Identification and characterization of IS1 transposition in plasmid amplification mutants of E. coli clones producing DNA vaccines

Merck Research Laboratories has developed a highly productive Escherichia coli fermentation process to produce plasmid DNA for use as vaccines. The process consists of a fed-batch fermentation in a chemically defined medium. Initiation of the feed stream precedes a growth-limited phase in which plasmid DNA is amplified. The fermentation is only maximally productive for a small fraction of E. coli transformants designated as high-producers, while the predominant low-producer population does not amplify plasmid DNA. In experiments undertaken to probe this phenomenon, transposition of the 768-bp E. coli insertion sequence IS1 into an HIV DNA vaccine vector was observed in several low-producer clones. IS1 was found to insert in or near the neomycin resistance gene in nearly a dozen unique sites from within a single population of plasmid molecules. The fraction of IS1-containing plasmids within several clones was determined by quantitative polymerase chain reaction and was found to increase with increasing cultivation time in the chemically defined medium. Because transposition into an antibiotic-resistance gene is unlikely to affect plasmid amplification, the genomes of high- and low-producers of three different HIV DNA vaccine vectors were subsequently profiled by restriction fragment length polymorphism analysis. In all three cases, IS1 insertional mutations were found in the genomes of the predominant low-producers, while the genomes of the high-producers were indistinguishable from untransformed cells. The insertions reside on similarly sized fragments for two of the low-producer clones, and the fragment size is smaller for the third clone. The third clone also produces much less plasmid DNA than a typical low-producer. The results suggest the presence of an IS1 insertional mutation that affects plasmid replication and amplification, possibly in a position-dependent manner.     

Mechanisms of metal ion action in Tn10 transposition

Tn10/IS10 transposition involves assembly of a synaptic complex (or transpososome) in which two transposon ends are paired, followed by four distinct chemical steps at each transposon end. The chemical steps are dependent on the presence of a suitable divalent metal cation (Me(2+)). Transpososome assembly and structure are also affected by Me(2+). To gain further insight into the mechanisms of Me(2+) action in Tn10/IS10 transposition we have investigated the effects of substituting Mn(2+) for Mg(2+), the physiologic Me(2+), in transposition. We have also investigated the significance of an Me(2+)-assisted conformational change in transpososome structure. We show that Mn(2+) has two previously unrecognized effects on the Tn10 donor cleavage reaction. It accelerates the rates of hairpin formation and hairpin resolution without significantly affecting the rate of the first chemical step, first strand nicking. Mn(2+) also relaxes the specificity of first strand nicking. We also show that Me(2+)-assisted transpososome unfolding coincides with a structural transition in the transposon-donor junction that may be necessary for hairpin formation. Possible mechanisms for these observations are considered.     

Helicobacter pylori mutagenesis by mariner in vitro transposition

We have developed a method for generating transposon insertion mutants using mariner in vitro mutagenesis. The gene of interest was PCR-amplified and cloned. A kanamycin-marked mariner transposon was randomly inserted into the purified plasmid in an in vitro transposition reaction. After repair and propagation in Escherichia coli, purified mutagenized plasmid was introduced into Helicobacter pylori by natural transformation. Transformants were selected by plating on kanamycin. Mutants were predominantly the result of double homologous recombination, and multiple mutants (with insertions in distinct positions) were often obtained. The site of insertion was determined by PCR or sequencing. We have made mutations in known or potential virulence genes, including ureA, hopZ, and vacA, using kanamycin- and kanamycin/lacZ-marked transposons. Colonies carrying a kanamycin/lacZ transposon appeared blue on medium containing the chromogenic agent X-gal, allowing discrimination of mutant and wild-type H. pylori in mixed competition experiments.     

Structural basis of transposon end recognition explains central features of Tn7 transposition systems

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Use of the plasmid pRK 2013 as a vehicle for transposition inAzotobacter vinelandii

Plasmid stability inAzotobacter vinelandii has been determined and a way to introduce transposon into these cells using the plasmid pRK 2013 has been devised. Transposition of both Tn3 and Tn10 has been attained.     

Spectrophotometric identification of 8-hydroxy-5-deazaflavin: NADPH oxidoreductase activity in streptomycetes producing tetracyclines

Abstract Cell-free extracts of vegetative mycelia of Streptomyces aureofaciens and Streptomyces rimosus were found to reduce streptomycete-origin 8-hydroxy-5-deazaisoalloxazine derivatives (SF₄₂₀) using NADPH as a dnor of hydrogen and electrons. 7,8-didemethyl-8-hydroxy-5-deazariboflavin (F₀) also was a substrate, although with a lower reaction rate than that for SF₄₂₀. NADH could not substitute for NADPH. The F₄₂₀-reductase activity was also observed in homogenates of S. aureofaciens spores.     

DNA recognition and the precleavage state during single-stranded DNA transposition in D. radiodurans

Bacterial insertion sequences (ISs) from the IS200/IS605 family encode the smallest known DNA transposases and mobilize through single-stranded DNA transposition. Transposition by one particular family member, ISDra2 from Deinococcus radiodurans, is dramatically stimulated upon massive γ irradiation. We have determined the crystal structures of four ISDra2 transposase/IS end complexes; combined with in vivo activity assays and fluorescence anisotropy binding measurements, these have revealed the molecular basis of strand discrimination and transposase action. The structures also show that previously established structural rules of target site recognition that allow different specific sequences to be targeted are only partially conserved among family members. Furthermore, we have captured a fully assembled active site including the scissile phosphate bound by a divalent metal ion cofactor (Cd2(+)) that supports DNA cleavage. Finally, the observed active site rearrangements when the transposase binds a metal ion in which it is inactive provide a clear rationale for metal ion specificity.     

The effect of dissolved oxygen and aeration rate on antibiotic production of Streptomyces fradiae

Different dissolved oxygen concentrations and aeration rates were imposed on a stable mutant of Streptomyces fradiae during the antibiotic-producing phase. At high aeration rate (1 vvm), the tylosin yield in the fermentor broth with dissolved oxygen (DO) concentrations controlled close to 100% saturation (6-8 ppm) increased 10% as against uncontrolled. The rates of cellular growth, oil consumption, and tylosin production were severely reduced when DO concentration fell below 25% saturation, but all resumed to their initial rates when DO was raised to saturation level again. The DO concentration in combination with air flow rate affected the pattern of the antibiotics produced. At high DO levels, an additional macrolide antibiotic, macrocin, was synthesized to more than one-third the amount of tylosin at high aeration rate (1 vvm). On the other hand, tylosin production rate remained constant and no significant amount of macrocin was produced at low aeration rate (0.2 vvm).     

Requirement of IS911 replication before integration defines a new bacterial transposition pathway

Movement of transposable elements is often accompanied by replication to ensure their proliferation. Replication is associated with both major classes of transposition mechanisms: cut-and-paste and cointegrate formation (paste-and-copy). Cut-and-paste transposition is often activated by replication of the transposon, while in cointegrate formation replication completes integration. We describe a novel transposition mechanism used by insertion sequence IS911, which we call copy-and-paste. IS911 transposes using a circular intermediate (circle), which then integrates into a target. We demonstrate that this is derived from a branched intermediate (figure-eight) in which both ends are joined by a single-strand bridge after a first-strand transfer. In vivo labelling experiments show that the process of circle formation is replicative. The results indicate that the replication pathway not only produces circles from figure-eight but also regenerates the transposon donor plasmid. To confirm the replicative mechanism, we have also used the Escherichia coli terminators (terC) which, when bound by the Tus protein, inhibit replication forks in a polarised manner. Finally, we demonstrate that the primase DnaG is essential, implicating a host-specific replication pathway.     

Protein secretion in streptomycetes

Some aspects of the current knowledge on protein secretion in streptomycetes are presented, including recent data on the identification of genes involved in the general secretory pathway, on the importance of the signal peptide structure and on the number of ribosome-binding sites inside signal peptides which can influence the production level of a gene product.     

Intrapopulation variability of antibiotic biosynthesis in streptomycetes

Spontaneous variants making up parallel series of hereditary variability inside the populations of antibiotic-producing actinomycetes differ in the level of their antibiotic activity. As a rule, spontaneous variants of the basic type possess the highest antibiotic activity. Other variants representing parallel series have a lower activity level. This raises the possibility to carry out a directed selection of previously known active colonies from populations on the basis of their easily discernible morphological properties. It enhances the efficiency of selection work both in the case of stabilization of the level of antibiotic activity and in the case of obtaining more productive commercial strains.     

Molecular structure and translocation of a multiple antibiotic resistance region of a Psychrobacter psychrophilus permafrost strain

A Psychrobacter psychrophilus strain resistant to tetracycline and streptomycin was isolated from a 15 000–35 000-year-old permafrost subsoil sediment sampled from the coast of the Eastern-Siberian Sea. The genes conferring antibiotic resistance were localized on an c . 30-kb pKLH80 plasmid. It was shown that the antibiotic resistance region of this plasmid has a mosaic structure and contains closely linked streptomycin resistance ( strA-strB ) and tetracycline resistance [ tetR-tet (H)] genes, followed by a novel IS element (IS Ppy1 ) belonging to the IS 3 family. Both the strA-strB and tetR-tet (H) genes of pKLH80 were highly similar to those found in modern clinical bacterial isolates. It was shown that the IS Ppy1 element of pKLH80 can direct translocation of the adjacent antibiotic resistance genes to different target plasmids, either by one-ended transposition or by formation of a composite transposon resulting from the insertion of the IS Ppy1 second copy at the other side of the antibiotic resistance region. Thus, our data demonstrate that clinically important antibiotic resistance genes originated long before the introduction of antibiotics into clinical practice and confirm an important role of horizontal gene transfer in the distribution of these genes in natural bacterial populations.     

Giant linear plasmids of β-lactam antibiotic producing Streptomyces

Abstract A survey of the total cellular DNA from five β-lactam antibiotic-producing Streptomyces spp. by pulsed field gel electrophoresis was conducted to investigate the presence of linear plasmids. Streptomyces clavuligerus NRRL 3585 contained two giant linear plasmids of 120 and 430 kb, in addition to the well-characterized 11.7 kb linear plasmid. Streptomyces griseus NRRL 3851 contained a single giant linear plasmid of 120 kb, and Streptomyces jumonjinensis NRRL 5741 contained two giant linear plasmids (220 and 280 kb), and two smaller linear plasmids. No plasmids were identified in Streptomyces cattleya NRRL 3841 or Streptomyces lipmannii NRRL 3584. Southern hybridization did not reveal any homology shared by these plasmids, and β-lactam antibiotic synthesis gene clusters were located on the chromosome.     

Population dynamics of phage-host interactions and phage conversion of streptomycetes in soil

Abstract The fate of Streptomyces lividans lysogens was studied in sterile and nonsterile soil microcosms. It was found that in sterile soil lysogens grew as well as the parental strain. However, in nonsterile soil, numbers of the lysogen decreased rapidly, indicating a decreased fitness when compared to the original organism. In addition, the release of this phage from a lysogen and its subsequent infection and lysogenisation of a recipient strain was demonstrated in sterile soil.     

Effects of Rapeseed Oil on Activity of Methylmalonyl-CoA Carboxyltransferase in Culture of Streptomyces fradiae

To investigate why more tylosin was produced when Streptomyces fradiae T1558 was cultured in a rapeseed oil medium than in a glucose or starch medium, we measured the activity of methylmalonyl-CoA carboxyltransferase (EC 2.1.3.1) and intracellular propionic acid. The activity of the enzyme, which catalyzes the formation of the precursor of tylosin, protylonolide, was 0.19 U/mg protein in 5 days of culture in rapeseed oil medium, which was 2.5- and 1.3-fold that with the glucose or starch medium, respectively. The intracellular propionic acid concentration was 1.2 g/g of dry weight, which was 4.3- and 2.1-fold that with the glucose or starch medium, respectively. The addition of propionic acid increased tylosin production in batch culture: when 0.2 g/l (final concentration) propionic acid was added to the glucose medium, 3.8 g/l tylosin was produced in 10 days of culture, 4.7-fold the amount without propionic acid. These findings suggest that in glucose medium, intracellular propionic acid is a limiting factor because of the low activity of methylmalonyl-CoA carboxyltransferase of the tylosin biosynthesis pathway.