Nucleotide sequence analysis of IS427 and its target sites in Agrobacterium tumefaciens T37

We have determined the nucleotide sequence of IS427, an insertion sequence from Agrobacterium tumefaciens T37, IS427 is 1271 bp long, contains 16-bp imperfect terminal inverted repeats, and generates a 2-bp target sequence duplication. It is present at three sites in the pTiT37 plasmid and is absent from the chromosome of A. tumefaciens T37. Each of the IS427 elements sequenced was near a site with sequence homology to integration host factor (IHF)-binding sites which suggested that IHF may be involved in IS427 transposition.     

Nucleotide sequence of the transposable element IS15

We have determined the complete nucleotide sequence of the right (R) copy of the insertion sequence IS15 which flanks, in direct orientation, the composite transposon Tn1525. IS15-R, which is capable of independent transposition, is 1648 bp long and has short (14 bp) perfect inverted repeats at its termini. Analysis of the nucleotide sequence indicates that IS15-R results from the transposition, in direct orientation, of a smaller (820 bp long) IS, designated IS15-Δ, into itself. This integration event is accompanied by the duplication of 8 bp in the target DNA. IS15-Δ possesses two large overlapping open reading frames (ORF) located on opposite strands. Because of this particular structure, IS15 possesses four large ORFs which, due to the integration event, exhibit some differences with those of the parental 1S15-Δ.     

Isolation of the mini insertions IS6 and IS7 of E. coli

Summary The mini IS elements IS6 and IS7 have been detected in constitutive gal ⁺ revertants of galOP-308::IS2 (I), in which the expression of the gal operon is turned off by IS2 in orientation I. Both, IS6 and IS7, are integrated into IS2 proximal to the gal structural genes. IS6 is 115 base pairs long and causes 50% constitutive expression of the gal genes. IS7 is only 65 base pairs long and the gal operon is expressed 20% constitutively compared to the gal ⁺ wild type operon. Both IS6 and IS7 are excised frequently, in the absence of selective pressure. These findings are discussed with respect to the evolution of gene expression.     

Characterisation of IS153, an IS3-family Insertion Sequence Isolated from Lactobacillus sanfranciscensis and its use for Strain Differentiation

An insertion sequence has been identified in the genome of Lactobacillus sanfranciscensis DSM 20451^T as segment of 1351 nucleotides containing 37-bp imperfect terminal inverted repeats. The sequence of this element encodes two out of phase, overlapping open reading frames, orfA and orfB, from which three putative proteins are produced. OrfAB is a transframe protein produced by –1 translational frame shifting between orf A and orf B that is presumed to be the transposase. The large orfAB of this element encodes a 342 amino acid protein that displays similarities with transposases encoded by bacterial insertion sequences belonging to the IS3 family.In L. sanfranciscensis type strain DSM 20451^T multiple truncated IS elements were identified. Inverse PCR was used to analyze target sites of four of these elements, but except of their highly AT rich character not any sequence specificity was identified so far. Moreover, no flanking direct repeats were identified. Multiple copies of IS 153 were detected by hybridization in other strains of L. sanfranciscensis. Resulting hybridization patterns were shown to differentiate between organisms at strain level rather than a probe targeted against the 16S rDNA. With a PCR based approach IS 153 or highly similar sequences were detected in L. acidophilus, L. casei, L. malefermentans, L. plantarum, L. hilgardii, L. collinoides L. farciminis L. sakei and L. salivarius, L. reuteri as well as in Enterococcus faecium, Pediococcus acidilactici and P. pentosaceus.     

Formation of the tandem repeat (IS30)2 and its role in IS30-mediated transpositional DNA rearrangements

Plasmids carrying two IS30 elements in the same orientation, as in the composite transposon Tn2706, are structurally unstable in Escherichia coli. A primary segregation product is formed by site-specific deletion of the sequences carried between the two IS30 elements. The resulting covalently closed replicon carries the two IS30 elements as tandem repeats separated by only 2 bp. This (IS30)₂ structure is extremely unstable, but it can nevertheless be isolated on its vector plasmid and, after purification, can be reintroduced into host cells by transformation. Among the descendants of transformants of recA ^– bacteria, replicated copies of the introduced (IS30)₂ structure are still present, together with various kinds of segregation products which provide evidence for the efficient generation of DNA rearrangements. Most abundant is the product of another site-specific recombination between two identical ends of the IS30 elements involved, which results in the presence of just one intact IS30 on the plasmid. Apart from this, and depending on the presence of appropriate targets for IS30 transposition, various transposition products of (IS30)₂ are also seen. Intramolecular reactions lead to DNA inversions and deletions with breakpoints other than IS30 ends. In intermolecular reactions inverse transposition occurs at high frequency and one also obtains simple transposition and cointegration. A mutational study revealed the requirement in cis of one intact IS30 transposase gene and of both proximal ends of the two IS30 elements concerned not only for the formation of (IS30)₂, but also for its further rearrangement reactions, including the efficient formation of site-specific deletions. A model is proposed, which postulates that (IS30)₂ intermediates play a key role in IS30 transposition pathways in which the formation of (IS30)₂ may be rate-limiting. Once this structure is formed, it gives rise to a burst of transpositional rearrangements in the subclone carrying (IS30)₂. Evolutionary implications of these findings are discussed.     

Identification of insertion sequence element IS427 in pTiT37 plasmid DNA of an Agrobacterium tumefaciens T37 isolate

The isolation and characterization of an insertion sequence (IS) element, IS427, from Agrobacterium tumefaciens T37 is described. IS427 is present in three nonidentical copies on the pTiT37 plasmid. The copy that was isolated through transposition on the entrapment vector pUCD800 contains at its ends a 16-bp imperfect inverted repeat and generates a 2-bp duplication of the target DNA. IS427 does not show homology with previously characterized IS elements of A. tumefaciens, based on hybridization experiments and/or sequence comparison.     

IS-like element IS8 in RP4 plasmid and its involvement in cointegration

The structure of the cointegrate plasmids formed by fusion of RP4 and the tumour-inducing plasmid (pTi) of Agrobacterium tumefaciens was analyzed. In all of the nine independently isolated pTi: :RP4 cointegrates, the integration occurred at the same site on the RP4 genome. Moreover, a 1.2 Md (1750 bp) RP4 sequence (IS8) was directly repeated at both junction sites of the two replicons. The insertion of RP4 generated deletions, starting from the IS8sequence and extending into the Ti part of the cointegrate. Dissociation of the cointegrates resulted in wild-type RP4 and Ti-plasmids with the IS8 sequence inserted at the original RP4 insertion site. The processes of integration and dissociation and the genetic properties of the cointegrates indicate that the IS8 sequence has unique characteristics defining a new insertion sequence.     

Nucleotide sequence and analysis of the mgl operon of Escherichia coli K12

Summary The nucleotide sequence of the Escherichia coli K12 -methylgalactoside transport operon, mgl, was determined. Primer extension analysis indicated that the synthesis of mRNA initiates at guanine residue 145 of the determined sequence. The operon contains three open reading frames (ORF). The operator proximal ORF, mglB, encodes the galactose binding protein, a periplasmic protein of 332 amino acids including the 23 residue amino-terminal signal peptide. Following a 62 nucleotide spacer, the second ORF, mglA, is capable of encoding a protein of 506 amino acids. The amino-terminal and carboxyl-terminal halves of this protein are homologous to each other and each half contains a putative nucleotide binding site. The third ORF, mglC, is capable of encoding a hydrophobic protein of 336 amino acids which is thought to generate the transmembrane pore. The overall organization of the mglBAC operon and its potential to encode three proteins is similar to that of the ara FGH high affinity transport operon, located approximately 1 min away on the E. coli K12 chromosome.     

New probes used for IS1245 and IS1311 restriction fragment length polymorphism of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. hominissuis isolates of human and animal origin in Norway

Background  

Mycobacterium avium is an environmental mycobacterium that can be divided into the subspecies avium, hominissuis, paratuberculosis and silvaticum. Some M. avium subspecies are opportunistic pathogens for animals and humans. They are ubiquitous in nature and can be isolated from natural sources of water, soil, plants and bedding material. Isolates of M. avium originating from humans (n = 37), pigs (n = 51) and wild birds (n = 10) in Norway were examined by IS1245 and IS1311 RFLP using new and specific probes and for the presence of IS901 and ISMpa1 by PCR. Analysis and generation of a dendrogram were performed with the software BioNumerics.     

Analysis of secondary, integration sites for IS117 in Streptomyces lividans and their role in the generation of chromosomal deletions

IS117, the 2.6 kb mini-circle of Streptomyces coelicolor A3(2), is a transposable element previously shown to be integrated into two distant sites in the chromosome. When introduced into S. lividans, IS117 integrates into one preferred chromosomal site, but when this site was artificially deleted, IS117 integrated into many secondary sites. Nucleotide sequence analysis of several secondary integration sites revealed varying degrees of similarity with the preferred site, but no consensus sequence. Nevertheless, sites more similar to the preferred site tended to be occupied more often than those that are less similar. Insertion of IS117 into secondary sites in the chromosome of S. lividans sometimes mediated chromosomal rearrangements. It was shown that some strains containing IS117 integrated into secondary sites had suffered deletions of chromosomal DNA. Deletions were adjacent to the inserted element and were at least several kilobases long. The proposed model implicates homologous recombination between IS117 copies integrated into two different secondary sites in the same chromosome as a cause of the deletions.     

Isolation and Characterization of IS1181, an Insertion Sequence from Staphylococcus aureus

The repeated nucleotide sequence isolated from a methicillin-resistant Staphylococcus aureus isolate displays the characteristic features of an insertion sequence and was named IS1181. It has a size of 1512 bp and consists of a 1359-bp open reading frame that encodes a 439-amino-acid protein which is predicted to be highly basic and 23-bp terminal inverted complementary repeated sequences exhibiting six mismatches. The three copies of IS1181 isolated from distinct parts of the chromosome of S. aureus, BM3121, are flanked at their ends by 8-bp direct repeats, suggesting a duplication of the target sequence. IS1181 exhibits similarities with IS1165 from Leuconostoc mesenteroides and IS1001 from Bordetella parapertusis. IS1181 was detected in at least two to eight copies in 41 of the 52 S. aureus isolates tested, whereas none of the 26 coagulase-negative staphylococci, 24 streptococci, or 11 enterococci analyzed carried nucleotide sequences hybridizing with IS1181.     

Isolation and Characterization of IS1416fromPseudomonas glumae,a New Member of the IS3Family

Isolation and characterization of four different insertion sequence (IS) elements fromPseudomonas glumaeMAFF 302744 through transposition into the entrapment vector pSHI1063 are described. One of the elements, IS1416,was further characterized. IS1416is 1322 bp long and carries 29-bp terminal inverted repeats flanked by a 3-bp direct duplication. IS1416contains three open reading frames (ORFs), which are designated ORFA1, ORFA2, and ORFB, on one strand. Both DNA sequence of IS1416and the deduced amino acid sequences of its ORFs strongly suggest that IS1416is a member of the IS3family, and is closely related to IS401fromPseudomonas cepaciaand IS51fromPseudomonas syringae.To our knowledge, IS1416is the first IS element isolated fromP. glumae.The gene organization and possible regulation of transposition functions of IS1416are also discussed.     

Nucleotide sequence of the tmr locus of Agrobacterium tumefaciens pTi T37 T-DNA.

The nucleotide sequence of the tmr locus from the nopaline-type pTi T37 plasmid of Agrobacterium tumefaciens was determined. Examination of this sequence allowed us to identify an open reading frame of 720 nucleotides capable of encoding a protein with a derived molecular weight of 27025 d. Comparison of the pTi T37 tmr sequence with the published sequence of the pTi Ach5 tmr locus shows over 88% homology in the 240 bases 5' to the translational initiation codon and over 91% homology in the coding sequences. The 3' nontranslated regions show less than 50% homology as expected for the 3' regions of divergent related genes. The possible significance of areas of conserved sequences, particularly in the 5' regulatory regions, is discussed.     

Nucleotide sequence of a recA operator mutation

Summary A single base pair change (AT to GC) is shown to be responsible for derepression of recA. The mutation (recAo281) alters the binding sequence for the LEXA repressor.Abbreviations bp base pair - kb kilobase pair