A cloned DNA fragment from bacteriophage P1 enhances IS2 insertion

Summary A 1.75 kb DNA segment of the bacteriophage P1 genome is known to serve as a preferred target for IS2 insertions. The presence of this fragment in a plasmid expressing the galK gene dramatically increases the proportion of IS2 insertions among spontaneous galK ^- mutants. Subfragments from two different parts of the 1.75 kb segment independently stimulate IS2 insertion, while another subfragment does not. In the plasmids studied IS2 elements not only insert into the cloned P1 fragment but also into parts of the galK gene with similar probability and mostly in one orientation. Many insertion sites are unique but several specific sites within the preferred target are repeatedly used for IS2 integration. The experimental data are compatible with a proposed cooperative mechanism, according to which more than one attracting sequence on the same plasmid might significantly enhance the probability of a particular target region to attract IS2.     

Electron microscopy of polar insertions in the lac operon of Escherichia coli

Summary Several strongly polar mutations in the omega region of the z gene of the lac operon result from insertions consisting of only two specific sequences of DNA, one about 870 and the other 1170 nucleotide pairs long (based on single-strand measurements). No sequence homology was detected between the shorter (IS1) and longer (IS3) insertions. The IS1 insertion was shown to possess a specific attachment site, but it can be inserted with either orientation at several sites in the z gene. Four insertion sites in the omega region of gene z were identified and the position of the lac5 substitution and the SR2 deletion in the plac DNA were determined by heteroduplex mapping.     

Insertions of transposable elements in the promoter proximal region of the gene cluster for Escherichia coli H+-ATPase: 8 base pair repeat generated by insertion of IS1

Summary A plasmid pKY159 (Yamaguchi and Yamaguchi 1983) carrying a promoter proximal portion of the gene cluster of the proton-translocating ATPase (H⁺-ATPase) of Escherichia coli causes growth inhibition of wild-type cells. Insertion of a transposable element in this plasmid released this inhibitory effect. In analyzing this inhibitory effect, we determined the insertion points at the nucleotidesequence level of transposable elements on 30 independent derivatives of pKY159. Insertions of IS1, IS5, and were found between the promoter and the gene for a possible component of 14,000 daltons of the H⁺-ATPase. Of 31 insertions, 26 were of IS1 and were located at the same site, indicating that this site is a hotspot for IS1 insertion and that IS1 insertion is much more frequent than that of IS5 of in this region. Four different sites for IS1 insertion were found; in two of these an 8 base pair (bp) duplicate of the target sequence (AAAAACGT and AAACGTTG) was generated, while in the other two a 9 bp duplicate was found. In all cases in this study the nucleotide sequence of IS1 was the same as that of IS1-K. In the two cases with an 8 bp duplicate in different sites, a common 6 bp sequence (AAACGT) was found. These results suggested that generation of the 8 bp duplicate is related to the common sequence rather than a mutation in IS1 suggested by Iida et al. (1981) and also suggested that the essential length of the duplicate is 8 bp or less than 8 bp. A 6 bp sequence (GTGATG) homologous to the end portion of IS1 was found at the hotspot, but not at other sites, suggesting that this homology contributed to the high frequency of IS1 insertion. The direction of IS1 insertion at the hotspot was the same in 25 of 26 instances, suggesting that the direction of IS1 insertion is determined by the structure of the target and/or its nearby sequence.Abbreviations bp base pairs - 14 K protein a possible component of the H⁺-ATPase with molecular weight of 14,000 (see Kanazawa and Futai 1982 for details) - EDTA ethylenediaminetetraacetate     

Factors determining the frequency of plasmid cointegrate formation mediated by insertion sequence IS3 from Escherichia coli.

Summary Transposition events mediated by plasmid-borne copies of the insertion sequence IS3 of Escherichia coli are difficult to detect because of a low frequency of cointegrate formation. We found that cointegration activity could be strongly enhanced by using plasmid constructions in which a second IS3 element, disabled by a large deletion, was placed adjacent to an intact IS3 copy. Attempts to construct plasmids containing two adjacent intact IS3 copies were unsuccessful, probably because of instability. Transpositional hyperactivity of tandemly duplicated IS sequences was previously described for spontaneous duplications of IS21 and IS30 and may well be a more general phenomenon. The frequency of cointegration events was also strongly increased in an E. coli strain deficient in Dam methylation, suggesting that IS3, like some other Dam site-containing IS elements, is regulated by the Dam methylation system. Insertion sites were strongly clustered within the target lambda repressor gene; however no sequence specificity determinants could be identified. All insertions analyzed carried the IS element in the same orientation; target sequence duplications were mostly 3 bp, but in some cases 4 by long. To obtain information about the roles of the open reading frames (ORFs) in IS3, we constructed plasmid-borne mutant elements in which potentially functional reading frames were inactivated by site-directed mutations; the mutants were introduced into partial tandem constructions and tested in cointegration assays. Mutations inactivating the putative initiation codons of ORF I and 11 in the intact element reduced insertion activity to less than 4% of the wild type, whereas the introduction of a termination codon into ORF IV had no effect on cointegration frequency. We conclude that translation of ORFs I and II is essential for cointegration activity and that the mutagenized ATG codons most probably serve as the normal initiation codons in the wild-type element. In contrast, ORF IV could either be non-functional or its gene product could be supplied in trans from chromosomal elements.     

Translocation specificity of the Tn3 element: characterization of sites of multiple insertions

247 independent events involving insertion of the TN3 transposable element into a 4 kb constructed plasmid (pTU4) of partially known DNA sequence were studied by restriction endonuclease mapping, and 65 of these insertion sites were examined further by DNA sequence analysis. Our results show that the previously proposed regional specificity for Tn3 insertion is associated with a strong preference for AT-rich segments as insertion sites. Moreover, multiple insertions of the Tn3 occurred at certain AT-rich nucleotide positions, and 23 of 26 independent insertion events at a single nucleotide position were found to be in the same orientation. A region of the recipient plasmid showing major homology with the terminal 18 bp of Tn3 was identified in the vicinity of an 11 nucleotide segment that included three insertional hot spots and 36 independent insertions. Our results indicate that the site and orientation of insertion of Tn3 are at least partly determined by the primary nucleotide sequence of the recipient genome, and suggest that insertional hot spots may result from the combined effects of AT richness plus homology of the recipient genome with the terminal sequences of Tn3.     

Characterization of IS1389, a new member of the IS3 family of insertion sequences isolated from Xanthomonas campestris pv. amaranthicola

IS1389, a new insertion sequence belonging to the IS3 family, has been identified in Xanthomonas campestris pv. amaranthicola. The genome of this bacterium contains at least 11 copies of the element, whereas no hybridizing sequences were detected in other Xanthomonas species [X. axonopodis, X. fragaridae, X. phaseoli, and X. (Stenotrophomonas) maltophila]. Two nearly identical copies of the element (IS1389-A and IS1389-B) were characterized. According to analysis of sequence alignments and similar structural features, IS1389 belongs to the IS407 subgroup of the IS3 family, which duplicates 4 bp of target DNA upon insertion. IS1389-A was found in the proximity of the modification gene of the XamI restriction-modification system. Received: 17 November 1998 / Accepted: 22 April 1999     

A New Insertion Variant, IS231I, Isolated from a Mosquito-Specific Strain of Bacillus thuringiensis

A new insertion variant belonging to the family IS231, designated IS231I, was isolated from a mosquito larvicidal strain of the Bacillus thuringiensis serovar sotto (H4ab). IS231I was 1653 bp long and delimited by two 20 bp inverted repeats with one mismatch, flanked by two perfect 11 bp direct repeats. The element contained a single open reading frame (ORF) encoding 478 amino acids and five conserved domains: N1, N2, N3, C1, and C2. The 5′ noncoding region upstream of the ORF, presumed to form a stable stem-and-loop structure, was highly conserved in IS231I. The secondary structure conformation had a deduced free energy (ΔG = 25°C) of −17.2 kcal/mol. Comparison of the IS231I amino acid sequence with those of the 10 existing IS variants revealed that the new variant shares 89% identity with IS231A and IS231B, 65–66% with IS231M and IS231N, and 38% with IS231W.     

Inactivation of the Serratia marcescens gene for the lipoprotein in Escherichia coli by insertion sequences, IS1 and IS5; sequence analysis of junction points

Summary A pBR322-derived plasmid pKEN221 carrying a Serratia marcescens lpp gene overproduces the outer membrane lipoprotein in an Escherichia coli lpp ^–cell. However, when this strain was continuously cultured in a rich medium for about thirty generations, many Lpp^– mutants were accumulated. Out of six mutants analyzed, three were found to carry insertion mutation in the lpp gene in pKEN221. From restriction enzyme mapping and hybridization analysis of the mutant plasmid DNA, it was found that two mutants were caused by insertion sequence IS1 and one by IS5. Nucleotide sequence analysis of these mutant DNAs revealed that both IS1 and IS5 insertions occured in the A-T rich 5 untranslated region of the lpp gene. While the IS1 insertion resulted in a direct duplication of a nine-base-pair sequence in the original pKEN221 DNA at the junction with IS1, the IS5 insertion resulted in a direct duplication of a four-base-pair sequence. IS5 was found to contain inverted-repeat sequences of twelve nucleotides at its exact ends. This is the first example of the nucleotide sequence analysis of an IS5 insertion mutation. By Southern blot hybridization, the E. coli chromosomal DNA was found to contain about ten copies of IS5.     

A selection cartridge for rapid detection and analysis of spontaneous mutations including insertions of transposable elements in Enterobacteriaceae

Summary We present a method that allows positive selection and rapid analysis of mutations in Enterobacteriaceae. Mutations are detected in a 2630 bp selection cartridge inserted in two different bacterial mutlicopy plasmid vectors. Spontaneous mutations in Escherichia coli, Enterobacter cloacae and Citrobacter freundii include insertions, deletions and point mutations. The small size of the target sequence facilitates rapid analysis of DNA rearrangements by cleavage with restriction enzymes and of any type of mutation by DNA sequence analysis. While in E. coli insertions of the mobile elements IS1, IS2 and IS5 were readily found, insertions of putative new transposable elements were detected in Enterobacter cloacae. The selection cartridge can thus serve as a tool for studying the spectrum of insertion mutations in Enterobacteriaceae and probably other Gramnegative bacteria, and the dependency of this spectrum on physiological and environmental factors and the host's genetic background can be investigated.     

Cloning, mapping and functional characterization of the hemB gene of Pseudomonas aeruginosa, which encodes a magnesium-dependent 5-aminolevulinic acid dehydratase

During tetrapyrrole biosynthesis 5-aminolevulinic acid dehydratase (ALAD) catalyzes the condensation of two molecules of 5-aminolevulinic acid (ALA) to form one molecule of the pyrrole derivative porphobilinogen. In Escherichia coli, the enzyme is encoded by the gene hemB. The hemB gene was cloned from Pseudomonas aeruginosa by functional complementation of an E. coli hemB mutant. An open reading frame of 1011?bp encoding a protein of 336 amino acids (M_r?=?37?008) was identified. The gene was mapped to SpeI fragment G and DpnI fragment G of the P. aeruginosa chromosome, corresponding to the 10 to 12?min region of the new map or 19 to 22?min interval of the old map. The 5′ end of the hemB mRNA was determined and the ?10 and ?35 regions of a potential σ⁷⁰-dependent promoter were localized. No obvious regulation of the hemB gene by oxygen, nitrate, heme or iron was detected. Alignment of the amino acid sequences deduced from hemB revealed a potential metal-binding site and indicated that the enzyme is Mg²⁺-dependent. P.?aeruginosa hemB was overexpressed in an E. coli hemB mutant using the phage T7 RNA polymerase system and its Mg²⁺-dependent activity was directly demonstrated.     

Cloning, mapping and functional characterization of the hemB gene of Pseudomonas aeruginosa , which encodes a magnesium-dependent 5-aminolevulinic acid dehydratase

During tetrapyrrole biosynthesis 5-aminolevulinic acid dehydratase (ALAD) catalyzes the condensation of two molecules of 5-aminolevulinic acid (ALA) to form one molecule of the pyrrole derivative porphobilinogen. In Escherichia coli, the enzyme is encoded by the gene hemB. The hemB gene was cloned from Pseudomonas aeruginosa by functional complementation of an E. coli hemB mutant. An open reading frame of 1011 bp encoding a protein of 336 amino acids (M_r = 37 008) was identified. The gene was mapped to SpeI fragment G and DpnI fragment G of the P. aeruginosa chromosome, corresponding to the 10 to 12 min region of the new map or 19 to 22 min interval of the old map. The 5′ end of the hemB mRNA was determined and the −10 and −35 regions of a potential σ⁷⁰-dependent promoter were localized. No obvious regulation of the hemB gene by oxygen, nitrate, heme or iron was detected. Alignment of the amino acid sequences deduced from hemB revealed a potential metal-binding site and indicated that the enzyme is Mg²⁺-dependent. P. aeruginosa hemB was overexpressed in an E. coli hemB mutant using the phage T7 RNA polymerase system and its Mg²⁺-dependent activity was directly demonstrated. Received: 11 July 1997 / Accepted: 9 October 1997     

Determination and Characterization of IS4Bsu1-Insertion Loci and Identification of a New Insertion Sequence Element of the IS256 Family in a Natto Starter

The insertion sequence IS4Bsu1 frequently causes Bacillus subtilis starters for the production of Japanese fermented soybean pasts (natto) to lose the ability to produce poly-γ-glutamate, the viscous material characteristic of natto. Bacillus subtilis NAFM5, a derivative of a natto starter, has six IS4Bsu1 copies on its chromosome. In this study, we determined all six insertion loci of the insertion sequence (IS). One was located in the coding region of yktD, a putative gene involved in polyketide synthesis. Four were located in non-coding regions between iolR and iolA, between tuaA and lytC, between rapI and orf1 (a potential gene of unknown function), and between ynaE and orf3 (a putative gene similar to thiF), and one resided in an intergenic region between divergent possible orf4 and orf5 genes of unknown function. Here we describe the structural features of these loci and discuss the effects of the IS4Bsu1 insertions on the functions of the target gene and the expression of the downstream genes. In addition, we found that strain NAFM5 and commercial natto starters possess eight to 10 loci of another IS of the IS256 family (designated IS256Bsu1) on their chromosomes. IS256Bus1 appeared active in transposition, potentially causing phenotypic alterations in natto starters like those induced by IS4Bsu1.     

Characterization of IS1676 from Rhodococcus erythropolis SQ1

To develop a transposable element-based system for mutagenesis in Rhodococcus, we used the sacB gene from Bacillus subtilis to isolate a novel transposable element, IS1676, from R. erythropolis SQ1. This 1693 bp insertion sequence is bounded by imperfect (10 out of 13 bp) inverted repeats and it creates 4 bp direct repeats upon insertion. Comparison of multiple insertion sites reveals a preference for the sequence 5′-(C/T)TA(A/G)-3′ in the target site. IS1676 contains a single, large (1446 bp) open reading frame with coding potential for a protein of 482 amino acids. IS1676 may be similar to an ancestral transposable element that gave rise to repetitive sequences identified in clinical isolates of Mycobacteriumkansasii. Derivatives of IS1676 should be useful for analysis of Rhodococcus strains, for which few other genetic tools are currently available. Received: 1 April 1999 / Received revision: 6 July 1999 / Accepted: 1 August 1999     

Nucleotide sequences at the boundaries between gene and insertion regions in the rDNA of Drosophila melanogaster

Ribosomal RNA genes interrupted by type 1 insertions of 1 kb and 0.5 kb have been sequenced through the insertion region and compared with an uninterrupted gene. The 0.5 kb insertion is flanked by a duplication of a 14 bp segment that is present once in the uninterrupted gene; the 1 kb insertion is flanked by a duplication of 11 of these 14 bp. Short insertions are identical in their entire length to downstream regions of long insertions. No internal repeats occur in the insertion. The presence of target site duplications suggests that type 1 insertions arose by the introduction of transposable elements into rDNA. Short sequence homologies between the upstream ends of the insertions and the 28S' boundaries of the rRNA coding region suggest that short type 1 insertions may have arisen by recombination from longer insertions.We have sequenced both boundaries of two molecules containing type 2 insertions and the upstream boundary of a third; the points of interruption at the upstream boundary (28S' site) differ from each other in steps of 2 bp. Between the boundary in the 0.5 kb type 1 insertion and the type 2 boundaries there are distances of 74, 76, and 78 bp. At the downstream boundary (28S' site) the two sequenced type 2 insertions are identical. The rRNA coding region of one molecule extends across the insertion without deletion or duplication, but a 2 bp deletion in the RNA coding region is present in the second molecule. Stretches of 13 or 22 adenine residues occur at the downstream (28S') end of the two type 2 insertions.     

Characterization and distribution of two insertion sequences,IS1191 and iso-IS981, in Streptococcus thermophilus: does intergeneric transfer of insertion sequences occur in lactic acid bacteria co-cultures?

A chromosomal repeated sequence from Streptococcus thermophilus was identified as a new insertion sequence (IS), IS1191. This is the first IS element characterized in this species. This 1313 bp element has 28 bp imperfect terminal inverted repeats and is flanked by short direct repeats of 8bp. The single large open reading frame of IS1191 encodes a 391-amino-acid protein which displays homologies with transposases encoded by IS1201 from Lactobacillus helveticus (44.5% amino-acid sequence identity) and by the other ISs of the IS256 family. One of the copies of IS 1191 is inserted into a truncated iso-IS981 element. The nucleotide sequences of two truncated iso-IS981 s from S. thermophilus and the sequence of IS981 element from Lactococcus lactis share more than 99% identity. The distribution of these insertion sequences in L. lactis and S. thermophilus strains suggests that intergeneric transfers occur during co-cultures used in the manufacture of cheese.     

A fine structure map of spontaneous and induced mutations in the lambda repressor gene, including insertions of IS elements

Summary Mutations at over 70 sites in the cI gene have been mapped by 4-factor crosses and assigned precise or approximate positions in the DNA sequence. 16 of 25 spontaneous mutations were insertions of IS1, IS3 or IS5 into AT-rich regions of cI. The 5-methylcytosine in the sequence Cm⁵CAGG is a hot spot for spontaneous cI amber mutations. Recombination frequencies between mutations were proportional to distance with the exception of amber mutations at 4 sites, including the host spot for spontaneous mutations. Mutations with a given phenotype are clustered on the genetic map. No missense mutations affecting repressor activity were found in the central one-third of cI, but 5 of 6 ind ^- mutations were located in this region. The aminoterminal third of the gene contains the sites of most trans-dominant cI^- mutations, and of all ts mutations that result in repressors that are reversibly inactivated at high temperatures.     

Analysis of relative reversion frequencies for IS2 insertion mutations in the regulatory region of the galOPETK operon of Escherichia coli

Two previously characterized mutations in the galOPETK operon of Escherichia coli, galOP-3 and galOPE-490, contain IS2 insertions only 1 bp apart in the gal regulatory region; yet only the former yields Gal⁺ phenotypic revertants at a detectable frequency. We have shown that the galOPE-490 allele comprises two mutations—an IS2(I) insertion at bp+(2–6) (relative to the gal mRNA start site) plus a C/G to A/T transversion at bp+59. The latter creates an ochre stop codon and lies within the internal site of the bipartite gal operator; it acts as an operator mutation in an in vivo repressor titration assay. Analysis of a newly isolated allele (galOP-490^*) which retains the IS2 of galOPE-490 but is galE⁺ reveals a reversion frequency approximately 30-fold higher than that of galOP-3. Reversion of galOPE-490 is at least 10,000-fold lower and has not been detectable even under conditions conducive to enhanced double mutations in other systems.     

Insertion sequence IS5 contains a sharply curved DNA structure at its terminus

Summary It was demonstrated that insertion sequence IS5 contains a sequence-directed bent (sharply curved) DNA structure at its terminus, close to one of its 16 bp terminal repeats. The minimal number of copies of IS5 related sequences and the locations of the latter on the Escherichia coli K12 W3110 chromosome were determined. Evidence is presented of the occurrence of IS5 mediated translocation and duplication of a large DNA segment on the E. coli chromosome.