A new insertion sequence, IS231M, in an autoagglutinable isolate of Bacillus thuringiensis

An insertion sequence was isolated from an autoagglutinable strain of Bacillus thuringiensis. Analysis of its DNA sequence revealed high homology to the IS231 family. The name IS231M is proposed for this new insertion sequence. IS231M is 1652 bp long and is delimited by two imperfect 20-bp inverted repeat sequences with two mismatches, which are flanked by two perfect 11-bp direct repeats (DRs). The region upstream of the open reading frame, presumed to be able to form a stable hairpin structure, is particularly well conserved in IS231M. Based on primary nucleotide sequences, IS231M is most homologous to IS231F and IS231G and most distant from IS231V and IS231W. However, as opposed to the single transposase A ORF found in IS231A, -B, -C, -D, -F, and -G, IS231M has two overlapping open reading frames, ORF1 and ORF2, that could code for polypeptides of 334 and 143 amino acids, respectively. Whether IS231M is a functional transposable element remains to be determined.     

IS231A from Bacillus thuringiensis is functional in Escherichia coli: transposition and insertion specificity.

A kanamycin resistance gene was introduced within the insertion sequence IS231A from Bacillus thuringiensis, and transposition of the element was demonstrated in Escherichia coli. DNA sequencing at the target sites showed that IS231A transposition results in direct repeats of variable lengths (10, 11, and 12 bp). These target sequences resemble the terminal inverted repeats of the transposon Tn4430, which are the preferred natural insertion sites of IS231 in B. thuringiensis.     

IS231-MIC231 elements from Bacillus cereus sensu lato are modular

Summary IS231A was originally discovered in Bacillus thuringiensis as a typical 1.6 kb insertion sequence (IS) displaying 20 bp inverted repeats (IR) flanking a transposase gene. A first major variation of this canonical organization was found in MIC231A1. This mobile insertion cassette (MIC), delineated by IS231A-related extremities, contained an active d-stereospecific endopeptidase (adp) gene instead of a transposase. Interestingly, it was shown that MIC231A1 can be mobilized in trans by the IS231A transposase. In this paper, we show that this family of IS231-MIC231 elements can be extended to a broad range of related entities displaying higher levels of structural complexity. Several IS231A-like elements contained, upstream of their transposase gene, passenger genes coding for putative antibiotic resistances or regulatory factors. Furthermore, the diversity of the MIC231 elements ranged from empty cassettes to structures carrying up to three passenger genes. Among these, MIC231V carried, in addition to an adp gene, an active fosfomycin resistance determinant. In vivo transposition assays showed that MIC231V is also trans-activated by the IS231A transposase. These results lend further support to the potential contribution of these modular mobile elements to the genome plasticity of the Bacillus cereus/B. thuringiensis group.     

Unusual organization associated to a tandem of IS231 may yield two peculiar cloverleaf secondary structures.

A 5.7-kb EcoRI fragment was cloned from plasmid DNA of Bacillus thuringiensis strain M15. It contains two insertion sequences (IS), IS231M2 and -M1 in the 5'-3' order, arranged in tandem, in same orientation, separated by a 540-bp region. The primary structure is typical of a composite transposon, here of 3847 bp in length, for which the name Tn231M is proposed. Each IS is delimited by 18-bp inverted repeats (IR), and flanked by 11-bp direct repeats (DR). Both IS share 99.3% nucleotide identities. IS231M1 has a single open reading frame (ORF) which encodes a putative 477-amino-acid transposase. IS231M2 has two smaller ORFs: ORF1 and ORF2, which could code for polypeptides of 329 and 118 amino acids in length, respectively. Further analysis reveals that the regions upstream of IS231M2, and downstream of -M1, and the 540-bp region, contain additional pairs of IR and DR. Interestingly, potential annealing between all pairs of IR and DR could generate two unusual cloverleaf secondary structures.     

Nucleotide sequence and characterization of a new insertion element, IS240, from Bacillus thuringiensis israelensis

The nucleotide sequence of two repeated sequences (RS) in opposite orientations flanking the 125-kDa toxin gene of Bacillus thuringiensis israelensis (C. Bourgouin et al., J. Bacteriol. 170, 3575-3583, 1988) is reported in this paper. The analysis of these sequences indicates that these two RS display characteristic features of bacterial insertion sequences (IS) and are therefore referred to as IS240. IS240 B is 865 bp long and has two perfect terminal-inverted repeats of 16 bp; IS240 A is 99% identical to IS240 B. A long open reading frame encoding a polypeptide of 235 amino acids spans almost the entire sequence of both IS240 elements. Both the sequence of the inverted repeats and the putative transposases are homologous to IS26 of Proteus vulgaris, IS15-delta of Salmonella panama, IS431 of Staphylococcus aureus, and ISS1 of Streptococcus lactis.     

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.     

The novel insertion sequences IS1417, IS1418, and IS1419 from Burkholderia glumae and their strain distribution

Three insertion sequences, IS1417, IS1418, and IS1419, were isolated from Burkholderia glumae (formerly Pseudomonas glumae), a gram-negative rice pathogenic bacterium, on the basis of their abilities to activate the expression of the neo gene of the entrap vector pSHI1063. The 1335-bp IS1417 element with 17-bp imperfect terminal inverted repeats was found to be flanked by 5-bp direct repeats of the vector sequence. IS1418 is 865 bp in length and carries 15-bp inverted repeats with a target duplication of 3 bp. The 1215-bp IS1419 sequence is bounded by the 36-bp terminal inverted repeats of the element and 7-bp direct repeats of the vector sequence. IS1417 and IS1418 belong to the IS2 subgroup of the IS3 family and the IS427 subgroup of the IS5 family, respectively, whereas IS1419 does not appear to be a member of any known IS family. Southern blot analysis of DNAs from B. glumae field isolates indicated that those IS elements are widely distributed, but the host range of the three IS elements appears to be limited to B. glumae and some other related species such as B. plantarii. The polymorphisms exhibited in B. glumae isolates suggest that those elements are useful for molecular epidemiological studies of B. glumae infections.     

Analysis of separate isolates of Bordetella pertussis repeated DNA sequences

Two independent isolates of a Bordetella pertussis repeated DNA unit were sequenced and shown to be an insertion sequence element with five nucleotide differences between the two copies. The sequences were 1053 bp in length with near-perfect terminal inverted repeats of 28 bp, had three open reading frames, and were each flanked by short direct repeats. The two insertion sequences showed considerable homology to two other B. pertussis repeated DNA sequences reported recently: IS481 and a 530 bp repeated DNA unit. The B. pertussis insertion sequence would appear to comprise a group of closely related sequences differing mainly in flanking direct repeats and the terminal inverted repeats. The two isolates reported here, which were from the adenylate cyclase and agglutinogen 2 regions of the genome, were numbered IS48lvl and IS48lv2 respectively.     

Differentiation of Bacillus anthracis and other 'Bacillus cereus group' bacteria using IS231-derived sequences

Abstract Sequences based on the conserved 20 bp inverted repeat of IS 231 variants were used as polymerase chain reaction-based fingerprinting primers of the member species of the Bacillus cereus group ( B. anthracis, B. cereus, B. thuringiensis and B. mycoides ), because of their close association with transposons, principally Tn 4430 in B. thuringiensis . Fingerprints of B. anthracis were simple, and specifically allowed its identification and sub-differentiation from other members of the group. Fingerprints for B. cereus were strain-specific; those for B. thuringensis gave a 1650 bp product, characteristic of 1S 231 variants A-F. The same reaction conditions gave one or two bands for both B. anthracis and B. cereus that differed by restriction endonuclease mapping from the B. thuringiensis PCR product and established IS 231 restriction maps; this does not preclude some kind of relationship between these products and IS 231 .     

Structural and genetic organization of IS232, a new insertion sequence of Bacillus thuringiensis.

In the Bacillus thuringiensis strains toxic for the lepidopteran larvae, the delta-endotoxin genes cryIA are frequently found within a composite transposonlike structure flanked by two inverted repeat sequences. We report that these elements are true insertion sequences and designate them IS232. IS232 is a 2,184-bp element and is delimited by two imperfect inverted repeats (28 of 37 bp are identical). Two adjacent open reading frames, overlapping for three codons, span almost the entire sequence of IS232. The potential encoded polypeptides of 50 and 30-kDa are homologous to the IstA and IstB proteins of the gram-negative insertion sequence IS21. The N-terminal part of the 50-kDa polypeptide contains a helix-turn-helix DNA-binding motif. The junctions at the insertion sites of three IS232 elements were analyzed. Each case was different, with 0, 4, or 6 bp of the target DNA being duplicated. Transposition of IS232 in Escherichia coli was demonstrated by using a genetic marker inserted upstream of the two open reading frames.     

IS902, an insertion element of the chronic-enteritis-causing Mycobacterium avium subsp. silvaticum.

An insertion sequence element of Mycobacterium avium subsp. silvaticum was isolated and its complete nucleotide sequence determined. IS902 is 1470 bp in size and is repeated 10-12 times per genome. An open reading frame of 1200 bp was identified, encoding a protein product of Mr 43932. This protein is highly similar to the predicted proteins of IS900 of Mycobacterium paratuberculosis, IS116 of Streptomyces clavuligerus and IS110 of Streptomyces coelicolor. IS902 lacks terminal inverted repeats and flanking direct repeats but displays insertion site specificity.     

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.     

Characterization of insertion sequence IS892 and related elements from the cyanobacterium Anabaena sp. strain PCC 7120.

IS892, one of the several insertion sequence (IS) elements discovered in Anabaena sp. strain PCC 7120 (Y. Cai and C. P. Wolk, J. Bacteriol. 172:3138-3145, 1990), is 1,675 bp with 24-bp near-perfect inverted terminal repeats and has two open reading frames (ORFs) that could code for proteins of 233 and 137 amino acids. Upon insertion into target sites, this IS generates an 8-bp directly repeated target duplication. A 32-bp sequence in the region between ORF1 and ORF2 is similar to the sequence of the inverted termini. Similar inverted repeats are found within each of those three segments, and the sequences of these repeats bear some similarity to the 11-bp direct repeats flanking the 11-kb insertion interrupting the nifD gene of this strain (J. W. Golden, S. J. Robinson, and R. Haselkorn, Nature [London] 314:419-423, 1985). A sequence similar to that of a binding site for the Escherichia coli integration host factor is found about 120 bp from the left end of IS892. Partial nucleotide sequences of active IS elements IS892N and IS892T, members of the IS892 family from the same Anabaena strain, were shown to be very similar to the sequence of IS892.     

The IS1111 family members IS4321 and IS5075 have subterminal inverted repeats and target the terminal inverted repeats of Tn21 family transposons

IS5075 and IS4321 are closely related (93.1% identical) members of the IS1111 family that target a specific position in the 38-bp terminal inverted repeats of Tn21 family transposons and that are inserted in only one orientation. They are 1,327 bp long and have identical ends consisting of short inverted repeats of 12 bp with an additional 7 bp (TAATGAG) or 6 bp (AATGAG) to the left of the left inverted repeats and 3 bp (AGA) or 4 bp (AGAT) to the right of the right inverted repeat. Circular forms of IS5075 and IS4321 in which the inverted repeats are separated by abutting terminal sequences (AGATAATGAG) were detected. A similar circular product was found for the related ISPa11. Transposition of IS4321 into the 38-bp target site was detected, but a flanking duplication was not generated. The precisely reconstituted target site was also identified. Over 50 members of the IS1111 family were identified. They encode related transposases, have related inverted repeats, and include related bases that lie outside these inverted repeats. In some, the flanking bases number 5 or 6 on the left and 4 or 3 on the right. Specific target sites were found for several of these insertion sequence (IS) elements. IS1111 family members therefore differ from the majority of IS elements, which are characterized by terminal inverted repeats and a target site duplication, and from members of the related IS110 family, which do not have obvious inverted repeats near their termini.     

Identification of a new insertion element, similar to gram-negative IS26, on the lactose plasmid of Streptococcus lactis ML3.

In Streptococcus lactis ML3, the lactose plasmid (pSK08) forms cointegrates with a conjugal plasmid (pRS01). It has been proposed that cointegration is mediated by insertion sequences (IS) present on pSK08 (D. G. Anderson and L.L. McKay, J. Bacteriol. 158:954-962, 1984). We examined the junction regions of the cointegrate pPW2 and the corresponding regions of pSK08 (donor) and pRS01 (target) and identified a new IS element on pSK08 (ISS1S) which was involved in and duplicated during formation of pPW2. ISS1S was 808 base pairs (bp) in size, had 18-bp inverted repeats (GGTTCTGTTGCAAAGTTT) at its ends, contained a single long open reading frame encoding a putative protein of 226 amino acids, and generated 8-bp direct repeats of target DNA during cointegrate formation. An iso-IS element, ISS1T, which is duplicated in some other cointegrate plasmids, was also found on pSK08. ISS1T was also 808 bp in size and was identical to ISS1S in sequence except for 4 bp, none of which altered the inverted repeats or amino acid sequence of the open reading frame. Comparison of ISS1 with gram-negative IS26 revealed strong homologies in size (820 bp), sequence of inverted repeats (GGCACTGTTGCAAA), size of direct repeats generated after cointegration (8 bp), and number, size, and amino acid sequence (44.5% identical) of the open reading of frame.     

Identification and characterization of IS 1138, a transposable element from Mycoplasma pulmonis that belongs to the IS3 family

Insertion sequence (IS) elements are mobile genetic elements found in prokaryotes. We have identified a repetitive element from Mycoplasma pulmonis, a murine pathogen, that is similar to eubacterial IS elements. By subcloning a single strain of M. pulmonis, we isolated a variant clone in which the IS element had undergone an apparent transposition event. The nucleotide sequences of the element, designated IS 1138, and the target site into which it inserted were determined. IS1138 consists of 1288bp with 18bp perfect terminal inverted repeats. Sequence analysis of the target site before and after insertion of IS1138 identified a 3bp duplication of target DNA flanking the element. The predicted amino acids encoded by the major open reading frame of IS 1138 share significant similarity with the transposases of the IS3 family. Southern hybridization analysis indicates that repetitive sequences similar to IS 1138 are present in most, if not all, strains of M. pulmonis, but Is1138–like sequences were not detected in other mycoplasmal species.     

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.     

A family of IS1031 elements in the genome of Acetobacter xylinum: nucleotide sequences and strain distribution

An insertion sequence (here called IS 1031A) from Acetobacter xylinum ATCC 23769 has recently been isolated. This study describes the complete nucleotide sequence of IS 1031A as well as the sequences of two novel iso-IS 1031 elements, IS1031C and IS1031D, from A. xylinum ATCC 23769. The three ISs are all exactly 930 bp long, have imperfect terminal inverted repeats of 24 bp for IS1031A and 21 bp for IS1031C and IS1031D, are flanked by three base pair direct repeats, and contain an open reading frame encoding a putative basic protein of 278 amino acids. Because of nucleotide substitutions, IS1031C and IS1031D differ from IS 1031A by 12.9% while IS1031C differs from IS1031D by only 0.6%. Hybridization analyses of total DNA from nine A. xylinum strains showed that all strains contained IS 1031-like elements varying in copy number from three to at least 16. None of three Acetobacter aceti strains examined contained IS1031-like elements. Taken together, the results suggest that A. xylinum contains a family of IS 1031 elements with considerably diversified nucleotide sequences.     

Identification, DNA sequence, and distribution of IS981, a new, high-copy-number insertion sequence in lactococci.

An insertion in the lactococcal plasmid pGBK17, which inactivated the gene(s) encoding resistance to the prolate-headed phage c2, was cloned, sequenced, and identified as a new lactococcal insertion sequence (IS). IS981 was 1,222 bp in size and contained two open reading frames, one large enough to encode a transposase. IS981 ended in imperfect inverted repeats of 26 of 40 bp and generated a 5-bp direct repeat of target DNA at the site of insertion. IS981 was present on the chromosome of Lactococcus lactis subsp. lactis LM0230 from where it transposed to pGBK17 during transformation. Twenty-three strains of lactococci examined for the presence of IS981 by Southern hybridization showed 4 to 26 copies per genome, with L. lactis subsp. cremoris strains containing the highest number of copies. Comparison of the DNA sequence and the amino acid sequence of the long open reading frame to other known sequences showed that IS981 is related to a family of IS elements that includes IS2, IS3, IS51, IS150, IS600, IS629, IS861, IS904, and ISL1.