| Summary: | 碩士 === 長庚大學 === 生物醫學研究所 === 99 === Streptococcus sanguinis is a primary colonizer of human tooth and an opportunistic pathogen for subacute endocarditis. A Type IV pili (Tfp) gene cluster was reported in the complete genome of Streptococcus sanguinis SK36 recently. The goal of this research aimed to analyze the expression and function of this gene cluster. The Tfp gene cluster is composed of total 16 genes, from pilB to pilD. A contiguous transcript was detected between pilD and the downstream lytB by RT-PCR, suggesting that lytB is also part of the operon. Three transcription initiation sites, 153- (P1), 536- (P2) and 837-base (P3) 5’ to the translation start site of pilB, respectively, were detected by rapid amplification of cDNA ends (5’ RACE) analysis. Both the P2 and P3 mapped to a σ70-like promoters (5’-TTGACA-N17-TATACT), whereas only an extended -10 sequence was observed with the P1. An anti-PilA antibody was generated and used to examine the structure of the pil cluster encoded products by transmission electron microscopy (TEM). A short hair-like structure was observed in the wild-type SK36 but not the Pil-deficient mutant strain, indicating that pil cluster is responsible for the synthesis of this structure. Furthermore, the pil-deficient mutant strains exhibited reduced biofilm formation. However, neither the adherence to Hela cells nor the twitching motility was affected by the deletion in pil cluster. Taken together, these results suggest that the pil cluster is responsible for the synthesis of a surface structure, and this structure is associated with biofilm formation. The multiple transcription initiation sties with long 5’ untranslated regions suggested the presence of a complex regulation system for the expression of the pil cluster.
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