Identification by subtractive hybridization of a novel insertion sequence specific for virulent strains of Porphyromonas gingivalis

Abstract

Subtractive hybridization was employed to isolate specific genes from virulent Porphyromonas gingivalis strains that are possibly related to abscess formation. The genomic DNA from the virulent strain P. gingivalis W83 was subtracted with DNA from the avirulent strain ATCC 33277. Three clones unique to strain W83 were isolated and sequenced. The cloned DNA fragments were 885, 369, and 132 bp and had slight homology with only Bacillus stearothermophilus IS5377, which is a putative transposase. The regions flanking the cloned DNA fragments were isolated and sequenced, and the gene structure around the clones was revealed. These three clones were located side-by-side in a gene reported as an outer membrane protein. The three clones interrupt the open reading frame of the outer membrane protein gene. This inserted DNA, consisting of three isolated clones, was designated IS1598, which was 1,396 bp (i.e., a 1,158-bp open reading frame) in length and was flanked by 16-bp terminal inverted repeats and a 9-bp duplicated target sequence. IS1598 was detected in P. gingivalis W83, W50, and FDC 381 by Southern hybridization. All three P. gingivalis strains have been shown to possess abscess-forming ability in animal models. However, IS1598 was not detected in avirulent strains of P. gingivalis, including ATCC 33277. The IS1598 may interrupt the synthesis of the outer membrane protein, resulting in changes in the structure of the bacterial outer membrane. The IS1598 isolated in this study is a novel insertion element which might be a specific marker for virulent P. gingivalis strains.

FIG. 1

Restriction map of the 2.1-kb PstI-digested DNA fragment containing IS1598. KIS-1, KIS-2, and KIS-3 are located in tandem on the 2.1-kb cloned DNA fragment (shaded bars). The 67-kDa OMP gene (pga67 [10]) is shown as a large open arrow. The pga67 is interrupted by the insertion of IS1598 at the DTS site and is shown as solid bars. The hatched bar indicates the ORF in IS1598.

FIG. 2

Nucleotide sequences and structure of the 2.1-kb neighbor region of IS fragment. Nucleotide numbers are indicated to the left. PstI sites at the both ends of the cloned 2.1-kb DNA fragment are shown in boldface. The locations of KIS-1, KIS-2, and KIS-3 are shown by indicating the 5′-end Sau3AI site of each fragment (vertical bar with arrow). pga67 is shown in lowercase letters (the start codon is shown by an arrow with ORF/pga67). The nucleotide sequence of the 5′-flanking region of the pga67 is partially omitted (indicated by dots). A DTS indicated by arrows above the sequence is found at both ends of IS1598, and the TIR indicated by lines above and below the sequence are found inside of each DTS. The ATG start codon and the TGA stop codon of the IS1598 are indicated by boxes. Putative translational products of IS1598 are given below the nucleotide sequence by a single uppercase letter. Upstream regulatory sequences are underlined and represent the −35 and −10 sequences and RBS (ribosome-binding site).

FIG. 3

Distribution of IS1598 in several gram-negative bacteria. Southern hybridization of PstI-digested genomic DNA from each strain was carried out with KIS-2 as a probe. Lanes: 1, P. gingivalis W83; 2, P. gingivalis ATCC 33277; 3, P. gingivalis W50; 4, P. gingivalis SU63; 5, P. gingivalis SUNY 1021; 6, P. gingivalis FDC 381; 7, P. gingivalis ESO 89; 8, P. asaccharolytica ATCC 25260; 9, P. loescheii ATCC 15930; 10, P. endodontalis ATCC 35406; 11, P. intermedia ATCC 25611; 12, C. ochracea S3; 13, F. nucleatum ATCC 25586; 14, A. actinomycetemcomitans Y4; 15, C. rectus ATCC 33238. P. gingivalis strains were classified based on AFNA and are indicated as “+” (AFNA positive) or “−” (AFNA negative) at the bottom of the panel.

FIG. 4

Transcripts from IS1598 and pga67. Total RNAs (10 μg/lane) isolated from W83 and ATCC 33277 strains were probed with IS1598 and pga67 by Northern hybridization. An asterisk shows a size difference of the hybridization signals between the two strains. Hybridization signal with the pga67 was detected at the 1.8-kb length in ATCC 33277 strain and was detected at the 3.0-kb length in W83. The hybridization signals in both strains corresponded well with the expected size (Fig. 1). Transcripts of IS1598 were detected only in W83 strain but varied in size.