The Determination Of The Promoters' Activity And The Terminal Sequence Of El Tor Vibrio Cholerea Typing Phage VP3

Cholera is an acute intestinal infectious disease caused by O1 and O139 Vibrio cholerae.There have been seven recorded cholera outbreaks caused by O1 serotype V. cholerae.The seventh and present pandemic of cholera begins in 1961 caused by El Tor strains.A useful phage-biotying approach was used in China.By this approach,V. cholerae El Tor strains is devided into two kinds of strains,i.e,epidemic strains and non-epidemic strains.We deal different group of strains with different processes in our daily work of diseases prevention and control.VP3 is one of the five typing phages used in phage-biotying approach.A shot-gun strategy has been used to determine the complete nucleotide sequence of VP3,combined with the primer walking method to close the sequence gaps.The genome sequence is 39,505bp,with a G+C content of 42.62%,which encodes 52 putative protein encoding open reading frames(CDs) and 17 T7-like promoters.In order to monitor the activity of VP3 promoters,we developed a real-time observation system by useing the rluc as a report gene,along with the VP3 infection of V. cholerae.The result of the test manifests that the 17 predicted VP3 promoters region have promoter activities,but the activity of VP3 promoters No.5,6,10and 15 is weaker than that of the other 13 promoters.After infection,the lysis of host strain is related with generous replication of the VP3 genome which depends on RNAP of V.cholerae and presumed RNAP of VP3. To further determine the role of the two RNAPs,we constructed the plasmid pBAD33-VP3-RNAP by cloning the VP3 RNAP gene to pBAD33.Then the plasmids containing the putative VP3 promoter and RNAP gene respectively were introduced into E.coli JM109 and the fluorescent signal was assayed with the VivRen^TM Live Cell Substrate.We find that the most of the VP3 promoters can be triggered by the RNAP of N16961 in V.cholerae,but not by RNAP of JM109 in JM109.We also found that some VP3 promoters could be triggered in JM109 by introducing VP3 RNAP gene on plasmid.Our results indicate that the infection pattern of VP3 maybe as same as T7 family,in which the host RNAP plays an important role in the transcription of the early genes of T7 phage.The transcription of VP3 genome in V.cholerae may be related to the VP3 DNA terminal sequence.Though the whole genome sequence of VP3 has been sequenced, the real end of it has not been deterimined.We got a single band by using two primers complementary to each end of the VP3 sequence.The results suggests that there maybe some circle or some unique struction at the end of VP3 DNA.In order to study the receptor of VP3 in host cell,we deleted the O antigen encoding gene VC 0237 by homologous recombination mediated by suicide plasmid pCVD442.The generated strain(named C37) can't be lysed by VP3.Our result indicated that the O antigen of V.cholerae is envolved in the VP3 infection.