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PubMed1978

Adjacent insertion sequences IS2 and IS5 in bacteriophage Mu mutants and an IS5 in a lambda darg bacteriophage.

Using electron microscopic heteroduplex analysis, we have demonstrated that an insertion found in a Mu prophage and in some infectious. Mu deletion-substitution mutants derived from it consists of bacterial insertion sequence IS2 linked directly to IS5. Other infectious Mu mutants derived from the same lysogen have only IS5 or a portion of IS2. In addition, we have found that an independent insertion in a transducing phage, lambda 13 dargB2, is IS5. The ends of IS5 are short, inverted duplications of each other. These observations support the notion that the DNA insertion previously designated IS5 on the basis of a single example in lambda KH100 is a bona fide bacterial insertion sequence.

Coliphages↗
PubMed1983

New class of mutations in Escherichia coli (uup) that affect precise excision of insertion elements and bacteriophage Mu growth.

We have used a papillation screening technique to isolate mutations that increase the precise excision of insertion elements. The three mutations isolated stimulated precise excision of Tn5, Tn10, and the IS elements. They had a large, 20- to 600-fold, effect on excision of Tn5 at various chromosomal sites. The varied stimulation for different Tn5 insertions showed that the mutations altered the relationship between a precise excision activity and the chromosomal sequence flanking an inserted Tn5. A much smaller stimulation was observed for insertions on the plasmid F'128. The stimulation was recA independent. The mutations also reduced the rate of production of bacteriophage Mu progeny. The mutations were mapped by two- and three-factor crosses with closely linked Tn10 insertions. They defined the uup locus, located at 21.3 min on the Escherichia coli map, next to pyrD.

Bacteriophage mu↗
PubMed1984

Plasmid insertion mutagenesis and lac gene fusion with mini-mu bacteriophage transposons.

Small bacteriophage Mu transposable elements containing the lac operon structural genes were constructed to facilitate the isolation and use of Mu insertions and lac gene fusions. These mini-Mu elements have selectable genes for either ampicillin or kanamycin resistance and can be used to form both transcriptional and translational lac gene fusions. Some of the mini-Mu-lac elements constructed are deleted for the Mu A and B transposition genes and form stable insertions that cannot undergo transposition unless complemented for these functions. A procedure was developed for selecting mini-Mu insertions specifically into plasmids, including commonly used high-copy-number cloning vectors such as pBR322. Mu insertions in pBR322 were found to be distributed around the plasmid, but insertions in certain regions occurred more frequently than in others.

Ampicillin↗
PubMed1984

Genetic analysis of the low calcium response in Yersinia pestis mu d1(Ap lac) insertion mutants.

Yersinia pestis strain KIM requires plasmid pCD1 for expression of the low calcium response, plague virulence antigen V, and virulence. We constructed Mu d1(Ap lac) insertion mutants of this plasmid which were unable to express the low calcium response. The insertions mapped to a 17-kilobase region of the plasmid. By determining the orientation of the insertions and examining beta-galactosidase production from the Mu d1 lac genes, we determined that this region contains three units of transcription, one of which is transcribed in a direction opposite the direction of transcription of the other two. Transcription of at least two of these units was induced significantly at 37 degrees C compared with 26 degrees C. Ca2+ (2.5 mM) and ATP (18 mM) had no significant effect on the level of expression of the Mu d1 lac genes of these mutants. All insertions in the region strongly reduced production of the V antigen. Insertions from each unit of transcription also reduced virulence in mice.

Antigens, Bacterial↗
PubMed1985

Vector insertion mutagenesis of Rhizobium sp. strain ORS571: direct cloning of mutagenized DNA sequences.

When the limited-host-range plasmid pVP2021 carrying Tn5 was mobilized into Rhizobium sp. strain ORS571 and stable acquisition of Tn5 was selected, ORS571 plasmid-genome cointegrates were exclusively obtained; direct Tn5 transposition was never observed. In every case, genomic cointegrates exhibited an additional (third) IS50 element that bordered VP2021 DNA sequences but maintained a single Tn5 element. Genomic cointegrates containing IS50 triplications were stable; neither phenotypic reversion nor resolution was detectable. Auxotrophic mutant strains (vector insertion mutants) were identified at expected frequencies among derivatives carrying ostensibly random genomic pVP2021 insertions; N2 fixation (Nif)-defective vector insertion mutants were observed among these derivatives at a frequency of 10(-3). The presence of integrated pVP2021 in ORS571 nif::VP2021 mutant genomes enabled VP2021 to constitute an endogenous cloning vector. After EcoRI or KpnI digestions, genomic nif::pVP2021 DNA sequences contiguous with integrated pVP2021 were directly cloned as new replicons without addition of an exogenous vector. Recombinant plasmids derived from two such nif::pVP2021 mutants hybridized to previously analyzed ORS571 Nif DNA sequences. Recombinant plasmid DNA and ORS571 Nif region DNA were found to be colinear; pVP2021 insertions could be accurately mapped. pVP2021 insertion-mutagenesis thus allows the direct cloning of ORS571 gene sequences for which mutant phenotypes can be selected or screened.

Base Sequence↗
PubMed1987

Instability of Tn5 inserts in cyanobacterial cloning vectors.

Transposon Tn5 was used to produce random insertions in two hybrid cloning vectors for the unicellular cyanobacterium Anacystis nidulans. The transposon-containing plasmids were used to localize essential replication functions and to characterize the stability of large inserts in these vectors. The effect of the insertions on plasmid function was tested by transformation into a derivative of A. nidulans that had been cured of the endogenous plasmid used to construct the vectors. A region of approximately 4 kilobases was essential for successful plasmid transformation and replication. This region has also been shown to be involved in plasmid replication by deletion analysis. High rates of excision of Tn5 inserts within this region and restoration of normal replication function were observed when transformants were selected by using a resistance marker outside the replication region in the absence of selection for the transposon-coded kanamycin resistance. Transposon inserts outside this region were not deleted.

Ampicillin↗
PubMed1988

Recognition of Escherichia coli attTn7 by transposon Tn7: lack of specific sequence requirements at the point of Tn7 insertion.

Transposon Tn7 inserts at high frequency into a specific site in the Escherichia coli chromosome called attTn7. We show that the point of Tn7 insertion in attTn7 lies within the transcriptional terminator of the bacterial glmS gene. We have exploited the glmS transcription terminator to isolate mutants with altered sequences at the point of Tn7 insertion and have used these mutants to show that the nucleotide sequence at the point of Tn7 insertion is irrelevant to attTn7 target activity. Thus, the nucleotides which provide attTn7 target activity are distinct from the point of Tn7 insertion. We have also examined the effect of transcription on the capacity of attTn7 to act as a target for Tn7 transposition. Our results suggest that transcription of attTn7 does not modulate its Tn7 target activity.

Base Sequence↗
PubMed1989

Nucleotide sequence of IS492, a novel insertion sequence causing variation in extracellular polysaccharide production in the marine bacterium Pseudomonas atlantica.

The complete nucleotide sequence of insertion element IS492, which causes reversible inactivation of extracellular polysaccharide production in the marine bacterium Pseudomonas atlantica, is presented. Insertion of IS492 results in the EPS- phenotype, and excision results in restoration of EPS+. DNA sequencing of the site of insertion in the eps locus showed that insertion of IS492 generates a 5-base-pair repeat and that its excision is precise. IS492 is 1,202 nucleotides in length and contains one large open reading frame encoding a protein of 318 amino acids, a candidate for transposition function. No similarity between IS492 and other transposable elements has been found. Unlike the situation with other insertion sequences, no direct or inverted repeats exist at the termini of IS492.

Amino Acid Sequence↗
PubMed1989

Temperature-dependent insertion of prolipoprotein into Escherichia coli membrane vesicles and requirements for ATP, soluble factors, and functional SecY protein for the overall translocation process.

The requirements for the translocation of prolipoprotein into membrane vesicles were examined in an in vitro system. As measured by the eventual modification and processing of the prolipoprotein to form mature lipoprotein, the overall translocation process was found to require ATP hydrolysis, the presence of some heat-labile soluble cytoplasmic translocation factors, and the function of a cytoplasmic membrane protein, SecY/PrlA. However, the initial step of complete insertion of prolipoprotein into the membrane vesicles occurred without apparent requirements of a nucleotide, cytoplasmic translocation factors, or a functional SecY/PrlA membrane protein. Immunopurified prolipoprotein spontaneously inserted into membrane vesicles at elevated temperatures and required ATP and cytoplasmic translocation factors to form mature lipoprotein. The prolipoprotein inserted most efficiently into liposomes made of negatively charged phospholipids, indicating the importance of phospholipids in protein translocation. These results suggest that ATP hydrolysis and the actions of both cytoplasmic translocation factors and a functional SecY/PrlA membrane protein occur at a step(s) after the insertion of the precursors into membrane vesicles. The initial step of spontaneous insertion of prolipoprotein into membranes is in good agreement with membrane trigger hypothesis proposed by W. Wickner (Annu. Rev. Biochem. 48:23-45, 1979) and the helical hairpin hypothesis proposed by D. M. Engleman and T. A. Steitz (Cell 23:411-422, 1981).

Adenosine Triphosphate↗
PubMed1990

Mutations that affect Tn5 insertion into pBR322: importance of local DNA supercoiling.

The major hot spot of transposon Tn5 insertion in plasmid pBR322 (hot spot I) is in the promoter for the tetracycline resistance gene (tet). We made a series of pBR322 derivatives with mutations in and around this promoter and assayed their effects on insertion of Tn5 into hot spot I. Those mutations which reduced transcription from the tet promoter also reduced the frequency of insertion into hot spot I. Transcription and translation of tet are thought to cause the formation of paired domains of negative and positive supercoiling in pBR322. An amber codon in tet, 345 base pairs from hot spot I, decreases the negative supercoiling of the DNA segment containing hot spot I because it terminates translation of tet prematurely. We report here that this amber mutation also reduces insertion into hot spot I. These results suggest that the ability of Tn5 to insert into its major hot spot in pBR322 depends directly on negative supercoiling of the target DNA.

Base Sequence↗
PubMed1990

Transposition in Shigella dysenteriae: isolation and analysis of IS911, a new member of the IS3 group of insertion sequences.

Twenty-nine clear-plaque mutants of bacteriophage lambda were isolated from a Shigella dysenteriae lysogen. Three were associated with insertions in the cI gene: two were due to insertion of IS600, and the third resulted from insertion of a new element, IS911. IS911 is 1,250 base pairs (bp) long, carries 27-bp imperfect terminal inverted repeats, and generates 3-bp duplications of the target DNA on insertion. It was found in various copy numbers in all four species of Shigella tested and in Escherichia coli K-12 but not in E. coli W. Analysis of IS911-mediated cointegrate molecules indicated that the majority were generated without duplication of IS911. They appeared to result from direct insertion via one end of the element and the neighboring region of DNA, which resembles a terminal inverted repeat of IS911. Nucleotide sequence analysis revealed that IS911 carries two consecutive open reading frames which code for potential proteins showing similarities to those of the IS3 group of elements.

Amino Acid Sequence↗
PubMed1991

Heterozygosity and instability of amplified chromosomal insertions in the radioresistant bacterium Deinococcus radiodurans.

Natural transformation, duplication insertion, and plasmid transformation in Deinococcus radiodurans, a bacterium that contains 4 to 10 chromosomes per cell, were studied. Duplication insertions were often heterozygous, with some chromosomes containing highly amplified insertions and others containing no insertions. Large amplified regions were apparently deleted by intrachromosomal recombination, generating as by-products extrachromosomal circles consisting of multiple tandem repeats of the amplified sequence. The circles were of heterogenous integer sizes, containing as many as 10 or more amplification units. Two strains that are defective in natural transformation and sensitive to DNA-damaging agents were further characterized. Both strains were defective in duplication insertion. While on strain was normal for plasmid transformation, the other was totally defective in this regard, suggesting that plasmid transfer in D. radiodurans may require recombinational functions.

Chromosomes, Bacterial↗
PubMed1992

Ty insertions upstream and downstream of native DUR1,2 promoter elements generate different patterns of DUR1,2 expression in Saccharomyces cerevisiae.

Expression of allantoin pathway genes is subject to induction and nitrogen catabolite repression. Two classes of cis-dominant mutations (DUR80 and DUR1,2-Oh) result in overproduction of DUR1,2 mRNA. In DUR80 mutants, DUR1,2 expression remained inducible, nitrogen catabolite repression sensitive, and unresponsive to cell ploidy, i.e., overproduction was superimposed on normal gene regulation. DUR1,2-Oh mutations, in contrast, generated a pattern of DUR1,2 expression similar to that often reported when a Ty element inserts upstream of a gene, the ROAM phenotype. We analyzed four independent DUR80 and DUR1,2-Oh alleles. The DUR1,2-Oh mutation was, as expected, a Ty insertion at -445 3' of the native DUR1,2 upstream activation sequences (UASs). All three DUR80 alleles were also Ty insertions between -644 and -653 immediately 5' of the native DUR1,2 USASs. We suggest that the difference in DUR1,2-Oh and DUR80 phenotypes depends on whether the native cis-acting elements and transcription factors associated with them can operate. If they can, enhancement of normally regulated DUR1,2 expression is observed. This is a novel phenotype for Ty insertions. If the native DUR1,2 cis-acting elements are not present, the case when Ty insertion occurs 3' of them, a ROAM phenotype is generated. Nitrogen-regulated upstream activation sequence (UASNTR)-homologous sequences present in the Ty delta elements rather than cis-acting elements required for Ty transcription are the most likely candidates to serve as the cis-acting elements mediating the DUR80 phenotype.

Base Sequence↗
PubMed1994

Isolation of a novel IS3 group insertion element and construction of an integration vector for Lactobacillus spp.

An insertion sequence (IS) element from Lactobacillus johnsonii was isolated, characterized, and exploited to construct an IS-based integration vector. L. johnsonii NCK61, a high-frequency conjugal donor of bacteriocin production (Laf+) and immunity (Lafr), was transformed to erythromycin resistance (Emr) with the shuttle vector pSA3. The NCK61 conjugative functions were used to mobilize pSA3 into a Laf- Lafs EMs recipient. DNA from the Emr transconjugants transformed into Escherichia coli MC1061 yielded a resolution plasmid with the same size as that of pSA3 with a 1.5-kb insertion. The gram-positive replication region of the resolution plasmid was removed to generate a pSA3-based suicide vector (pTRK327) bearing the 1.5-kb insert of Lactobacillus origin. Plasmid pTRK327 inserted randomly into the chromosomes of both Lactobacillus gasseri ATCC 33323 and VPI 11759. No homology was detected between plasmid and total host DNAs, suggesting a Rec-independent insertion. The DNA sequence of the 1.5-kb region revealed the characteristics of an IS element (designated IS1223): a length of 1,492 bp; flanking, 25-bp, imperfect inverted repeats; and two overlapping open reading frames (ORFs). Sequence comparisons revealed 71.1% similarity, including 35.7% identity, between the deduced ORFB protein of the E. coli IS element IS150 and the putative ORFB protein encoded by the Lactobacillus IS element. A putative frameshift site was detected between the overlapping ORFs of the Lactobacillus IS element. It is proposed that, similar to IS150, IS1223 produces an active transposase via translational frameshifting between two tandem, overlapping ORFs.

Amino Acid Sequence↗
PubMed1997

In vivo nickel insertion into the carbon monoxide dehydrogenase of Rhodospirillum rubrum: molecular and physiological characterization of cooCTJ.

The products of cooCTJ are involved in normal in vivo Ni insertion into the carbon monoxide dehydrogenase (CODH) of Rhodospirillum rubrum. Located on a 1.5-kb DNA segment immediately downstream of the CODH structural gene (cooS), two of the genes encode proteins that bear motifs reminiscent of other (urease and hydrogenase) Ni-insertion systems: a nucleoside triphosphate-binding motif near the N terminus of CooC and a run of 15 histidine residues regularly spaced over the last 30 amino acids of the C terminus of CooJ. A Gm(r)omega-linker cassette was developed to create both polar and nonpolar (60 bp) insertions in the cooCTJ region, and these, along with several deletions, were introduced into R. rubrum by homologous recombination. Analysis of the exogenous Ni levels required to sustain CO-dependent growth of the R. rubrum mutants demonstrated different phenotypes: whereas the wild-type strain and a mutant bearing a partial cooJ deletion (of the region encoding the histidine-rich segment) grew at 0.5 microM Ni supplementation, strains bearing Gm(r)omega-linker cassettes in cooT and cooJ required approximately 50-fold-higher Ni levels and all cooC insertion strains, bearing polar or nonpolar insertions, grew optimally at 550 microM Ni.

Aldehyde Oxidoreductases↗
PubMed2003

ISCce1 and ISCce2, two novel insertion sequences in Clostridium cellulolyticum.

Two new insertion sequences, ISCce1 and ISCce2, were found to be inserted into the cipC gene of spontaneous mutants of Clostridium cellulolyticum. In these insertional mutants, the cipC gene was disrupted either by ISCce1 alone or by both ISCce1 and ISCce2. ISCce1 is 1,292 bp long and has one open reading frame. The open reading frame encodes a putative 348-amino-acid protein with significant levels of identity with putative proteins having unknown functions and with some transposases belonging to the IS481 and IS3 families. Imperfect 23-bp inverted repeats were found near the extremities of ISCce1. ISCce2 is 1,359 bp long, carries one open reading frame, and has imperfect 35-bp inverted repeats at its termini. The open reading frame encodes a putative 398-amino-acid protein. This protein shows significant levels of identity with transposases belonging to the IS256 family. Upon transposition, both ISCce1 and ISCce2 generate 8-bp direct repeats of the target sequence, but no consensus sequences could be identified at either insertion site. ISCce1 is copied at least 20 times in the genome, as assessed by Southern blot analysis. ISCce2 was found to be mostly inserted into ISCce1. In addition, as neither of the elements was detected in seven other Clostridium species, we concluded that they may be specific to the C. cellulolyticum strain used.

Amino Acid Sequence↗
PubMed2004

Increased motility of Escherichia coli by insertion sequence element integration into the regulatory region of the flhD operon.

The flhD operon is the master operon of the flagellar regulon and a global regulator of metabolism. The genome sequence of the Escherichia coli K-12 strain MG1655 contained an IS1 insertion sequence element in the regulatory region of the flhD promoter. Another stock of MG1655 was obtained from the E. coli Genetic Stock Center. This stock contained isolates which were poorly motile and had no IS1 element upstream of the flhD promoter. From these isolates, motile subpopulations were identified after extended incubation in motility agar. Purified motile derivatives contained an IS5 element insertion upstream of the flhD promoter, and swarm rates were sevenfold higher than that of the original isolate. For a motile derivative, levels of flhD transcript had increased 2.7-fold, leading to a 32-fold increase in fliA transcript and a 65-fold increase in flhB::luxCDABE expression from a promoter probe vector. A collection of commonly used lab strains was screened for IS element insertion and motility. Five strains (RP437, YK410, MC1000, W3110, and W2637) contained IS5 elements upstream of the flhD promoter at either of two locations. This correlated with high swarm rates. Four other strains (W1485, FB8, MM294, and RB791) did not contain IS elements in the flhD regulatory region and were poorly motile. Primer extension determined that the transcriptional start site of flhD was unaltered by the IS element insertions. We suggest that IS element insertion may activate transcription of the flhD operon by reducing transcriptional repression.

Base Sequence↗
PubMed1983

Acquisition of Host Cell DNA Sequences by Baculoviruses: Relationship Between Host DNA Insertions and FP Mutants of Autographa californica and Galleria mellonella Nuclear Polyhedrosis Viruses.

Mutants of Autographa californica and Galleria mellonella nuclear polyhedrosis viruses, which produce an altered plaque phenotype as a result of reduced numbers of viral occlusions in infected cells, were isolated after passage in Trichoplusia ni (TN-368) cells. These mutants, termed FP (few-polyhedra) mutants, had acquired cell DNA sequences ranging from 0.8 to 2.8 kilobase pairs in size. The insertions of cell DNA occurred in a specific region between 35.0 and 37.7 map units of the A. californica viral genome. A cloned viral fragment containing one of the host DNA inserts was homologous to host DNA inserts in two other mutant viruses and to dispersed, repetitious sequences in T. ni cell DNA. Most of the homology between the cloned insert and cell DNA was contained within a 1,280-base-pair AluI fragment. Marker rescue studies and analysis of infected-cell-specific proteins suggested that the insertion of cell DNA into the viral genomes resulted in the FP plaque phenotype, possibly through the inactivation of a 25,000-molecular-weight protein.

Journal Article↗