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Identification by subtractive hybridization of a novel insertion sequence specific for virulent strains of
K Sawada1, S Kokeguchi, H Hongyo
1Department of Periodontology, Okayama University Dental School, Okayama 700-8525, Japan.
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.
Insights
A novel insertion element, IS1598, was identified in virulent Porphyromonas gingivalis strains. This element may be a specific marker for abscess formation in these bacteria.
Area of Science:
- Microbiology
- Genetics
- Molecular Biology
Background:
- Porphyromonas gingivalis is a key pathogen in periodontitis and abscess formation.
- Identifying virulence factors is crucial for understanding and combating P. gingivalis infections.
Purpose of the Study:
- To isolate and characterize specific genes associated with abscess formation in virulent P. gingivalis strains.
- To identify potential genetic markers for P. gingivalis virulence.
Main Methods:
- Subtractive hybridization was used to compare genomic DNA from virulent (W83) and avirulent (ATCC 33277) P. gingivalis strains.
- Sequencing and Southern hybridization were employed to identify, characterize, and detect the insertion element IS1598.
Main Results:
- Three unique DNA fragments were identified in virulent strain W83, forming a 1,396 bp insertion element named IS1598.
- IS1598 was found in virulent P. gingivalis strains (W83, W50, FDC 381) associated with abscess formation but absent in avirulent strains.
- IS1598 interrupts an outer membrane protein gene, potentially altering bacterial outer membrane structure.
Conclusions:
- IS1598 is a novel insertion element in P. gingivalis.
- IS1598 is strongly associated with virulent, abscess-forming P. gingivalis strains.
- IS1598 may serve as a specific genetic marker for P. gingivalis virulence.
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