IS1630 of Mycoplasma fermentans, a novel IS30-type insertion element that targets and duplicates inverted repeats of variable length and sequence during insertion

Abstract

A new insertion sequence (IS) of Mycoplasma fermentans is described. This element, designated IS1630, is 1,377 bp long and has 27-bp inverted repeats at the termini. A single open reading frame (ORF), predicted to encode a basic protein of either 366 or 387 amino acids (depending on the start codon utilized), occupies most of this compact element. The predicted translation product of this ORF has homology to transposases of the IS30 family of IS elements and is most closely related (27% identical amino acid residues) to the product of the prototype of the group, IS30. Multiple copies of IS1630 are present in the genomes of at least two M. fermentans strains. Characterization and comparison of nine copies of the element revealed that IS1630 exhibits unusual target site specificity and, upon insertion, duplicates target sequences in a manner unlike that of any other IS element. IS1630 was shown to have the striking ability to target and duplicate inverted repeats of variable length and sequence during transposition. IS30-type elements typically generate 2- or 3-bp target site duplications, whereas those created by IS1630 vary between 19 and 26 bp. With the exception of two recently reported IS4-type elements which have the ability to generate variable large duplications (B. B. Plikaytis, J. T. Crawford, and T. M. Shinnick, J. Bacteriol. 180:1037-1043, 1998; E. M. Vilei, J. Nicolet, and J. Frey, J. Bacteriol. 181:1319-1323, 1999), such large direct repeats had not been observed for other IS elements. Interestingly, the IS1630-generated duplications are all symmetrical inverted repeat sequences that are apparently derived from rho-independent transcription terminators of neighboring genes. Although the consensus target site for IS30 is almost palindromic, individual target sites possess considerably less inverted symmetry. In contrast, IS1630 appears to exhibit an increased stringency for inverted repeat recognition, since the majority of target sites had no mismatches in the inverted repeat sequences. In the course of this study, an additional copy of the previously identified insertion sequence ISMi1 was cloned. Analysis of the sequence of this element revealed that the transposase encoded by this element is more than 200 amino acid residues longer and is more closely related to the products of other IS3 family members than had previously been recognized. A potential site for programmed translational frameshifting in ISMi1 was also identified.

FIG. 1

Nucleotide sequence and selected features of IS1630. The nucleotide sequence of IS1630A and flanking regions is shown, with the nucleotide numbers at left. Nucleotide 1 is the first nucleotide of the left IR of the IS. The deduced amino acid sequence encoded by the single ORF is shown in single-letter code below the DNA sequence, and each of the three acidic residues that comprise the highly conserved active-site motif (DDE) is enclosed within a box. The two candidate methionine start codons are underlined. The limits of IS1630 are indicated by square brackets, and the IR sequences at the left (IR-L) and right (IR-R) termini are highlighted by solid black lines. IR sequences within IS1630 are indicated by opposing arrows. The DR sequences that flank IS1630A are highlighted with asterisks below the corresponding sequence. The nucleotide sequence is presented in the orientation opposite that in GenBank AF100324.

FIG. 2

Identification of multiple copies of IS1630 in M. fermentans strains. Genomic DNA from M. fermentans PG18 (lanes 2 and 4) or II-29/1 (lanes 3 and 5) was digested with EcoRI (lanes 2 and 3) or HindIII (lanes 4 and 5), transferred to a nylon membrane, and hybridized with DIG-labeled oligonucleotide probe 5 (see Materials and Methods). Lane 1 contains DIG-labeled lambda HindIII markers (Boehringer), the sizes of which are indicated in kilobase pairs.

FIG. 3

Location of indels within IS1630 variants. Each cloned copy of IS1630 is shown as a thick horizontal line. IS1630A and IS1630C lack nucleotide (nt) indels. The locations of indels within the other IS1630 variants are shown by a nucleotide number above the horizontal line (numbered according to the IS1630A sequence). The nature of the indel is shown below the line. The positions of two candidate methionine start codons (M), the stop codon (stop), and the residues that comprise the DDE motif are shown below the horizontal line representing IS1630A. The 10 nucleotides immediately downstream of each IS copy are shown at the right, except for the truncated IS1630F, which lacks the distal portion of IS1630.

FIG. 4

Gene organization of cloned IS1630 flanking regions. The location and direction of ORFs flanking each cloned IS1630 element are shown by open arrows. IS1630 is represented by a grey arrow (rectangle in the case of the truncated IS1630F), and ISMi1 is shown as a solid black arrow. The number of base pairs between the IS1630 termini and the adjacent ORFs is shown between the arrows. Two tRNA genes downstream of IS1630C are also shown (T). When an ORF could not be given an unambiguous gene assignment, even if it exhibited homology to genes of unknown function in other bacteria, a designation that indicated to which IS1630 copy the ORF was linked was given (e.g. orfG1 and orfG2 flank IS1630G).

FIG. 5

Determination of insertion site occupancy in two M. fermentans strains by PCR. Amplicons generated by PCR on genomic DNA from M. fermentans PG18 (lanes 1, 4, 7, 11, and 14) or II-29/1 (lanes 2, 5, 8, 12, and 15) or water-containing negative controls (lanes 3, 6, 9, 13, and 16) were separated by agarose gel electrophoresis. Each series of three reactions contained a pair of opposing oligonucleotide primers that were derived from sequences flanking a specific IS1630 copy: IS1630A (lanes 1 to 3), IS1630B (lanes 4 to 6), IS1630H (lanes 7 to 9), IS1630C (lanes 11 to 13), and IS1630E (lanes 14 to 16). For any primer pair, a size difference of 1.4 kb between strains indicates that a site is occupied in one strain and empty in the other. When a site is occupied in both strains, the resulting PCR products are equal in size. Lanes 10 and 17 contain a 1-kb ladder (Promega Corporation, Madison, Wis.). The positions of the 1-kb (open triangle) and 3-kb (solid triangle) size markers are indicated.

FIG. 6

Features of IS1630 insertion sites. Predicted RNA secondary structures are shown for putative transcription terminators encoded by DNA sequences that are IS1630 target sites. The C-terminal amino acid (single-letter code: K, E or N) and stop codon (*) are shown at the left of each secondary structure. Three empty sites [(E)] were sequenced to confirm the stem-loop sequence. For orfE1, orfG2, and orfC1, the structures shown were derived by deleting the sequence of IS1630 and one copy of the DR. For orfD2 and orfF1, the appropriate IS1630 flanking sequences are not present to allow empty target sites to be deduced. The sites of insertion are shown by the solid triangles. The sequences duplicated as DRs are the DNA sequences that encode the stem-loop structures shown above the pairs of triangles. nt, nucleotides.

FIG. 7

Features of ISMi1. (A) Alignment of the deduced amino acid sequences encoded by ORF2* from the copy of ISMi1 inserted into IS1630I and ORF2 from IS150. Identical amino acid residues are highlighted with a black background. The numbers at the right indicate the positions of the amino acid residues for the respective ORFs (residue 1 for ORF2* is the first amino acid for the −1 reading frame after the stop codon for ORF1). A black vertical arrow indicates the originally proposed (13) position of the methionine start site for ORF2, and an open vertical arrow indicates the start site for the C-terminal region encoded by ORF2*, which is absent in ORF2. The residues comprising the DDE motif are indicated by asterisks. Gaps in the alignment are shown by dashes. (B) Proposed model for a putative frameshifting window in ISMi1 and comparison of the revised (upper cartoon) and originally proposed (lower cartoon) ORF arrangements in this element. A stem-loop structure for the ISMi1 mRNA region in which ribosomal frameshifting is proposed to occur is shown, together with the 0 and −1 reading frames. The ORF1 stop codon (UAA) is highlighted with asterisks in the secondary structure model and in the 0-frame sequence. The same three nucleotides are underlined in the −1 reading frame, in which they are components encoding consecutive Asn codons. In the schematic diagram showing the features of ISMi1, ORF1 is shown as a black arrow and ORF2 and ORF2* are represented by open arrows. The left (IR-L) and right (IR-R) IRs are shown as grey vertical bars at the IS termini. The location of the proposed frameshifting window is indicated by a triangle. The horizontal lines indicate untranslated regions. (C) Features of the frameshift window that has been established for IS150, highlighted as in panel B. The ORF1 stop codon in this case is a UGA codon. The features of IS150 are shown in the schematic diagram, following the conventions used in panel B.