Study on function and interaction of GTF and ORF4 coded by fap1-orf4 gene locus of Streptococcus parasanguis

Yi Rong Li, Li Hua Hu, Xiang Huang, Hui Wu

Research output: Contribution to journalArticlepeer-review

Abstract

Objective: To investigate whether glucosyltransferase(GTF) and open reading frame 4 (ORF4) coded by fap1-orf4 gene locus of Streptococcus parasanguis was involved in the regulation of Fap1 glycosylation, and mature and to determine whether there was interaction between GTF and ORF4. Methods: A gene replacement strategy was adapted to construct gtf and orf4 alleic replace mutant of 5. parasanguis. Complementation assay and Western blot were used to test fap1 expression levels. Yeast two-hybrid analysis and GST pull down assays were adapted to determine the interaction between GTF and ORF4. Results: (1) Compared with wild S. parasanguis, mature Fap1 (M r about 220 × 10 3) disappeared and were substituted with high molecular weight Fap1 (M r about 360 × 10 3) in gtf or orf4 alleic replace mutants of S. parasanguis. Complementation assay showed that pVPT-GFP-gtf and pVPT-GFP-or/4 restored mature fap1 expression in gtf or or/4 alleic replace mutants, respectively. (2) With Yeast two-hybrid analysis, the cotransformants, AH109/pAD-Gtf + pBD-orf4 and AH109/pAD-orf4 + pBD-gtf growed on SD-LTHA selective agar plate after streaked, reversely, the cotransformants, AH109/pAD + pBD-or/4, AH109/pAD + pBD-gtf, AH109/pBD + pAD-orf4, AH109/pBD + pAD-gtf did not grow on SD-LTHA selective agar plate, furthermore, the cotransformants , AH109/pAD + pBD-or/4 and AH 109/pAD-orf4 + pBD-gtf showed blue during X-α-gal assay. (3) CST pull down assay confirmed the direct interaction between GTF and ORF4. Conclusion: There is interaction between GTF and ORF4 coded by fap1-orf4 gene locus of S. parasanguis and the formation of the GTF and ORF4 complex was required for the glycosylation and mature of Fap1 in S. parasanguis.

Original languageEnglish (US)
Pages (from-to)771-776
Number of pages6
JournalChinese Journal of Microbiology and Immunology
Volume28
Issue number9
StatePublished - 2008
Externally publishedYes

Keywords

  • Fimbriae-associated protein 1
  • Glucosyltransferase
  • Glycosylation
  • Open reading frame 4
  • Streptococcus parasanguis

ASJC Scopus subject areas

  • Microbiology
  • Immunology
  • Virology

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