Determination of groESL Gene Sequences of Streptococcus gordonii and Streptococcus oralis and Use as Targets for Species Identification
| Autor: | Jia-Chuan Hsu, 許家銓 |
|---|---|
| Rok vydání: | 1999 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 87 The oral or viridans group streptococci form a significant part of the normal flora of the human oral cavity and are associated with several disease conditions including dental caries, infective endocarditis and septicemia as well as purulent infections. Currently the viridans group streptococci taxonomically could be divided into five major clusters which included at least 20 species. These are S. mutans group, S. mitis group, S. milleri group, S. salivarius group and S. bovis group. No single system of classification suffices for the identification of this heterogeneous of organisms. Instead, classification depends on a combination of features including pattern of hemolysis observed on blood agar plates, antigenic composition, growth characteristics, biochemical reactions, and more recently, genetic analysis. The groEL gene, which encodes 60-kDa heat shock protein (GroEL), is ubiquitous and highly conserved among bacteria. It has been recently reported by using groEL gene as an alternative target for species-specific identification of staphylococci or mycobacteria. In this study, the S. gordonii groESL operon containing groES (282 bp) and groEL (1623 bp) was cloned and sequenced. The GroEL (groEL) of S. gordonii had 91% (81%) homology to S. pneumoniae and 60% (62%) to E. coli while GroES (groES) had 73% (71%) homology to S. pneumoniae and 39% (45%) to E. coli. Like other Gram-positive bacteria, a putative transcriptional promoter upstream of groES that was comprised of -35 and -10 hexamers franked downstream by the conserved Gram-positive heat shock gene regulatory sequence, CIRCE. A large inverted repeat that may function as a rho-independent transcriptional terminator was located downstream of groEL gene. In addition, the gene encoded ATP-binding cassette (ABC) transporter protein located upstream of groES and another gene located downstream of groEL were found. Degenerate PCR primers derived from conserved regions of the groESL operon of S. gordonii were used to amplify groESL of other species of viridans group streptococci. Sequences were determined for entire groESL of S. oralis and partial groEL fragment of the other species. The GroEL (groEL) homology of S. orals with S. gordonii, S. pneumoniae and E. coli were 93% (82%), 98% (91%) and 60% (62%), respectively. Comparing the sequence of GroEL and groEL among viridans group streptococci in this study, there were 75% and 85% homology among them. For application of species identification, the dot blot hybridization, direct sequencing and PCR-RFLP were used. Our results demonstrate that groEL-based identification method has the potential to be an alternative method for identification of the viridans group streptococci. Further work is required to test more strains of bacteria for designing a simple performing method with both high specificity and sensitivity. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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