Nucleotide sequence of IS26, a new prokaryotic mobile genetic element.

The DNA sequence of a new IS element, the IS26, is 820 bp long and carries 14 bp perfect terminal inverted repeats. Upon integration, IS26 generates an 8 bp duplication of its target sequence. A large open reading frame within IS26 could code for a protein of 234 amino acids. On its reverse strand, IS26 also carries one large open reading frame, 591 bp long, which contains no stop codon within IS26.     

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.     

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.     

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.     

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.     

IS431, a staphylococcal insertion sequence-like element related to IS26 from Proteus vulgaris

We present the nucleotide sequence of IS431, a new staphylococcal insertion sequence-like element flanking the mercury-resistance determinant of pI524 and associated with the methicillin-resistance determinant. IS431 left is 800 bp long and has a perfect terminal inverted repeat (IR) of 22 bp; IS431 right is 786 bp long and has a terminal IR homologous to the IR of IS431 left except that the terminal 8 bp are absent. Both IRs share a 10-bp homology with the IR of IS26 from Proteus vulgaris. No directly repeated sequences were detected immediately adjacent to the IRs. An open reading frame (ORF) of 675 bp spans most of the IS431 sequence. Its deduced amino acid (aa) sequence shows 40% homology to the 234-aa-long putative transposase coded by ORFI of IS26.     

Isolation of a new insertion element of Yersinia intermedia closely related to remnants of mobile genetic elements present on Yersinia plasmids harboring the Yop virulon

A new insertion element present in two alleles, designated IS1635.1 and IS1635.2, was identified on a plasmid of a Yersinia intermedia strain by hybridization with the Yersinia enterocolitica pYV virulence plasmid. IS1635.1 and IS1635.2 are 861 bp long, carry imperfect inverted terminal repeats and possess a single open reading frame encoding a putative transposase of the IS6 family. A truncated IS1635 element is present immediately downstream of element IS1635.2. The capacity of the IS1635 elements to mediate transposition in Yersinia was demonstrated with a R6K-derived suicide vector, where a kanamycin resistance gene had been inserted between IS1635.1 and IS1635.2. Hybridization and sequence alignments showed that remnants of IS1635-like insertion elements harboring large deletions and point mutations are present on the Yop virulon harboring plasmids of pathogenic Yersinia strains. In a few cases, the IS1635 element has also been found on plasmids of apathogenic Yersinia strains.     

ISR1, a transposable DNA sequence resident in Rhizobium class IV strains, shows structural characteristics of classical insertion elements

ISR1 is a small transposable element, identified in Rhizobium class IV strains by its high frequent mutagenic insertion into plasmid RP4. Hybridization studies showed that ISR1 is present in, multiple copies in Rhizobium class IV strains. Nucleotide sequence analysis revealed that ISR1 has a length of 1260 bp and is characterized by perfect inverted repeats of 13 nucleotides followed by a stretch of 28/29 nucleotides with imperfect homology. The insertion under study generated a target site duplication of 4 bp. ISR1 carries a large open reading frame, encoding a putative polypeptide of 278 amino acids (ORFA*), and three smaller ones in antiparallel direction (ORFs A1, A2, A3). Two of them are completely covered by the large open reading frame. No significant homology to 17 other known insertion sequence elements could be detected, either at nucleotide or at amino acid levels.     

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.     

IS870 requires a 5'-CTAG-3' target sequence to generate the stop codon for its large ORF1.

The TB regions of the Agrobacterium vitis octopine/cucumopine Ti plasmids constitute a family of related structures. All contain a bacterial insertion element downstream of the TB-iaaM gene, IS870.1. Whereas 43 isolates with octopine/cucumopine Ti plasmids carry only one IS870 copy, strain Ag57 carries a second copy (IS870.2) 3.9 kb to the right of IS870.1 and part of the same TB region. Two other octopine/cucumopine strains carry an IS870 copy on their chromosome (IS870.3). A study of the unmodified insertion sites of IS870.2 and IS870.3, cloned from closely related strains, enabled us to delimit the IS870 elements. IS870 has a size of 1,152 bp and is terminated by inverted repeats. It contains a large open reading frame without a stop codon. However, a stop codon is generated by insertion into the target sequence 5'-CTAG-3'. IS870 is related to five other insertion sequence elements. For two of these, the stop codon of the largest open reading frame is also created by insertion into a CTAG target site.     

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.     

Isolation and characterization of IS elements repeated in the bacterial chromosome

Shigella sonnei contains repetitive sequences, including an insertion element IS1, which can be isolated as double-stranded DNA fragments by DNA denaturation and renaturation and by treatment with S1 nuclease. In this paper, we describe a method of cloning the IS1 fragments prepared by the S1 nuclease digestion technique into phage M13mp8 RFI DNA. Several clones contained IS1, usually with a few additional bases. We isolated and characterized five other repetitive sequences using this method. One sequence, 1264 base-pairs in length, had terminal inverted repeats and contained two open reading frames. This sequence, called IS600, showed about 44% sequence homology with IS3 and was repeated more than 20 times in the Sh. sonnei chromosome. Another sequence (named IS629, 1310 base-pairs in length), which was repeated six times, was found also to be related to IS3 and thus IS600. Two other sequences (named IS630 and IS640, 1159 and 1092 base-pairs in length, respectively), which were repeated approximately ten times, had characteristic terminal inverted repeats and contained a large open reading frame coding for a protein. The inverted repeat sequences of IS630 were similar to the sequence at one end of IS200, a Salmonella-specific IS element. The fifth sequence, repeated ten times in Sh. sonnei, had about 98% sequence homology with a portion of IS2. The method described here can be applied to the isolation of IS or iso-IS elements present in any other bacterial chromosome.     

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.     

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.     

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.     

An insertion element prevents phycobilisome synthesis in N2-fixing Synechocystis sp. strain BO 8402.

The unicellular diazotrophic cyanobacterium Synechocystis sp. strain BO 8402, isolated from Lake Constance, contains a novel insertion sequence, IS8402, in the apcA gene encoding a pigmented protein of phycobilisomes. IS8402 comprises 1,322 bp, flanked by two inverted repeats of 15 bp. Upon insertion in the target DNA, direct duplications of 8 nucleotides were generated. One open reading frame, potentially coding for a protein of 399 amino acids, was found. The deduced amino acid sequence shows homology to putative transposases of the IS4 family. Precise excision of the insertion element resulted in a spontaneous revertant, Synechocystis sp. strain BO 9201, that had regained the ability to form hemidiscoidal phycobilisomes. Apart from the unique insertion of IS8402 into apcA in strain BO 8402 both strains contain at least 12 further homologous insertion elements at corresponding sites in the genomes. The unique insertion in strain BO 8402 prevents the expression of apcABC operon and hence abolishes the formation of intact phycobilisomes. This decreases the quantum efficiency of photosystem II and promotes anaerobic N2 fixation in a unicellular cyanobacterium with a highly oxygen-sensitive nitrogenase.     

Nucleotide sequence of IS110, an insertion sequence of Streptomyces coelicolor A3(2).

The nucleotide sequences of the Streptomyces transposable element IS110 and its insertion site in the DNA of a derivative of the temperate phage luminal diameter C31 were determined. The element is inserted about 460 bp from the right-hand end of luminal diameter C31 DNA, in a region of apparently non-coding DNA. The target site (in a run of seven C residues) is within an 11 bp sequence homologous with one end of IS110. The inserted element is flanked by runs of 11 and 15 C residues which form part of more extensive regions of homology between the left and right junction regions. Imperfect inverted repeats (10 matches out of 15 bp) are present near (but not at) the ends of IS110. The whole IS110 element contains about 1550 bp of which 71% are G-C bp. One major potentially protein-coding region (ORF 1215) was detected, of 1215 bp, the product of which, a presumptively soluble protein of MR 43,563, was not overtly related to any entry in a protein sequence database. A smaller open reading frame (ORF 330) was tentatively identified in the opposite strand of the ORF 1215 region.