Study on function and interaction of GTF and ORF4 coded by fap1-orf4 gene locus of Streptococcus parasanguis
- VernacularTitle:副血链球菌fap1-orf4基因座位编码的GTF和ORF4的功能与相互作用的研究
- Author:
Yirong LI
;
Lihua HU
;
Xiang HUANG
;
Hui WU
- Publication Type:Journal Article
- Keywords:
Streptococcus parasanguis;
Glycosylation;
Glueosyltransferase;
Open reading frame 4;
Fimbriae-associated protein 1
- From:
Chinese Journal of Microbiology and Immunology
2008;28(9):771-776
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate whether glucosyltransfernse(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 allele replace mutant of S. 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 Fapl (Mr about 220×103) disappeared and were substituted with high molecular weight Fapl (Mr about 360×103) in gtf or orf4 alleie replace mutants of S. Parasan-guis. Complementation assay showed that pVPT-GFP-gtf and pVPT-GFP-orf4 restored mature fap1 expression in gtf or orf4 alleie replace mutants, respectively. (2) With Yeast two-hybrid analysis, the eotransformants, AH109/pAD-Gtf+pBD-orf4 and AHlOg/pAD-orf4+pBD-gtf growed on SD-LTHA selective ngar plate after streaked, reversely, the eotransformants, AH109/pAD+pBD-orf4,AH109/pAD+pBD-gtf、AH109/pBD+ pAD-orf4、AH109/pBD+pAD-gtf did not grow on SD-LTHA selective agar plate, furthermore, the cotrans-formants, AH109/pAD+pBD-orf4 and AH109/pAD-orf4+pBD-gtfshowed blue during X-α-gal assay. (3) GST pull down assay confirmed the direct interaction between GTF and ORF4. Conclusion There is inter-action between GTF and ORF4 coded byfapl-orf4 gene locus of S. Parasangnis and the formation of the GTF and ORF4 complex was required for the glycosylation and mature of Fapl in S. Parasanguis.
