Cloning and sequence of IS1000, a putative insertion sequence from Thermus thermophilus HB8

The complete nucleotide sequences of two copies of a putative insertion sequence IS1000 from Thermus thermophilus HB8 are presented. IS1000 is 1196 base pairs long, contains a long open reading frame which could code for a protein of 317 amino acids, and has imperfect terminal inverted repeats of 6 base pairs (confirmed by the terminal sequencing of 4.5 copies of IS1000), but does not cause a target site duplication. There are at least 6 copies of IS1000 in the genome of T. thermophilus HB8. A search of the GEN-EMBL data base revealed that the putative 317 amino acid protein had significant homology with open reading frames in the transposable elements IS110 of Streptomyces coelicolor and IS492 of Pseudomonas atlantica.     

Complete nucleotide sequence of insertion element IS4351 from Bacteroides fragilis.

The nucleotide sequence and genetic analyses of one of the directly repeated sequences flanking the macrolide-lincosamide-streptogramin B drug resistance determinant, ermF, from the Bacteroides fragilis R plasmid, pBF4, suggested that this region is an insertion sequence (IS) element. This 1,155-base-pair element contained partially matched (20 of 25 base pairs) terminal-inverted repeats, overlapping, anti-parallel open reading frames, and nine promoterlike sequences, including three that were oriented outward. Analysis of this sequence revealed no significant nucleotide homology to 13 other known IS elements. Inasmuch as Southern blot hybridization analysis detected homologous sequences in chromosomal DNA and its G+C content (42 mol%) was similar to that of B. fragilis, the data suggested that this element is of Bacteroides origin. Transposition promoted by this element was demonstrated in recA E. coli. Recombinants were recovered by selecting for the activation of a promoterless chloramphenicol resistance gene on the plasmid pDH5110 and were characterized by restriction endonuclease mapping and Southern blot hybridization. We propose that this IS element be designated IS4351.     

Characterization of an insertion sequence, IS12528, from Gluconobacter suboxydans.

A novel insertion sequence element, IS12528, was found to be associated with inactivation of the alcohol dehydrogenase by insertion in the adhA gene, which encodes the primary dehydrogenase subunit of the three-component membrane-bound alcohol dehydrogenase complex in Gluconobacter suboxydans. Cloning and sequencing analyses revealed that IS12528 was 905 bp in length and had a terminal inverted repeat of 18 bp. In addition, IS12528 was found to generate a 3-bp duplication (TMA, where M represents C or A) at the inserted site upon transposition. IS12528 encoded one long product of 274 amino acids that was rich in basic amino acids. This protein showed significant homology with putative transposases of the IS1031 family isolated from Acetobacter xylinum, which belongs to another genus of acetic acid bacteria. IS12528-like sequences were distributed in a wide variety of acetic acid bacteria, as determined by Southern hybridization and PCR. These observations suggest that IS12528 is one of the insertion sequences that are responsible for genetic instability leading to deficiencies in various physiological properties in a variety of acetic acid bacteria.     

IS200: a Salmonella-specific insertion sequence

A new IS element (IS200) has been identified in Salmonella. The sequence was identified as an IS element by the following criteria: its insertion caused the mutation hisD984; six copies of the sequence are present in strain LT2 of S. typhimurium; and transposition of the sequence has been observed on several occasions. IS200 is found in almost all Salmonella species examined but is absent from most other enteric bacteria. The specificity of this element for Salmonella (and the absence of IS1-IS4 from Salmonella) suggest that transfer of insertion sequences between bacterial groups may be less extensive than is commonly believed. Alternatively, the distribution may suggest that these elements play a selectively important role in bacteria.     

Cloning and sequence analysis of the muramidase-2 gene from Enterococcus hirae.

Extracellular muramidase-2 of Enterococcus hirae ATCC 9790 was purified to homogeneity by substrate binding, guanidine-HCl extraction, and reversed-phase chromatography. A monoclonal antibody, 2F8, which specifically recognizes muramidase-2, was used to screen a genomic library of E. hirae ATCC 9790 DNA in bacteriophage lambda gt11. A positive phage clone containing a 4.5-kb DNA insert was isolated and analyzed. The EcoRI-digested 4.5-kb fragment was cut into 2.3-, 1.0-, and 1.5-kb pieces by using restriction enzymes KpnI, Sau3AI, and PstI, and each fragment was subcloned into plasmid pJDC9 or pUC19. The nucleotide sequence of each subclone was determined. The sequence data indicated an open reading frame encoding a polypeptide of 666 amino acid residues, with a calculated molecular mass of 70,678 Da. The first 24 N-terminal amino acids of purified extracellular muramidase-2 were in very good agreement with the deduced amino acid sequence after a 49-amino-acid putative signal sequence. Analysis of the deduced amino acid sequence showed the presence at the C-terminal region of the protein of six highly homologous repeat units separated by nonhomologous intervening sequences that are highly enriched in serine and threonine. The overall sequence showed a high degree of homology with a recently cloned Streptococcus faecalis autolysin.     

Nucleotide sequence of Rhizobium meliloti insertion sequence ISRm1: homology to IS2 from Escherichia coli and IS426 from Agrobacterium tumefaciens.

Nucleotide sequencing of Rhizobium meliloti insertion sequence ISRm1 showed that it is 1319 nucleotides long and includes 32/31 nucleotide terminal inverted repeats. Analysis of five different insertion sites using sequencing primers complementary to sequences within the left and right ends demonstrated that ISRm1 generates five bp direct repeats at the sites of insertion. Although ISRm1 has shown a target preference for certain short regions (hot spots), there was no apparent similarity in the DNA sequences near the insertion sites. On one strand ISRm1 contains two contiguous open reading frames (ORFs) spanning most of its length. ISRm1 was found to have over 50% sequence homology to insertion sequences IS2 from Escherichia coli and IS426 from Agrobacterium tumefaciens. Their sizes, the sequences of their inverted repeats, and the characteristics of their insertion sites are also comparable, indicating that ISRm1, IS2 and IS426 belong to a class of related insertion sequences. Comparison of the proteins potentially encoded by these insertion sequences showed that the two ORFs found in ISRm1 are also present in IS2 and IS426, suggesting that they may be functional genes.     

A new insertion sequence from Sinorhizobium meliloti with homology to IS1357 from Methylobacterium sp. and IS1452 from Acetobacter pasteurianus

The insertion sequence ISRm8 was identified by sequence analysis of the cryptic plasmid pRmeGR4b of Sinorhizobium meliloti GR4. ISRm8 is 1451 bp in length and carries 22/24-bp terminal imperfect inverted repeats with seven mismatches and a direct target site duplication of 3 bp. ISRm8 carries a unique open reading frame whose putative protein showed significant similarity to the insertion sequences IS1357 and IS1452, isolated from Methylobacterium sp. and Acetobacter pasteurianus, respectively. Two copies of this IS element were found in strain GR4; one of them is linked to plasmid pRmeGR4b, whereas the other is localized out of the non-pSym plasmids. In S. meliloti field populations ISRm8 shows a limited distribution (50% of the strains tested carry the IS element), with a copy number ranging from 1 to 6.     

Linearization and transposition of circular molecules of insertion sequence IS3.

IS3 transposase has been shown to promote production of characteristic circular and linear IS3 molecules from the IS3-carrying plasmid; IS3 circles have the entire IS3 sequence with terminal inverted repeats, IRL and IRR, which are separated by a three base-pair sequence originally flanking either end in the parental plasmid, whereas linear IS3 molecules have three nucleotide overhangs at their 5' ends. Here, we showed that a plasmid carrying an IS3 derivative, which is flanked by different sequences at both ends, generated IS3 circles and linear IS3 molecules owing to the action of transposase. Cloning and sequencing analyses of the linear molecules showed that each had the same 5'-protruding three nucleotide overhanging sequences at both ends, suggesting that the linear molecules were not generated from the parental plasmid by the two double-strand breaks at both end regions of IS3. The plasmid carrying IS3 with a two base-pair mutation in the terminal dinucleotide, which would be required for transposase to cleave the 3' end of IS3, could still generate linear molecules as well as circles. Plasmids bearing an IS3 circle were cleaved by transposase and gave linear molecules with the same 5'-protruding three nucleotide overhanging sequences. These show that the linear molecules are generated from IS3 circles via a double-strand break at the three base-pair intervening sequence. Plasmids carrying an IS3 circle with the two base-pair end mutation still were cleaved by transposase, though with reduced efficiencies, suggesting that IS3 transposase has the ability to cleave not only the 3' end of IS3, but a site three nucleotides from the 5' end of IS3. IS3 circles also were shown to transpose to the target plasmids. The end mutation almost completely inhibited this transposition, showing that the terminal dinucleotides are important for the transfer of the 3' end of IS3 to the target as well as for the end cleavage.     

Transposase-induced excision and circularization of the bacterial insertion sequence IS911.

We have investigated the role of three IS911-specified proteins in transposition in vivo: the products of the upstream (OrfA) and downstream (OrfB) open reading frames, and a transframe protein (OrfAB) produced by -1 translational frameshifting between orfA and orfB. The production of OrfAB alone is shown to lead both to excision and to circularization of the element and to be sufficient for intermolecular transposition into a plasmid target. Simultaneous and independent production of OrfA is shown to stimulate OrfAB-mediated intermolecular transposition while greatly reducing the appearance of transposon circles. We have not been able to detect a role for OrfB. Although under certain conditions, the vector plasmid undergoes precise resealing after IS911 excision, the data suggest that this is not normally the case and that the donor plasmid is not generally conserved. The use of IS911 derivatives carrying mutations in the terminal 2 bp suggested that circle formation represents a site-specific intramolecular transposition event. We present a model which explains both intra- and intermolecular transposition events in terms of a single reaction mechanism of the 'cut and paste' type.     

Cloning and sequencing of IS1086, an Alcaligenes eutrophus insertion element related to IS30 and IS4351.

A new insertion sequence (IS), designated IS1086, was isolated from Alcaligenes eutrophus CH34 by being trapped in plasmid pJV240, which contains the Bacillus subtilis sacB and sacR genes. The 1,106-bp IS1086 element contains partially matched (22 of 28 bp) terminal-inverted repeats and a long open reading frame. Hybridization data suggest the presence of one copy of IS1086 in the strain CH34 heavy-metal resistance plasmid pMOL28 and at least two copies in its chromosome. Analysis of the IS1086 nucleotide sequence revealed striking homology with two other IS elements, IS30 and IS4351, suggesting that they are three close members in a family of phylogenetically related insertion sequences. One open reading frame of the Spiroplasma citri phage SpV1-R8A2 B was also found to be related to this IS family but to a lesser extent. Comparison of the G+C contents of IS30 and IS1086 revealed that they conform to their respective hosts (46 versus 50% for IS30 and Escherichia coli and 64.5% for IS1086 and A. eutrophus). The pressure on the AT/GC ratio led to a very different codon usage in these two closely related IS elements. Results suggesting that IS1086 transposition might be activated by some forms of stress are discussed.     

Novel insertion sequence IS1380 from Acetobacter pasteurianus is involved in loss of ethanol-oxidizing ability.

Acetobacter pasteurianus NCI1380, a thermophilic strain isolated from the surface culture of acetic acid fermentation, showed genetic instability to produce at high frequency spontaneous mutants which were deficient in ethanol oxidation because of the loss of alcohol dehydrogenase activity. Southern hybridization experiments with the cloned alcohol dehydrogenase-cytochrome c gene cluster as the probe showed insertion of an unknown DNA fragment into a specific position in the cytochrome c gene in most of the mutant strains. Cloning and sequencing analyses revealed that the inserted sequence was 1,665 bp in length and had a terminal inverted repeat of 15 bp. In addition, this inserted sequence was found to generate a 4-bp duplication at the inserted site upon transposition. The target site specificity was not very strict, but a TCGA sequence appeared to be preferentially used. The inserted sequence contains two long open reading frames of 461 and 222 amino acids which are overlapped and encoded by different strands. Although these open reading frames showed no homology to any protein registered in the DNA data bases, the longer open reading frame contained many basic amino acids (87 of 461), as was observed with transposases of so-called insertion sequence (IS) elements. All of these characteristics are typical of IS elements, and the sequence was named IS1380. The copy number of IS1380 in a cell of A. pasteurianus NCI1380 was estimated to be about 100. Several strains of acetic acid bacteria also contained IS1380 at high copy numbers. These results suggest that IS1380 is associated with the genetic loss of ethanol-oxidizing ability as well as the genetic instability of acetic acid bacteria in general.     

Nucleotide sequence and structural organization of Yersinia pestis insertion sequence IS100

Abstract The L1 major protein of human papillomavirus type 16 was expressed in Sf-21 insect cells with a recombinant baculovirus vector. Virus-like particles similar in appearance to empty verions were identified by electron microscoy at densities of 1.29–1.30. Purified particles reacted with monoclonal anti-HPV-16-L1 antibody in Western blot and immuno dot blot suggesting that conformational epitopes are present in the recombinant particles. Immunodot blot assays using human sera correlated with the detection of HPV-16 DNA by the polymerase chain reaction. The results suggest that HPV-16-L1 virions produced by the baculovirus system might be useful for developing serologic tests to measure antibodies to conformational epitopes and may offer potential for vaccine development.     

IS861, a group B streptococcal insertion sequence related to IS150 and IS3 of Escherichia coli.

A 1,442-base-pair (bp) insertion sequence (IS861) was identified in the type III group B streptococcal (GBS) strain COH-1. It is flanked by 26-bp imperfect inverted repeats and contains two open reading frames, 1 and 2, encoding 141- and 277-amino-acid proteins, respectively. A 3-bp target sequence, ACA, is duplicated and flanks each inverted repeat. IS861 shares greater than 30% homology with IS3 and IS150 of Escherichia coli, primarily in the region of their putative transposases. Northern (RNA) analysis revealed that RNA is actively transcribed in vivo by IS861 and 17- and 36-kilodalton proteins were synthesized in E. coli maxicell assays. Multiple copies of IS861 were observed throughout the chromosome of COH-1, and one of the copies is located near genes involved in GBS capsule synthesis. IS861 is the first insertion sequence identified in GBS. Its role in GBS and the significance of its relationship to the phylogenetically similar insertion sequences typified by IS150 and IS3 of E. coli are unknown.     

Characterization of IS1001, an insertion sequence element of Bordetella parapertussis.

By analysis of repetitive DNA in Bordetella parapertussis, an insertion sequence element, designated IS1001, was identified. Sequence analysis revealed that IS1001 comprised 1,306 bp and contained inverted repeats at its termini. Furthermore, several open reading frames that may code for transposition functions were identified. The largest open reading frame coded for a protein comprising 406 amino acid residues and showed homology to TnpA, which is encoded by an insertion sequence element (IS1096) found in Mycobacterium smegmatis. Examination of flanking sequences revealed that insertion of IS1001 occurs preferentially in stretches of T's or A's and results in a duplication of target sequences of 6 to 8 bases. IS1001 was found in about 20 copies in 10 B. parapertussis strains analyzed. No restriction fragment length polymorphism was observed in B. parapertussis when IS1001 was used as a probe. An insertion sequence element similar or identical to IS1001 was found in B. bronchiseptica strains isolated from pigs and a rabbit. In these strains, about five copies of the IS1001-like element were present at different positions in the bacterial chromosome. Neither B. pertussis nor B. bronchiseptica strains isolated from humans and dogs contained an IS1001-like element. Therefore, IS1001 may be used as a specific probe for the detection of B. parapertussis in human clinical samples.     

IS801, an insertion sequence element isolated from Pseudomonas syringae pathovar phaseolicola

A transposable element, designated IS801, was isolated from strain LR781 of Pseudomonas syringae pathovar phaseolicola in two independent events using the entrapment plasmid, pUCD800. IS801 is 1517 base pairs in length and contains open reading frames that potentially encode proteins of 311 and 172 amino acids, as well as smaller proteins. Unlike most other prokaryotic transposable elements, IS801 lacks terminal repeats. Sequence analysis revealed two target pentamers for IS801 insertion that differ by one base pair. One copy of IS801 generated a perfect duplication of its target, TGAAC. The second copy of IS801 was flanked by the target, TGGAC, at one end, and TGAAC at the other end. A third copy of IS801 was cloned from pMMC7105, an indigenous plasmid of strain LR781, and it was flanked by copies of the pentamer TGAAC.     

DNA sequence at the integration sites of the insertion element IS1.

We have detected two independent occurrences of insertion mutations in the lacl gene of E. Coli, and have used small plasmids carrying the l gene to purify large amounts of DNA containing these insertions. Analyses with restriction endonucleases and DNA sequencing techniques establish that both insertions involve the previously characterized element IS1. In each case, the integration of IS1 into the l gene DNA is associated with a directly repeated sequence of 9 nucleotides appearing at each end of the insertion element. Since one of these sequences was present in the wild-type gene, the second sequence either preexisted in the IS1 before integration, or else was generated by the process of insertion itself. The 9 base repeat is different in both cases. We discuss the relevance of these findings to the mechanism of integration of transposable elements.     

Identification of an insertion sequence, IS1081, in Mycobacterium bovis.

An insertion sequence, IS1081, in the genome of Mycobacterium bovis has been identified and sequenced. It is 1324 bp long with 15 bp inverted repeat ends and contains a large ORF. There are six copies of IS1081 in the genome of M. bovis and the element is also present in Mycobacterium tuberculosis. IS1081 is not closely related to other DNA elements described in actinomycetes but its putative transposase bears some resemblance to that of IS256 from Staphylococcus aureus. IS1081 may be useful for genetic manipulations and for developing a diagnostic test for bovine tuberculosis based on the polymerase chain reaction.