Califano J V, Kitten T, Lewis J P, Macrina F L, Fleischmann R D, Fraser C M, Duncan M J, Dewhirst F E
Department of Periodontics, Virginia Commonwealth University, Richmond, Virginia 23298, USA.
Infect Immun. 2000 Sep;68(9):5247-53. doi: 10.1128/IAI.68.9.5247-5253.2000.
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.
牙龈卟啉单胞菌是一种产黑色素的革兰氏阴性厌氧菌，存在于牙周炎病变中，其在龈下菌斑中的存在会显著增加患牙周炎的风险。与许多细菌病原体不同，牙龈卟啉单胞菌菌株表现出相当大的变异性，这可能是由于基因交换和基因组内变化所致。为了探究后一种可能性，我们利用一种便捷快速的捕获类插入序列（IS）的方法，并通过对牙龈卟啉单胞菌W83菌株的基因组序列分析，研究了牙龈卟啉单胞菌W83中类插入序列元件的出现情况。我们采用了Matsutani等人（S. Matsutani, H. Ohtsubo, Y. Maeda, and E. Ohtsubo, J. Mol. Biol. 196:445 - 455, 1987）的方法，来分离和克隆基因组内重复区域特有的快速退火DNA序列。我们发现，在牙龈卟啉单胞菌W83菌株中，此类序列包括：（i）与tRNA基因具有同源性的核苷酸序列；（ii）一个先前描述过的IS元件；（iii）一个新型类IS元件。通过分析牙龈卟啉单胞菌基因组序列中使用频率最低的四核苷酸CTAG的分布情况，在最初的218个重叠群中的许多重叠群中确定了含有CTAG簇的区域。对这些CTAG簇的检查导致发现了11个与Matsutani等人的重复序列捕获方法鉴定出的相同新型类IS元件的拷贝。这个新的1512 bp类IS元件，命名为ISPg5，具有IS元件IS3家族的特征。当使用含有大部分ISPg5的重组质粒对包括临床分离株在内的几种牙龈卟啉单胞菌菌株进行Southern分析时，菌株间的差异很明显。这表明ISPg5和其他IS元件可能有助于菌株的多样性，并且可用于菌株指纹分析。
