Tn5-induced Xenorhabdus bovienii lecithinase mutants demonstrate reduced virulence for Galleria mellonella larvae

A derivative of Tn5 was used to construct a variety of stable insertion mutations in the entomopathogenic bacterium, Xenorhabdus bovienii T228/1. Mutants which had altered expression of Congo Red binding ability, ampicillin resistance, haemolytic activity and lecithinase were isolated. Isolates with altered lecithinase activity had either lost ability to produce this enzyme or showed reduced expression. The role of lecithinase in pathogenesis of X. bovienii T228/1 for Galleria mellonella larvae was examined by LD₅₀ analysis. Maximum killing of G. mellonella was observed at 72 h post infection for both the wild-type parent strain and a lecithinase mutant 34(45). However, the LD₅₀ value for the wild-type parent strain (8·7 cells per larva) was significantly less than that calculated for the lecithinase mutant (35·5 cells per larva). The data suggested that although lecithinase is a virulence factor produced by X. bovienii , lecithinase activity alone is not sufficient for killing of G. mellonella larvae.     

Properties of the Translocatable Tetracycline-Resistance Element Tn10 in ESCHERICHIA COLI and Bacteriophage Lambda

A number of independent insertions into bacteriophage lambda of the translocatable tetracycline-resistance element Tn10 have been isolated and characterized. The physical positions and relative orientations of several such insertions were determined. Two independent insertions appear to lie in the same orientation at or very near the same site in the cI gene, and two more lie in opposite orientations at or near the same position in or near the rex gene. Insertions in or near genes cI, rex, and cIII have been characterized genetically for their effects on expression of nearby genes. Tn10 appears to exert a polar effect on expression of distal genes when it is inserted within an operon, even when expression of that operon is under the influence of lambda N-function. In addition, Tn10 insertions in rex appear to influence in some way expression of an "upstream" gene, cI. Lambda derivatives carrying Tn10 give rise to spontaneously occurring, tetracycline-sensitive deletions at high frequencies. It is likely that formation of these deletions is promoted in some way by the Tn10 element. Lambda::Tn10 phages carrying a Tn10 element that has undergone several successive cycles of translocation since its first isolation and characterization have been analyzed. The results confirm that Tn10 often retains its physical and functional integrity during many cycles of translocation. Lambda derivatives carrying Tn10 have been used to generate insertions of Tn10 in the chromosome of Escherichia coli. This process is independent of recA function, and seems to be quite analogous to the translocation of Tn10 in Salmonella typhimurium as studied previously.     

Comparative analysis of P2-type remnant prophage loci in Xenorhabdus bovienii and Xenorhabdus nematophila required for xenorhabdicin production

The xnp1 remnant P2-type prophage of Xenorhabdus nematophila produces xenorhabdicin that is active against closely related species. Xenorhabdicin had not been characterized previously in other Xenorhabdus species. Here, we show xenorhabdicin production in six different strains of Xenorhabdus bovienii. The sequenced genome of X. bovienii SS-2004 was found to possess a highly conserved remnant P2-type cluster (xbp1). Inactivation of the xbpS1 sheath gene resulted in loss of bacteriocin activity, indicating that the xbp1 locus was required for xenorhabdicin production. xbp1 and xnp1 contain a CI-type repressor, a dinI gene involved in stabilization of ssDNA-RecA complexes and are inducible with mitomycin C, suggesting that both loci are regulated by cleavage of the CI repressor. Both xnp1 and xbp1 lack typical P2-type lysis genes but contain a predicted endolysin gene (enp) that may be involved in cell lysis. The main tail fibers of xnp1 and xbp1 are mosaic structures with divergent C-terminal regions suggesting they differ in host specificity. Several genes encoding C-terminal tail fiber fragments are present in the same position in xnp1 and xbp1. Recombination between the main fiber genes and the C-terminal fragments could potentially expand the host range specificity of xenorhabdicin in the respective strains.     

Adenovirus Dodecahedron,as a Drug Delivery Vector

Background

Bleomycin (BLM) is an anticancer antibiotic used in many cancer regimens. Its utility is limited by systemic toxicity and dose-dependent pneumonitis able to progress to lung fibrosis. The latter can affect up to nearly 50% of the total patient population, out of which 3% will die. We propose to improve BLM delivery by tethering it to an efficient delivery vector. Adenovirus (Ad) dodecahedron base (DB) is a particulate vector composed of 12 copies of a pentameric viral protein responsible for virus penetration. The vector efficiently penetrates the plasma membrane, is liberated in the cytoplasm and has a propensity to concentrate around the nucleus; up to 300000 particles can be observed in one cell in vitro.

Principal Findings

Dodecahedron (Dd) structure is preserved at up to about 50°C at pH 7–8 and during dialysis, freezing and drying in the speed-vac in the presence of 150 mM ammonium sulfate, as well as during lyophilization in the presence of cryoprotectants. The vector is also stable in human serum for 2 h at 37°C. We prepared a Dd-BLM conjugate which upon penetration induced death of transformed cells. Similarly to free bleomycin, Dd-BLM caused dsDNA breaks. Significantly, effective cytotoxic concentration of BLM delivered with Dd was 100 times lower than that of free bleomycin.

Conclusions/Significance

Stability studies show that Dds can be conveniently stored and transported, and can potentially be used for therapeutic purposes under various climates. Successful BLM delivery by Ad Dds demonstrates that the use of virus like particle (VLP) results in significantly improved drug bioavailability. These experiments open new vistas for delivery of non-permeant labile drugs.     

Evolution of Transposons: Natural Selection for Tn5 in ESCHERICHIA COLI K12

A novel in vivo effect of the transposable element Tn5 has been observed in chemostats when certain isogenic Tn5 and non-Tn5 strains of Escherichia coli compete for a limiting carbon source in the absence of kanamycin. The Tn5-bearing strain has a more rapid growth rate and increases in frequency from 50% to 90% within the first 15 to 20 generations. The effect occurs when Tn5 is inserted at a variety of chromosomal locations or when the element is carried by an episome, but it is strain specific, having been observed in two out of three strains examined. (For reasons unknown, the effect has not been observed with derivatives of strain CSH12.) Although the growth-rate advantage of Tn5 is independent of nutrient concentration and generation time, it can be reduced by prior adaptation of the strains to limiting conditions, and the amount of reduction is proportional to the length of prior adaptation. The growth-rate effect is evidently not caused by beneficial mutations induced by Tn5 transposition, as Tn5-bearing strains selected in chemostats retain their initial Tn5 position and copy number. However, the effect does not occur in Tn5-112, a transpositionless deletion mutation missing the transposase-coding region of the right-hand IS sequence flanking the element. Since Tn5-112 retains a functional kanamycin-phosphotransferase gene, this gene is not responsible for the growth-rate effect. Thus, the effect evidently requires transposase function, but it does not involve actual transposition of the intact element. Altogether, these data provide a mechanism for the maintenance of Tn5 in bacterial populations in the absence of kanamycin, and they suggest a model for the proliferation and the maintenance of IS sequences and transposable elements in the absence of other identifiable selection pressures.     

壳聚糖作为基因药物载体的研究进展

以壳聚糖及其衍生物作为基因的载体的转染效率受到许多因素的影响, 如复合物粒子大小、壳聚糖/DNA的比值、壳聚糖的分子量、脱乙酰度、转染过程中血清的浓度、介质的pH值等。对壳聚糖进行一定程度的修饰, 可以改变壳聚糖的转染效率。介绍了壳聚糖作为基因转移载体的转染条件, 转染效率和转染机制的研究情况及研究进展。     

Prophage Induction by High Temperature in Thermosensitive dna Mutants Lysogenic for Bacteriophage Lambda

High-temperature treatment of thermosensitive dna mutants lysogenic for phage lambda leads to prophage induction and release of phage (at the permissive temperature) in elongation-defective mutants of the genotypes dnaB, dnaE, and dnaG. In initiation-defective mutants no prophage induction occurs at 42 C in mutants of the genotype dnaA, whereas with a dnaC mutant as well as with strain HfrH 252 (map position not yet known) phages are released at 42 C. DNA degradation at the replication fork at 42 C is observed in all dnaB(lambda) mutants tested, but not in mutants of the genotypes dnaE(lambda) and dnaG(lambda). Therefore, degradation of replication fork DNA is not a prerequisite for prophage induction.     

Transposons as tools for enhancer trap screens in vertebrates

DNA transposons are efficient tools in transgenesis and have therefore become popular in the analysis of the regulatory genome in vertebrates via enhancer trap screens. Here, I discuss recent progress in this field of research, with a focus on the application of one of these transposons, namely the medaka fish derived Tol2, to enhancer trapping in zebrafish, and how this approach compares with others that have a similar objective.     

噬菌体phi29 pRNA载体在RNA干扰中的初步应用研究

RNA干扰是在细胞胞质中双链RNA(dsR-NA)介导的序列特异性mRNA的降解[1]。这个过程是由21~25个被称为小干扰RNA(si RNA)形成的dsRNA完成[2]。目前,这一技术已经广泛应用于研究基因的功能,病毒感染治疗等方面。但是,si RNA在体内容易降解,干扰作用持续的时间不长。新的研究表明枯     

Evidence for a common mechanism for the insertion of the Tn10 transposon and for the generation of Tn10-stimulated deletions

Summary Mutations in and near the Salmonella typhimurium histidine transport operon were generated by insertion of the translocatable tetracycline-resistance element Tn10. Deletion mutants affecting histidine transport genes were subsequently isolated in several of the Tn10-containing strains. Tn10 insertions in hisJ occurred preferentially at one site, designated site A. This same site was also the preferential endpoint of deletions originating from Tn10 insertions at two neighboring sites. Thus, Tn10 insertion and Tn10-stimulated deletion formation appear to involve a common DNA-recogition step.     

Development of a Tn10 Vehicle for Insertion Mutagenesis in Rhizobium

A transposon Tn10 vehicle was developed using a self transmissible (Tra⁺) plasmid pRK2013 having narrow host range ori of replication (ColEl). The construct pSA10-3 carrying Tn10 was useful in efficiently transferring transposon Tn10 from E. coli into various rhizobia. The ColEl replicon conferred suicidal property to vector in Rhizobium background where it falls to replicate stably. Thus this plasmid can be employed to cause independent insertion mutations in rhizobia by Tn10 transposition. The frequency of tetracycline resistant colonies of Rhizobium (Tn10 mutants) was approximately 10⁵ folds higher than the spontaneous Tet^R mutants. Reversion frequency of these mutants was less than 10^?8 indicating adequate stability of Tn10 mutations.     

Determinants for hairpin formation in Tn10 transposition

Tn10 transposition involves the formation of a hairpin intermediate at the transposon termini. Here we show that hairpin formation exhibits more stringent DNA sequence requirements at the terminal two base pairs than either transpososome assembly or first strand nicking. We also observe a significant DNA distortion at the terminal base pairs upon transpososome assembly by chemical nuclease footprinting. Interestingly, mutations at these positions do not necessarily inhibit the formation of the distortion. However, it remains a possibility that the inhibitory effect of these mutations is due to a defect in protein-DNA interactions subsequent to this deformation. Terminal base pair mutations also inhibited strand transfer, providing evidence that transposase interactions with the terminal residues on both 'transferred' and 'non-transferred' strands are important for hairpin formation. We also demonstrate that mutation of a highly conserved tyrosine residue that is a component of the YREK motif, Y285, results in a phenotype comparable to that of the terminal base pair mutations. In contrast, a mutation at another conserved position, W265, is shown to relax the specificity of the hairpin formation reaction.     

Rearrangement of the Bacterial Chromosome Using Tn10 as a Region of Homology

The transposable tetracycline resistance element, Tn10, can serve as a region of homology to promote rec-dependent deletion, duplication and directed transposition of bacterial genes. Tn10 insertions in regions of the chromosome near the histidine operon (his) have been isolated and characterized in Salmonella typhimurium. When strains are constructed containing two Tn10 insertions flanking the his operon in the same orientation (Tn10-his-Tn10), recombination can occur between Tn10 sequences resulting in the deletion of the intervening his region. The sites of the Tn10 insertions determine the endpoints of the deletion. In crosses designed to construct strains carrying Tn10-his-Tn10, another class of unstable recombinants arises in which the his region exists in tandem duplication, with a Tn10 insertion joining the duplicated copies (his-Tn10-his). The sites of the parental Tn10 insertions mark the endpoints of the duplication. When a strain carrying Tn10-his-Tn10 is used as a donor of his⁺ in conjugation or P22-mediated transduction, recombinants can arise in which the his region has been transposed to the site of any Tn10 insertion, far from the normal location of his in the recipient chromosome. In this manner, the his operon has been moved to the site of a pyrB::Tn10 insertion and has been placed on F'' plasmids. At these new locations, the his⁺ character shows the rec-dependent deletion of his⁺ expected for a Tn10-his-Tn10 duplication. These methods should be generally useful for the manipulation of bacterial genes.     

Isolation of a Specialized Lambda Transducing Bacteriophage Carrying the Beta Subunit Gene for Escherichia coli Ribonucleic Acid Polymerase

A heat-inducible lysis-defective lambda prophage has been integrated directly into the E. coli chromosome at a site (bfe) very closely linked to the ribonucleic acid polymerase mutation rif(d), a dominant rifampin resistance allele. This unusual lysogen has facilitated the isolation of specialized transducing phages conferring rifampin resistance to sensitive cells, and carrying at least the beta subunit gene of RNA polymerase in intact form.     

Rec-Mediated Recombinational Hot Spot Activity in Bacteriophage Lambda II. a Mutation Which Causes Hot Spot Activity

Crosses have been performed which identify phage mutants (chi) which cause recombinational hot spot activity in lambda. The hot spot activity is found in crosses of red(-) gam(-) chi(-) strains in rec(+) hosts; in the crosses reported here, both the chi(-) mutations and the hot spot are located near the right end of the chromosome. The hot spot occurs in standard crosses as well as under conditions which block DNA synthesis, and is dependent on a functional host recB gene.-The chi mutation is shown to be dominant, but the tests do not show whether chi is a gene or a site.     

Controlled Expression in Klebsiella pneumoniae and Shigella flexneri Using a Bacteriophage P1-Derived C1-Regulated Promoter System

The utility of promoters regulated by the bacteriophage P1 temperature-sensitive C1 repressor was examined in Shigella flexneri and Klebsiella pneumoniae. Promoters carrying C1 operator sites driving LacZ expression had induction/repression ratios of up to 240-fold in S. flexneri and up to 50-fold in K. pneumoniae. The promoters exhibited remarkably low basal expression, demonstrated modulation by temperature, and showed rapid induction. This system will provide a new opportunity for controlled gene expression in enteric gram-negative bacteria.     

Amblyomma americanum as a Bridging Vector for Human Infection with Francisella tularensis

The γ-proteobacterium Francisella tularensis causes seasonal tick-transmitted tularemia outbreaks in natural rabbit hosts and incidental infections in humans in the south-central United States. Although Dermacentor variabilis is considered a primary vector for F. tularensis, Amblyomma americanum is the most abundant tick species in this endemic region. A systematic study of F. tularensis colonization of A. americanum was undertaken to better understand its potential to serve as an overwintering reservoir for F. tularensis and as a bridging vector for human infections. Colony-reared A. americanum were artificially fed F. tularensis subspecies holarctica strain LVS via glass capillaries and colonization levels determined. Capillary-fed larva and nymph were initially infected with 10⁴ CFU/tick which declined prior to molting for both stages, but rebounded post-molting in nymphs and persisted in 53% at 10³ to 10⁸ CFU/nymph at 168 days post-capillary feeding (longest sampling time in the study). In contrast, only 18% of adults molted from colonized nymphs maintained LVS colonization at 10¹ to 10⁵ CFU/adult at 168 days post-capillary feeding (longest sampling time). For adults, LVS initially colonized the gut and disseminated to salivary glands by 24 h and had an ID₅₀ of <5CFU in mice. Francisella tularensis infected the ovaries of gravid females, but transmission to eggs was infrequent and transovarial transmission to hatched larvae was not observed. The prolonged persistence of F. tularensis in A. americanum nymphs supports A. americanum as an overwintering reservoir for F. tularensis from which seasonal epizootics may originate; however, although the rapid dissemination of F. tularensis from gut to salivary glands in adults A. americanum is compatible with intermittent feeding adult males acting as bridging vectors for incidental F. tularensis infections of humans, acquisition of F. tularensis by adults may be unlikely based on adult feeding preference for larger mammals which are not involved in maintenance of sylvatic tularemia.     

Tn5 as a model for understanding DNA transposition

Tn5 is an excellent model system for understanding the molecular basis of DNA-mediated transposition. Mechanistic information has come from genetic and biochemical investigations of the transposase and its interactions with the recognition DNA sequences at the ends of the transposon. More recently, molecular structure analyses of catalytically active transposase; transposon DNA complexes have provided us with unprecedented insights into this transposition system. Transposase initiates transposition by forming a dimeric transposase, transposon DNA complex. In the context of this complex, the transposase then catalyses four phosphoryl transfer reactions (DNA nicking, DNA hairpin formation, hairpin resolution and strand transfer into target DNA) resulting in the integration of the transposon into its new DNA site. The studies that elucidated these steps also provided important insights into the integration of retroviral genomes into host DNA and the immune system V(D)J joining process. This review will describe the structures and steps involved in Tn5 transposition and point out a biologically important although surprising characteristic of the wild-type Tn5 transposase. Transposase is a very inactive protein. An inactive transposase protein ensures the survival of the host and thus the survival of Tn5.