Detection Of Mutation In The Acid-resistant Gene Ffh Of The Fluoride-resistant Strain Of Streptococcus Mutans

Streptococcus mutans is recognized cariogenic bacteria, its cariogenic ability is related not only with coherence, and acidogenic ability, but also closely with acid resistance. Acid resistance is that bacteria can continue producing acid and performing its function under low pH value, which is regarded as an important determinant factor for survival of cariogenic bacteria in the biofilm on teeth. It inchudes constitutive acid tolerance and acid tolerance response. Its Acid resistance is decided by many genetic factors. Some acid-resistant genes of streptococcus mutans have been separated and cloned now. the acid-resistant gene ffh is closely concerned by international researchers. The product of the gene is a kind of bioactive protein, which is the homologue of the 54-kDa subunit of the eukaryotic signal recognition particle. And an essential protein translocation pathway involves the signal recognition particle, of which the 54-kDa homologue(Ffh) is an essential component. Fluoride is extensively used for caries preventive. Its effect of cariogenic bacteria is among important mechanisms for the caries preventive of fluoride. Many studies have proved that hypersensitive streptococcus mutans for fluoride may produce mutation because of long contact with fluoride, even may lead to appearance of fluoride-resistant strain. The fluoride-resistant strain of streptococcus mutans has not been separated in normal people now, but the fact that high consistence of fluoride lead to appearance of fluoride-resistant strain , the success of inducement of fluoride-resistant strain in vitro, and using the fluoride for long-term all suggest the possibility of appearance of fluoride-resistant strain in normao people. Many research have proved that the fluoride-resistant strain of streptococcus mutans has stronger cariogenic ability, include its acidogenic ability and acid resistance and the genetypes has been changed, but idiographic part of gene mutation is not known. In this study, First Fluoride-resistant strain of streptococcus mutans was obtained in vitro and the genome was obtained by V-gene bacterial Gennomic DNA Mini-prep kit. according to the principle of primer design, we designed a pair of primers in the most reserved region of the sequences from GenBank database in order to obtain the unique polymerase chain reaction product. We found that we could obtain the unique product in the fluoride-resistant strain of streptococcus mutans. We cloned the PCR product of the fluoride-resistant strain of streptococcus mutans. into the TA vector and selected the white clones. The recombinant plasmid was digested by Pst I and Sph I. Among those, there were two including the fragment of 936bp and 1033bp. Thenwe cultured the clone to extract the genome abundantly. After polymerase chain reaction, we obtained PCR product and sequenced it. We compared the product sequence with those from GenBank database. Compared by BLASTN, we found that the acid-resistant gene ffh of the fluoride-resistant strain of streptococcus mutans has been changed. It has sixting point mutations.These changes: 1214(T→C), 1281(T→C) , 1420(A→G) , 1820(T→C) ,1892(A→T) , 1904(G→A) , 1916(T→C) , 2004(A→G) ,2045(G→A) , 2067(C→T) , 2148(G→A) , 2406(C→T) ,2442(C→A),2488(C→T),2510(A→G),2512(T→C),seven purins is 44%,nine pyrimidines is 56%.The result was submitted to American GenBank, and the accession number was AY857409. And the PCR reponse includes partial satC gene sequence, We find it has six point mutations. They are: 2540(A→T) ,2550(T→G) , 2554(G→T) , 2555(T→C) , 2558(G→A) ,2562(A→G)。We found that a transposon insertion in the ylxM-ffh intergenic region of the designated secretion and acid tolerance (sat) operon in an acid-sensitive S. mutans AS17. sat operon include s five genes : ylxM, ffh, satC, satD and satE. Except for Ffh, the functions of the sat operon gene products are unknown. SatC, SatD, and SatE have no homology to proteins with known functions, although YlxM may function as a transcriptional regulator linked to genes encoding SRP pathway proteins. The molecular mass of Ffh is...