Characterization of Porphyromonas gingivalis insertion sequence-like element ISPg5

Abstract

Porphyromonas gingivalis, a black-pigmented, gram-negative anaerobe, is found in periodontitis lesions, and its presence in subgingival plaque significantly increases the risk for periodontitis. In contrast to many bacterial pathogens, P. gingivalis strains display considerable variability, which is likely due to genetic exchange and intragenomic changes. To explore the latter possibility, we have studied the occurrence of insertion sequence (IS)-like elements in P. gingivalis W83 by utilizing a convenient and rapid method of capturing IS-like sequences and through analysis of the genome sequence of P. gingivalis strain W83. We adapted the method of Matsutani et al. (S. Matsutani, H. Ohtsubo, Y. Maeda, and E. Ohtsubo, J. Mol. Biol. 196:445-455, 1987) to isolate and clone rapidly annealing DNA sequences characteristic of repetitive regions within a genome. We show that in P. gingivalis strain W83, such sequences include (i) nucleotide sequence with homology to tRNA genes, (ii) a previously described IS element, and (iii) a novel IS-like element. Analysis of the P. gingivalis genome sequence for the distribution of the least used tetranucleotide, CTAG, identified regions in many of the initial 218 contigs which contained CTAG clusters. Examination of these CTAG clusters led to the discovery of 11 copies of the same novel IS-like element identified by the repeated sequence capture method of Matsutani et al. This new 1,512-bp IS-like element, designated ISPg5, has features of the IS3 family of IS elements. When a recombinant plasmid containing much of ISPg5 was used in Southern analysis of several P. gingivalis strains, including clinical isolates, diversity among strains was apparent. This suggests that ISPg5 and other IS elements may contribute to strain diversity and can be used for strain fingerprinting.

FIG. 1

The Matsutani et al. (28) method for isolating repeated DNA. Genomic DNA was subjected to hydrodynamic shearing, alkali denaturation, neutralization, and a brief period of reannealing followed by quick cooling on ice to favor intra- and intermolecular hybridization of repetitive DNA. Single-stranded DNA was digested with S1 nuclease, and remaining double-stranded DNA was cloned into pUC18. Arrows in bold indicate repetitive DNA.

FIG. 2

Agarose gel electrophoresis of DNA prepared by the method of Matsutani et al. (28). Arrows on the right indicate distinct bands visible by ethidium bromide staining. MW STD, molecular weight standards.

FIG. 3

(A) Agarose gel of BamHI/EcoRI digest of clone 14, containing IS5. Upper band is pUC18 vector; lower band is insert DNA. MW, molecular weight standards. (B) Southern blot of a BamHI/EcoRI digest of nine P. gingivalis strains probed with the ISPg5 clone. Strains in lanes 1 to 9: W83, W50, V2299, V2300, V2302, V2305, V2306, V2307, and V2308, respectively. The figure is a composite photograph of different exposures of the same blot. The positions of the bands are in register to the original blot.

FIG. 4

Organization of ISPg5. The position of the insert DNA in clone 14 is shown, along with the following features of ISPg5: DR (3-bp direct repeat), LIR (left inverted repeat), RIR (right inverted repeat), ORF1, and three possible versions of ORF2, including a fusion with ORF1 caused by frameshifting. The nucleotide and amino acid sequences of the fusion region are shown, with the frameshift-promoting region and the stop codon for ORF1 indicated in bold.