ABSTRACT
Objective To study whether ORF3 coded by fap1-orf4 gene locus of Streptococcus pa-rasanguis is involved in the regulation of Fap1 glycosylation and maturation and to investigate whether ORF3 influences Streptococcus parasanguis adhesion. Methods A gene replacement strategy was adapted to con-struct orf3 alleic replace mutant of Streptococcus parasanguis. Complementation assay and Western blot were used to test Fap1 expression levels. Whole saliva-coated hydroxyapatite (SHA) adhesion assay was adapted to determine Streptococcus parasanguis adhension. Results (1) Non-polar was found in strain VT1774, the orf3 alleic replace mutant of Streptococcus parasanguis. (2) Western blot showed that mature Fapl (Mr about 220 × 103) disappeared and were substituted with high molecular weight Fapl (Mr about 470 × 103) in strain VT1774, furthermore, complementation assay showed VT1775, the complementation strain of VT1774, re-stored mature Fapl expression. (3) The binding ability reduced significantly in strain VT1774. Conclusion ORF3 coded byfapl-orf4 gone locus was required for Fap1 glycosylation and maturation in Streptococcus pa-rasanguis, orf3 alleic replacment resulted in Fap1 glycosylation and mature disorder and decreasing of adhen-sion ability of Streptococcus parasanguis.
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.