Complete genomic sequence of the temperate bacteriophage ΦAT3 and functional assay of the circular structure in the transposition of insertion sequence ISLC3

• Main Authors: Lo, Ta-Chun, 羅大鈞
• Other Authors: 林志侯
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/71824028782208876735

博士 === 國立清華大學 === 生命科學系 === 94 === The complete genomic sequence of a temperate bacteriophage ΦAT3 from Lactobacillus casei ATCC 393 was reported. The newly isolated phage consisted of a linear dsDNA genome of 39,166 bp (accession no. AY605066), an isometric head of 53 nm in diameter, and a flexible noncontractile tail of approximately 200 nm in length. The morphology of ΦAT3 examined resembled that of Siphoviridae, the number of potential open reading frames determined on the phage genome is 53. The ΦAT3 genome was grouped into five distinct functional clusters: DNA packaging, morphogenesis, lysis, lysogenic/lytic switch, and replication. The amino acid sequences at the NH2-termini of some major proteins were determined. An in vivo integration assay for the ΦAT3 integrase (Int) protein in several lactobacilli was conducted by constructing an integration vector including ΦAT3 int gene and the attP (int-attP) region. It was found that the ΦAT3 genome integrated at the tRNAArg gene locus of Lb. rhamnosus HN 001 (also known as DR 20), similar to that observed in its native host, Lb. casei ATCC 393. An insertion sequence ISLC3 of 1,351 bp has been isolated from ΦAT3. The distribution of ISLC3 in several lactobacilli has been analyzed by Southern hybridization. The transposition of ISLC3 in all the lactobacilli studied produces a truncated junction of right and left inverse repeat where a direct repeat and 25 bp in the left inverse repeat are deleted. However, both the complete and truncated circles are detected in an Escherichia coli system studied. The activities of promoters formed by both the complete and truncated circles are analyzed using an assay system of β-galactosidase. That the promoter activity formed by the circle junction region of ISLC3 was nearly the same as the indigenous one and the activities of both are much stronger than that formed by the truncated one. However, the transposition will not occur in 25 bp truncated junction circles, indicating that formation of the truncated circles in the host is to suppress the effect of high frequent transposition which may be detrimental to the host.