Isolation of E. coli mutants containing multiple transpositions of IS sequences

Summary The characterization of three E. coli mutants that appeared to have unselected IS1 insertions on the chromosome are described. One had a single new IS1 sequence. The second had three new IS1 sequences. The third had two new IS1 sequences and one of the IS1 sequences in the parent was missing. These mutants were found in a collection of strains that contained IS insertions in the spc operon. The frequency of finding mutants with unselected IS1 transpositions was at least 100 times greater than expected. The results suggest several transposition events may frequently occur in the same cell.     

Genetic evidence for absence of transposition functions from the internal part of Tn981 a relative of Tn9

Summary Inverse transposition of the DNA of pBR322 was found to be mediated by the small transposon Tn981 a relative of Tn9 flanked by direct repeats of IS1. Since the resulting structure IS1:: pBR322::IS1 (Tn983) is transposed in a second step in the absence of Tn981, it is concluded that all the functions necessary for transposition of IS1 flanked transposons are coded for by IS1 itself or the E. coli chromosome, respectively.     

The regulatory role of the IS 1-encoded InsA protein in transposition

We show here that the protein InsA, which is encoded by IS 1 and binds specifically to the terminal inverted repeats of this insertion sequence, negatively regulates IS 1 transposition activity. We demonstrate that it inhibits both IS 1-mediated cointegrate formation and transposition of a synthetic IS 1-based transposon (‘omegon’Ω-on). These results also indicate that the Ω-on which does not itself encode IS 1 transposition functions can be complemented in trans, presumably by the copies of IS 1 resident in the Escherichia coli chromosome. Using insA-lacZ gene fusions, we show that at least part of this effect can be explained by the ability of InsA to repress expression of IS 1-encoded genes both in cis or in trans. The experiments involving Ω-on transposition raise the possibility that InsA inhibits transposition directly by competition with the transposase for their cognate site within the ends of IS 1.     

Thermosensitive vector derived from RP1 plasmid for detection of transposable elements

The involvement of the transposable DNA element of E. coli K12 chromosome in integrative recombination of RP1 plasmid was studied. Using temperature sensitive for replication plasmid RP1ts12--the derivative of RP1 which contains mutated transposon Tnl, it was shown that integration of RP1 into host chromosome and Hfr formation may occur according to a mechanism mediated by chromosome IS-elements. Plasmids that are desintegrated from the chromosome of these Hfrs contain discrete DNA segments (IS-elements) and possess elevated frequency of integration into chromosome of rec+ cells. The latter was used for selection of RP1ts12 recombinants carrying chromosome IS. For identification of IS involved in RP1 integration the number of independent RP1ts 12 recombinants was subjected to restriction and heteroduplex analysis. By analysing recombinants integrated into bacterial chromosome with frequency 5 X 10(-3), a new IS-element of E. coli K12 designated IS111 was discovered. IS111-element is about 1500bp of length, contains Smal, Pst1 and BamH1 restriction endonuclease sites and was found in the same position on the plasmid RP1 in two different orientations. IS-elements that have been revealed in a number of other RP1ts12 recombinants were preliminary identified as IS1-like elements. One recombinants plasmid was found to have an IS5-like elements. The activity of IS-elements inserted into RP1ts12 in recA-dependent integrative recombination was estimated. From the data of absolute and relative RP1ts12 integration frequencies mediated by IS111, IS1- and IS5-like elements a conclusion was made about the absence of E. coli K12 chromosome IS-elements in RP1 plasmid. The Hfr-formation and chromosomal gene transfer by recombinant plasmids RP1ts12: IS111 were studied. The possibility to use insertion RP1ts12 derivatives for the estimation of copies number, mapping and definition of orientation of IS-elements in bacterial chromosome and the possibilities for detection of transposable DNA elements using RP1ts12 in a wide range of gram-negative bacteria are discussed.     

IS<Emphasis Type="Italic">Ppy1</Emphasis>, a novel mobile element of the ancient <Emphasis Type="Italic">Psychrobacter maritimus</Emphasis> permafrost strain: Translocations in <Emphasis Type="Italic">Escherichia coli</Emphasis> K-12 cells and formation of composite transposons

It was shown that IS element ISPpy1 isolated earlier in the permafrost strain Psychrobacter maritimus MR29-12 has a high level of functional activity in cells of the heterologous host Escherichia coli K-12. ISPpy1 can be translocated in E. coli cells by itself and mobilize adjacent genes and can also form composite transposons flanked by two copies of this element. Apart from translocations between different plasmids, the composite ISPpy1-containing transposon Tn5080a is capable of translocation from the plasmid into the E. coli chromosome with high frequency and from the chromosome into the plasmid. Among products of Tn5080a transposition into plasmid R388, simple insertions were predominantly formed together with cointegrates. Upon mobilization of adjacent genes with the use of one ISPpy1 copy, only cointegrates arise.     

IS30, a new insertion sequence of Escherichia coli K12

Summary Three independent spontaneous mutations of prophage P1 affecting the ability of the phage to reproduce vegetatively are due to the insertion of a mobile genetic element, called IS 30. The same sequence is also carried in the R plasmid NR 1-Basel, but not in the parental plasmid NR 1. Southern hybridisation study indicates that the Escherichia coli K 12 chromosome carries several copies of IS 30 as a normal resident. IS 30 is 1.2 kb long and contains unique restriction cleavage sites for Bg/II, ClaI, HindIII, NciI and HincII, and it is cleaved twice by the enzymes HpaII and TaqI. The ends of IS 30 are formed by 26 bp long inverted repeats with 3 bases mismatched. Upon transposition IS 30 generates a duplication of only 2 bp of the target. The following observations suggest a pronounced specificity in target selection by IS 30. In transposition to the phage P 1 genome a single integration site was used three times independently, and in both orientations. A short region of sequence homology has been identified between the P 1 and NR 1-Basel insertion sites. IS 30 has mediated cointegration as well as deletion. The entire IS 30 sequences were duplicated in the cointegrates between a pBR 322 derivative containing IS 30 and the genome of phage P 1–15, and several loci on the P1–15 genome served as fusion sites, some of which were used more than once.     

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.     

Absence of insertions among spontaneous mutants of Salmonella typhimurium

Summary While insertion sequences (IS) in Escherichia coli transpose frequently to generate spontaneous insertion mutants, such mutations are rare in Salmonella typhimurium: the only documented insertion mutation is a hisD mutation caused by the Salmonella-specific IS element IS200. To obtain more examples of IS200 insertion mutations and to seek additional types of IS elements in Salmonella, we selected and characterized 422 independent, spontaneous His^– mutants and some 2100 additional mutants that are not necessarily independent. None of the mutants showed the absolute polar effect characteristic of insertion mutations or the reversion properties characteristic of insertions (low spontaneous reversion frequency and no reversion induction by chemical mutagens). A few mutants, showing a high spontaneous reversion frequency, were screened physically. No insertion mutations were found. Thus insertion mutations appear to be rare in S. typhimurium, in strong contrast to E. coli and despite the possession in Salmonella of at least one type of insertion element (IS200). These results suggest that in Salmonella transposition of the endogenous elements has been controlled. The transposition ability of the elements may have been reduced or favored target sites removed from the host genome.     

The frequency of P1 transduction of the genes of Escherichia coli as a function of chromosomal position: preferential transduction of the origin of replication.

Summary The frequencies with which the generalized transducing phage P1 transduced 26 selected markers on the E. coli. chromosome were measured. The frequencies were found to vary relative to argH ⁺=1 from a maximum of 6.8 near the origin of replication to a minimum of 0.23 for a marker not far from the terminus. The low frequencies obtained for some markers were shown not to result from poor expression under the selective conditions employed. When plotted as a function of marker position on the chromosome the frequencies were found to exhibit a series of peaks and troughs which correspond to those in gene density noted by Bachmann et al. (1976). The possible relationship of these results to the structure of the E. coli chromosome and to the mechanism of generalized transduction are discussed.     

Integration and Intramolecular Transposition of the TnBP3 Transposon ofBordetella pertussis in the Escherichia coliK12 Cells Mutant for the Hpr Protein of the Phosphoenolpyruvate-Dependent Phosphotransferase System

Integration of a plasmid carrying the TnBP3 transposon of Bordetella pertussisinto the chromosome of Escherichia coliand transpositions of the integrated structure within a chromosome in the wild-type and mutant cells ptsHdevoid of the major Hpr protein of the phosphoenolpyruvate-dependent phosphotransferase system were studied. When transposed to a new chromosome site, the integrated structure was precisely (or almost precisely) excised from the metYgene sequence, which resulted in restoration of the Met⁺phenotype. The integration and transposition events were only observed in the E. colicells carrying the ptsH ⁺allele. The ptsHmutations inhibited integration and intramolecular transposition, which were restored after phenotypic or genetic suppression of the ptsHmutation. The intensity of the processes studied were suggested to depend on the integrity of a chain that ensures transferring of the phosphoryl residue by proteins of the phosphotransferase system in E. coliK12.     

Transposition of IS 117, the 2.5 kb Streptomyces coelicolor A3(2) 'minicircle': roles of open reading frames and origin of tandem insertions

IS 117 is a 2527 bp transposable element from Streptomyces coelicolor A3(2) with a circular transposition intermediate. Disruption of 0RF1 of IS 117, presumed to encode a transposase, abolished transposition. Deletion or mutation of 0RF2 and 0RF3, which overlap each other on opposite strands of IS 117, caused a c. 20-fold reduction in integration frequency of the circular form of IS 117 into the Streptomyces lividans chromosome or into the preferred chromosomal target site cloned on a plasmid in transformation experiments. In contrast, inactivation of ORF2/3 did not significantly influence transposition of IS 117 derivatives from an already integrated state in the chromosome to the preferred target site cloned on a plasmid. 0RF2 mutants apparently excised readily from the S. lividans chromosome, whereas excision of integrated wild-type IS 117 derivatives to yield the unoccupied site was not detected; presumably, therefore, the circular transposition intermediate normally arises replicatively. Attempts to promote integration of a plasmid carrying the attachment site of IS 117 by providing the ORF1 product in trans were unsuccessful. Most transformation of S. lividans with circular IS 117 derivatives yielded tandem chromosomal insertions, which arose by co-transformation rather than dimerization of a monomeric insert. Typically, two to three transforming elements gave a transformed strain, suggesting a local concentration of transposase as a limit on integration.     

Multiple copies of the insertion-DNA sequences IS1 and IS2 in the chromosome of E. coli K-12

Summary About 8 copies of the DNA sequence IS1 (which consists of 800 nucleotide pairs) and about 5 copies of the DNA sequence IS2 (1400 nucleotide pairs) have been detected by DNA-DNA hybridization in the chromosome of E. coli K-12. No homology is observed between IS1 and IS2. Both IS1 and IS2 are also found in the DNA of the F plasmid.     

High level expression of human lipocortin (annexin) 1 in Escherichia coli

Summary Human lipocortin (annexin) 1, a member of the annexin family of phospholipid binding proteins, has previously been expressed in E. coli (Huh et al., 1990). To improve the expression level of lipocortin 1 in E. coli, several expression vectors containing either the P_L or the P_trc promoter were constructed. The highest expression level, up to 20 % of the total E. coli proteins, was obtained with plasmid pHT3. Plasmid pHT3 contains the pUC origin, the lipocortin 1 cDNA under P_trc promoter, and the lacI gene.     

Genetic and physical characterization of putP, the proline carrier gene of Escherichia coli K12

Summary Two new mutants of E. coli K12, strains PT9 and PT32 were isolated, that were defective in proline transport. They had no high affinity proline transport activity, but their cytoplasmic membranes retained proline binding activity with altered sensitivity to inhibition by p-chloromercuribenzoate(pCMB). The lesion was mapped at the putP gene, which is located at min 23 on the revised E. coli genetic map (Bachmann 1983) as a composite gene in the proline utilization gene cluster, putP, putC, and putA, arranged in this order. The putC gene was shown to regulate the synthesis of proline dehydrogenase (putA gene product).Hybrid plasmids carrying the put region (Motojima et al. 1979; Wood et al. 1979) were used to construct the physical map of the put region. The possible location of the putP gene in the DNA segment was determined by subcloning the putP gene, genetic complementation, and recombination analyses using several proline transport mutants.Abbreviations pCMB p-chloromercuribenzoate - DM Davis and Mingioli - Ap ampicillin - NTG N-methyl-N-nitro-N-nitrosoguanidine - EMS ethylmethane sulfonate - Str streptomycin - Tet tetracycline - Ac l-azetidine-2-carboxylic acid - DHP 3, 4-dehydro-d,l-proline - MTT 3-(4,5-dimethyl-2)2,5-diphenyl tetrazolium bromide - Tris tris(hydroxymethyl)aminomethane - EDTA ethylenediamine tetraacetic acid - Kan kanamycin - Spc spectinomycin     

Physical characterisation of the "Rac prophage" in E. coli K12.

Summary We confirm the hypothesis of Low (1973) that many E. coli K 12 strains contain a prophage (the Rac prophage) located a few minutes clockwise of the trp operon on the genetic map. We have used restriction endonucleases and ³²P-labelled probes to construct a physical map of this prophage. Some E. coli K 12 strains, including AB1157, have lost the entire prophage, apparently by a specific deletion. This is consistent with prophage excision by site-specific recombination. reverse (rev) phages (Zissler et al., 1971) are recombination proficient derivatives of phage in which the phage recombination functions have been replaced by analogous functions (RecE) derived from the host chromosome (Gottesman et al., 1974; Gillen et al., 1977). Our data support the origin of rev phages by recombination between and the Rac prophage following excision of the Rac prophage from the E. coli chromosome.Important experimental data are included in the Figure legends.     

An Escherichia coli mutant thermosensitive in the B subunit of DNA gyrase: effect on the structure and replication of the colicin E1 plasmid in vitro

Summary An E. coli strain which carries a mutation conferring clorobiocin resistance and temperature sensitivity for growth has recently been described and evidence has been presented suggesting that the mutation is located in the gyrB gene (Orr et al. 1979). The replication of the ColE1 plasmid was analysed in cell-free extracts from this thermosensitive strain. These extracts were totally deficient in the replication of exogenous plasmid DNA and were unable to maintain the superhelical structure of the plasmid DNA. Both defects could be fully complemented by addition of purified gyrB protein.     

Rates of transposition in Escherichia coli

The evolutionary role of transposable elements (TEs) is still highly controversial. Two key parameters, the transposition rate (u and w, for replicative and non-replicative transposition) and the excision rate (e) are fundamental to understanding their evolution and maintenance in populations. We have estimated u, w and e for six families of TEs (including eight members: IS1, IS2, IS3, IS4, IS5, IS30, IS150 and IS186) in Escherichia coli, using a mutation accumulation (MA) experiment. In this experiment, mutations accumulate essentially at the rate at which they appear, during a period of 80 500 (1610 generations × 50 lines) generations, and spontaneous transposition events can be detected. This differs from other experiments in which insertions accumulated under strong selective pressure or over a limited genomic target. We therefore provide new estimates for the spontaneous rates of transposition and excision in E. coli. We observed 25 transposition and three excision events in 50 MA lines, leading to overall rate estimates of u ∼ 1.15 × 10^–5, w ∼ 4 × 10⁻⁸ and e ∼ 1.08 × 10⁻⁶ (per element, per generation). Furthermore, extensive variation between elements was found, consistent with previous knowledge of the mechanisms and regulation of transposition for the different elements.     

Conjugative Interaction Induces Transposition of ISPst9 in Pseudomonas stutzeri AN10

ISPst9 is an ISL3-like insertion sequence (IS) that was recently described in the naphthalene-degrading organism Pseudomonas stutzeri strain AN10. In this paper we describe a novel strong IS regulation stimulus; transposition of ISPst9 is induced in all P. stutzeri AN10 cells after conjugative interaction with Escherichia coli. Thus, we observed that in all P. stutzeri AN10 cells that received genetic material by conjugation the ISPst9 genomic dose and/or distribution was changed. Furthermore, ISPst9 transposition was also observed when P. stutzeri AN10 cells were put in contact with the plasmidless conjugative strain E. coli S17-1λ_pir, but not when they were put in contact with E. coli DH5α (a nonconjugative strain). The mechanism of ISPst9 transposition was analyzed, and transposition was shown to proceed by excision from the donor DNA using a conservative mechanism, which generated 3- to 10-bp deletions of the flanking DNA. Our results indicate that ISPst9 transposes, forming double-stranded DNA circular intermediates consisting of the IS and a 5-bp intervening DNA sequence probably derived from the ISPst9 flanking regions. The kinetics of IS circle formation are also described.     

Escherichia coli K-12 F′ plasmids carrying insertion sequences IS1 and IS5

A simple method is described for the detection of the insertion elements IS1 and IS5 in Escherichia coli F′ plasmids. Several of these insertion elements are normal constituents of the E. coli chromosome and are located on chromosomal regions carried by the F′ plasmids, while several others were probably acquired during the isolation or propagation of the F′ plasmids. The F′ plasmids carrying copies of IS1 or IS5 have been transferred into Salmonella (a host lacking chromosomal copies of IS1 and IS5) where individual copies can be examined for a variety of properties, including structural similarities and ability to transpose to new sites.     

Organization of chimeras between filamentous bacteriophage f1 and plasmid pSC101

Two recombinants formed in vivo between the filamentous phage f1 and the tetracycline-resistance-conferring plasmid pSC101 are capable of transducing sensitive cells to Tet^r. These chimeric filamentous phage, VO-1 and VO-2, were previously shown to contain the entire f1 and pSC101 genomes (Vovis et al., 1977; Ohsumi et al., 1978). The genomes of VO-1 and VO-2 are unstable in vivo; VO-1 breaks down to yield a molecule similar to pSC101 and an f1-like species, f1′. f1′ was previously shown to differ from f1 by the presence of 209 additional nucleotides inserted in the carboxy-terminal portion of gene IV (Ravetch et al., 1979). We have found by hybridization analysis and direct DNA sequencing that this 209-nucleotide segment is present in one copy in pSC101, and that it has properties similar to known transposable elements. Therefore, we have called this sequence IS101. We have characterized the structures of both VO-1 and VO-2 in greater detail by restriction mapping and DNA sequence analysis. Both chimeras contain two copies of IS101, which are present as direct repeats and form the junctions between the f1 and pSC101 genomes. The IS101 elements in VO-1 and VO-2 are flanked by a five-base direct repeat of f1 sequence that is not repeated in wild-type f1. The junction between f1 and pSC101 in VO-1 is located at the same point as the IS101 element in f1′, while in VO-2 the junction between the two genomes is at a point in f1 located between the promoter and ribosome binding site for gene VIII. The pSC101-like molecules derived from the breakdown of VO-1 in vivo are identical to the original pSC101 in the region of IS101. The IS101 elements in the original and derived pSC101 plasmids are not flanked by any repeated sequence. Attempts to regenerate VO-1 from f1′ and pSC101, both of which contain one IS101 element, indicate that the breakdown of VO-1 is irreversible. These results are discussed in terms of current models for transposition, which postulate structures similar to VO-1 and VO-2 as intermediates in transposition.