination of suitable indene feeding rates in the stationary phase and operating with a high biomass concentration to limit the effects of toxicity. In addition, the yield of cis-indandiol on indene (g/g) was higher at 0.48 in the single phase system compared to 0.20 in the two-liquid phase system. However, the final concentration of cis-indandiol was considerably lower, possibly as a result of higher dehydrogenase activity resulting in an increased transformation of cis-indandiol to 1-keto-2-hydroxy indan. This study has demonstrated that it is feasible to feed indene directly in the stationary phase of the bioconversion using high biomass concentrations to obtain enhanced cis-indandiol formation rates as well as yields based on indene utilisation compared to a two-phase silicone oil system.     

Efficient in vitro synthesis of cis-polyisoprenes using a thermostable cis-prenyltransferase from a hyperthermophilic archaeon Thermococcus kodakaraensis

The Tk-idsB encoding cis-prenyltransferase which catalyzes consecutive cis-condensation of isopentenyl diphosphate to allylic diphosphate was isolated from a hyperthermophilic archaeon Thermococcus kodakaraensis, and enzymatic characteristics of the recombinant Tk-IdsB were examined. Tk-IdsB was not fully denatured even at 90 °C and preferably utilizes both C₁₀ and C₁₅ allylic diphosphates to yield mainly the C₆₀–C₆₅ products. Based on structural models, single alanine-substitution mutants at Glu68, Lys109, or Leu113 were constructed, showing that all the three produced longer chains (C₆₅–C₇₀) than the wild-type and the substitution at 109 (K109A) was the most effective. Tk-IdsB was applied to an organic-aqueous dual-phase system and more than 90% of the products were recovered from the organic phase when 1-butanol or 1-pentanol was overlaid. When 1-octanol was overlaid, 70% of the products were obtained from the upper organic phase. The product distributions were changed depending on the hydrophobicity of organic solvents used. Tk-IdsB was then immobilized onto silica beads to make Tk-IdsB more tolerant, showing that half-life of enzyme at 80 °C was prolonged by immobilization. When the immobilized Tk-IdsB was applied in the organic-aqueous dual-phase system, immobilized Tk-IdsB catalyzed consecutive condensation more efficiently than the unimmobilized one.     

Conjugative transposons: the tip of the iceberg

Elements that excise and integrate, such as prophages, and transfer by conjugation, such as plasmids, have been found in various bacteria. These elements appear to have a diversified set of characteristics including cell-to-cell contact using pili or cell aggregation, transfer of single-stranded or double-stranded DNA, low or high specificity of integration and serine or tyrosine recombinases. This has led to a highly heterogeneous nomenclature, including conjugative transposons, integrative 'plasmids', genomic islands and numerous unclassified elements. However, all these elements excise by site-specific recombination, transfer the resulting circular form by conjugation and integrate by recombination between a specific site of this circular form and a site in the genome of their host. Whereas replication of the circular form probably occurs during conjugation, this replication is not involved in the maintenance of the element. In this review, we show that these elements share very similar characteristics and, therefore, we propose to classify them as integrative and conjugative elements (ICEs). These elements evolve by acquisition or exchanges of modules with various transferable elements including at least ICEs and plasmids. The ICEs are probably widespread among the bacteria.     

Genomic analysis of insertion behavior and target specificity of mini-Tn7 and Tn3 transposons in Saccharomyces cerevisiae

Transposons are widely employed as tools for gene disruption. Ideally, they should display unbiased insertion behavior, and incorporate readily into any genomic DNA to which they are exposed. However, many transposons preferentially insert at specific nucleotide sequences. It is unclear to what extent such bias affects their usefulness as mutagenesis tools. Here, we examine insertion site specificity and global insertion behavior of two mini-transposons previously used for large-scale gene disruption in Saccharomyces cerevisiae: Tn3 and Tn7. Using an expanded set of insertion data, we confirm that Tn3 displays marked preference for the AT-rich 5 bp consensus site TA[A/T]TA, whereas Tn7 displays negligible target site preference. On a genome level, both transposons display marked non-uniform insertion behavior: certain sites are targeted far more often than expected, and both distributions depart drastically from Poisson. Thus, to compare their insertion behavior on a genome level, we developed a windowed Kolmogorov–Smirnov (K–S) test to analyze transposon insertion distributions in sequence windows of various sizes. We find that when scored in large windows (>300 bp), both Tn3 and Tn7 distributions appear uniform, whereas in smaller windows, Tn7 appears uniform while Tn3 does not. Thus, both transposons are effective tools for gene disruption, but Tn7 does so with less duplication and a more uniform distribution, better approximating the behavior of the ideal transposon.     

Real-time three-dimensional imaging of lipid signal transduction: apical membrane insertion of epithelial Na(+) channels

In the distal tubule, Na⁺ resorption is mediated by epithelial Na⁺ channels (ENaC). Hormones such as aldosterone, vasopressin, and insulin modulate ENaC membrane targeting, assembly, and/or kinetic activity, thereby regulating salt and water homeostasis. Insulin binds to a receptor on the basal membrane to initiate a signal transduction cascade that rapidly results in an increase in apical membrane ENaC. Current models of this signaling pathway envision diffusion of signaling intermediates from the basal to the apical membrane. This necessitates diffusion of several high-molecular-weight signaling elements across a three-dimensional space. Transduction of the insulin signal involves the phosphoinositide pathway, but how and where this lipid-based signaling pathway controls ENaC activity is not known. We used tagged channels, biosensor lipid probes, and intravital imaging to investigate the role of lipids in insulin-stimulated Na⁺ flux. Insulin-stimulated delivery of intracellular ENaC to apical membranes was concurrent with plasma membrane-limited changes in lipid composition. Notably, in response to insulin, phosphatidylinositol 3,4,5-trisphosphate (PIP₃) formed in the basolateral membrane, rapidly diffused within the bilayer, and crossed the tight junction to enter the apical membrane. This novel signaling pathway takes advantage of the fact that the lipids of the plasma membrane's inner leaflet are not constrained by the tight junction. Therefore, diffusion of PIP₃ as a signal transduction intermediate occurs within a planar surface, thus facilitating swift responses and confining and controlling the signaling pathway. phosphatidylinositol 3,4,5-trisphosphate; insulin-stimulated Na⁺ transport; metabolic syndrome; real-time confocal imaging     

Towards a genome-wide reconstruction of cis-regulatory networks in the human genome

The vast amount of recent progress made on the sequence of the human genome has allowed an unprecedented examination of cis-regulatory networks. These networks consist of functional elements such as promoters, enhancers, silencers, and insulators, and their coordinated activity is responsible for regulation of gene expression. Recent studies surveyed the entire genome, identifying novel elements and evaluating functional differences in respect to development. These investigations present the first steps towards a global regulatory map for expression in the human genome.     

Naphthalene-dioxygenase-catalysed cis-dihydroxylation of azaarene derivatives: Part 1: 2-Pyridones and 2-quinolones

The scope of the biotransformation of 2-pyridone- and 2-quinolone-derived compounds by recombinant whole-cells of E. coli JM109(DE3)(pDTG141) expressing the naphthalene-dioxygenase system from Pseudomonas sp. NCIB 9816-4 was explored, using a series of N- and C-substituted derivatives. Among them, only the N-methyl substituted compounds were good substrates for a regio- and stereoselective dihydroxylation reaction leading to cis-dihydroxydihydro pyridone derivatives, corresponding to the general pattern expected for this enzyme. In the absence of dihydroxylation reactions, N-dealkylations and monohydroxylations on external methyl groups were observed.     

Immune signal transduction in leishmaniasis from natural to artificial systems: Role of feedback loop insertion

Background

Modulated immune signal (CD14–TLR and TNF) in leishmaniasis can be linked to EGFR pathway involved in wound healing, through crosstalk points. This signaling network can be further linked to a synthetic gene circuit acting as a positive feedback loop to elicit a synchronized intercellular communication among the immune cells which may contribute to a better understanding of signaling dynamics in leishmaniasis.

Methods

Network reconstruction with positive feedback loop, simulation (ODE 15s solver) and sensitivity analysis of CD14–TLR, TNF and EGFR was done in SimBiology (MATLAB 7.11.1). Cytoscape and adjacency matrix were used to calculate network topology. PCA was extracted by using sensitivity coefficient in MATLAB. Model reduction was done using time, flux and sensitivity score.

Results

Network has five crosstalk points: NIK, IκB–NFκB and MKK (4/7, 3/6, 1/2) which show high flux and sensitivity. PI3K in EGFR pathway shows high flux and sensitivity. PCA score was high for cytoplasmic ERK1/2, PI3K, Atk, STAT1/3 and nuclear JNK. Of the 125 parameters, 20% are crucial as deduced by model reduction.

Conclusions

EGFR can be linked to CD14–TLR and TNF through the MAPK crosstalk points. These pathways may be controlled through Ras and Raf that lie upstream of signaling components ERK ½ (c) and JNK (n) that have a high PCA score via a synthetic gene circuit for activating cell–cell communication to elicit an inflammatory response. Also a disease resolving effect may be achieved through PI3K in the EGFR pathway.

General significance

The reconstructed signaling network can be linked to a gene circuit with a positive feedback loop, for cell–cell communication resulting in synchronized response in the immune cell population, for disease resolving effect in leishmaniasis.     

Localization of insertion into E. coli chromosome of Tn7-like transposons controlling resistance to trimethoprim and streptothricin

To localize the insertion sites for Tn7-like transposons Tn1824, Tn1825 and Tn1826 the EcoRI-cleaved fragments of E. coli recA+ and recA- strains chromosome carrying the transposons were hybridized. It was shown that transposition of Tn7-like elements takes place in the strictly defined region of E. coli chromosome, like it had been previously described for transposon Tn7.     

Kinetically stable high-energy isomers of C14H12 and C12H10N2 derived from cis-stilbene and cis-azobenzene

Following on from our recent enforced geometry optimization (EGO) investigation of isomerization in cis-stilbene (J Comput Chem, in press) we report the discovery of two interesting new, symmetrical “fused sandwich” isomers of both cis-stilbene and the related cis-azobenzene. The isomers were obtained by applying external forces to pairs of carbon atoms from each of the benzene rings in cis-stilbene and cis-azobenzene simultaneously, and are all at least 100 kcal mol^-1 higher in energy than the starting material. Each new structure was characterized as a minimum by vibrational analysis. Despite their high energy, all of the new isomers appear to be kinetically stable with respect to rearrangement back to cis-stilbene or cis-azobenzene, respectively.     

cis-Inhibition of Notch by Endogenous Delta Biases the Outcome of Lateral Inhibition

Lateral inhibition mediated by Delta/Notch (Dl/N) signaling is used throughout development to limit the number of initially equivalent cells that adopt a particular fate [1], [2] and [3]. Although adjacent cells express both Dl ligand and N receptor, signaling between them ultimately occurs in only one direction. Classically, this has been explained entirely by feedback: activated N can downregulate Dl, amplifying even slight asymmetries in the Dl or N activities of adjacent cells [1], [2], [3], [4] and [5]. Here, however, we present an example of lateral inhibition in which unidirectional signaling depends instead on Dl's ability to inhibit N within the same cell, a phenomenon known as cis-inhibition [6], [7], [8], [9], [10] and [11]. By genetically manipulating individual R1/R6/R7 photoreceptor precursors in the Drosophila eye, we show that loss of Dl-mediated cis-inhibition reverses the direction of lateral signaling. Based on our finding that Dl in R1/R6s requires endocytosis to trans-activate but not to cis-inhibit N, we reexamine previously published data from other examples of lateral inhibition. We conclude that cis-inhibition generally influences the direction of Dl/N signaling and should therefore be included in standard models of lateral inhibition.     

Stereocontrolled routes to cis-hydroxyamino sugars, part VII: synthesis of daunosamine and ristosamine

The nitrogen of an allylic amine can serve as the fulcrum for stereocontrolled delivery of oxygen to an adjacent trigonal site, and cis-hydroxyamino sugars can thus be prepared. Methods for achieving the complementary procedure, namely, control of the delivery of nitrogen to an adjacent site by an allylic oxygen, are described. For example, treatment of methyl 2,3,6-trideoxy-- -erythro-hex-2-enopyranoside with trichloroacetonitrile gave an imidate ester which reacted with iodonium dicollidine perchlorate to give 2-trichloromethyl-(methyl 2,3,4,6-tetradeoxy-2-iodo-- -altropyranoside)-[3,4-d]-2-oxazoline. Exhaustive reductive dehalogenation of this product followed by hydrolysis led to methyl N-acetyl-- -ristosaminide. An analogous series of reactions was used to prepare the corresponding daunosaminide.     

Positive and negative cis-regulatory elements in the bithoraxoid region of the Drosophila Ultrabithorax gene

Summary The Ultrabithorax (Ubx) gene is required during embryogenesis and larval development to specify the third thoracic and first abdominal segments of Drosophila melanogaster. Mutations in the bithoraxoid (bxd) region, a 40 kb DNA stretch upstream of the Ubx promoter, affect cis-regulatory elements responsible for the ectodermal expression of the Ubx gene in the posterior compartment of the third thoracic segment and anterior compartment of the first abdominal segment. Our genetic data and the available molecular information are used to map the adult epidermal cis-regulatory elements within the bxd region. Genetic combinations involving mutations affecting the bxd region show that (1) redundant or cooperatively acting sequences are required for Ubx gene expression in the anterior compartment of the first abdominal segment, and (2) the expression of Ubx in the posterior compartment of the third thoracic segment is modulated by positive and negative cis-regulatory elements.The Wellcome Trust CRC Institute for Cancer Research and Developmental Biology, Tennis Court Road Cambridge, CB2 1QR, UKDivision de Genética, Departamento de Genética Molecular y Microbiología, Campus de San Juan, Apdo. 374, 03080 Alicante, Spain