Characterization of IS1515, a functional insertion sequence in Streptococcus pneumoniae

Abstract

We describe the characterization of a new insertion sequence, IS1515, identified in the genome of Streptococcus pneumoniae I41R, an unencapsulated mutant isolated many years ago (R. Austrian, H. P. Bernheimer, E. E. B. Smith, and G. T. Mills, J. Exp. Med. 110:585-602, 1959). A copy of this element located in the cap1EI41R gene was sequenced. The 871-bp-long IS1515 element possesses 12-bp perfect inverted repeats and generates a 3-bp target duplication upon insertion. The IS encodes a protein of 271 amino acid residues similar to the putative transposases of other insertion sequences, namely IS1381 from S. pneumoniae, ISL2 from Lactobacillus helveticus, IS702 from the cyanobacterium Calothrix sp. strain PCC 7601, and IS112 from Streptomyces albus G. IS1515 appears to be present in the genome of most type 1 pneumococci in a maximum of 13 copies, although it has also been found in the chromosome of pneumococcal isolates belonging to other serotypes. We have found that the unencapsulated phenotype of strain 141R is the result of both the presence of an IS1515 copy and a frameshift mutation in the cap1EI41R gene. Precise excision of the IS was observed in the type 1 encapsulated transformants isolated in experiments designed to repair the frameshift. These results reveal that IS1515 behaves quite differently from other previously described pneumococcal insertion sequences. Several copies of IS1515 were also able to excise and move to another locations in the chromosome of S. pneumoniae. To our knowledge, this is the first report of a functional IS in pneumococcus.

FIG. 1

Genetic organization of the cap1 cluster of S. pneumoniae 13868 containing the genes coding for type 1 capsular polysaccharide synthesis (A) and the cap1E_I41R gene (B). The partial restriction maps of the corresponding regions are also shown. Thick and thin arrows represent complete or interrupted ORFs, respectively. Some of the plasmids used in this study are indicated, as are relevant restriction sites (B, BglII; E, EcoRI; Ec, Eco47III; H, HindIII; and S, ScaI). The location of the promoter of the cap1 cluster is also shown (p). Ovals represent putative transcription terminators (22). Facing solid triangles indicate the locations and directions of pairs of oligonucleotide primers.

FIG. 2

Nucleotide and deduced amino acid sequences of the cap1E_I41R gene containing the IS1515 element. The sequence of the cap1E₁₃₈₆₈ gene (22) is shown for comparison. The sequence corresponding to the I41R strain is shown in italics, whereas that for the IS1515 element is represented in boldface italics. When nucleotides (or amino acid residues) coincide, that corresponding to strain 13868 is substituted for by a colon. The nucleotide differences between both cap1E genes are highlighted as boldface ellipses. The repeated target sequence AAT is shown inside a white box, whereas the terminal inverted repeat sequences of IS1515 are inside black boxes. Upstream of the tnp₁₅₁₅ gene coding for the IS1515 transposase, putative extended −10 (✚) and −35 promoter (•) regions are located. The locations and directions of oligonucleotide primers are indicated, as are EcoRI-ApoI restriction sites. One of the ends of the EcoRI insert of plasmid pCMM6 is also shown. Asterisks indicate stop codons.

FIG. 3

Alignment of the deduced amino acid sequences of transposases of several IS1515-related elements. The multiple alignment was carried out with the PILEUP program. Identical amino acid residues in at least four of the proteins are shown in black boxes, and conserved substitutions in all of the transposases are shown in shaded boxes. Pairwise comparisons of the transposases were done with the BESTFIT program. The percentages of identities and similarities (in parentheses) are indicated.

FIG. 4

IS1515 distribution among strains of S. pneumoniae belonging to different serogroups. Southern blotting was performed with EcoRI-digested genomic DNAs from the indicated independent isolates, which were electrophoresed on agarose gels. The hybridization was carried out at 65°C with biotin-labeled pRMM34 as the probe. S5, S19, and S25 represent isolates belonging to serogroups 5, 19, and 25, respectively. M22 is a rough derivative of a type 2 strain. All other strains are type 1 isolates. λφX indicates a mixture of HindIII-digested λ DNA and the replicative form of φX174 DNA digested with HaeIII.

FIG. 5

IS1515 distribution in encapsulated transformants derived from the I41R strain. Competent cells of I41RΔlytA32 were transformed with pCMM6, and encapsulated cells were isolated as described in the text. Chromosomal DNA prepared from four independently isolated transformants (1 through 4) was digested with HindIII (A) or ScaI (B), and the fragments were separated by agarose gel electrophoresis. Southern blotting was performed at 65°C with biotin-labeled pRMM34 as the probe. The profiles of the chromosomal DNAs prepared from the parental strains I41R and I41ΔlytA32 digested with the same restriction enzymes are also shown. The DNA bands (5.2-kb HindIII and 3.7-kb ScaI fragments, respectively) corresponding to the IS1515 copy located in the cap1E_I41R gene are indicated by arrowheads. λ indicates HindIII-digested λ DNA.

FIG. 6

IS1515 distribution in streptomycin (Str^R)- or lincomycin (Lin^R)-resistant transformants of the I41R strain. Competent cells of I41R were transformed for the LytA⁻ phenotype with pGL32, for streptomycin resistance with M22 chromosomal DNA, or for lincomycin resistance with pLSE1. Total DNA from one transformant of each class was digested with EcoRI, and the fragments were separated by agarose gel electrophoresis, blotted, and hybridized at 65°C with biotin-labeled pRMM34. The molecular sizes (in kilobases) of the standards (a mixture of HindIII-digested λ DNA and the replicative form of φX174 DNA digested with HaeIII) are indicated to the left.