Objective:To study the effects of RNA polymerase(RNAP) from different sources on the predicted promoters of Vibrio cholerae typing phage VP3.Methods:T7 promoter of plasmid pRL-null was replaced by the fragments containing the predicted VP3 promoters,and Rluc was used as the report gene.The resulting plasmids were transformed into V.cholerae N16961,and the effect of N16961 RNAP on VP3 promoters was observed.These plasmids were transformed into Escherichia coli JM109 as well as a plasmid expressing VP3 RNAP,and the effect of JM109 and VP3 RNAP on VP3 promoters was observed.Results:Promoters P1,P2,P5,P6,P10 and P12 couldn't be used by N16961 RNAP,promoters P7 and P11 could be used by JM109 RNAP.In JM109,promoters P2,P7,P8,P9,P13,P16 and P17 could be used by VP3 RNAP.Conclusion:RNAP from host N16961 and non-host JM109 have different capability in transcribing from VP3 promoters,probably due to the specificity of phage for host cells.VP3 RNAP can transcribe from most VP3 promoters directly,but part promoters may need the help of other VP3 or host proteins to show stronger activities.VP3 promoters showed different specificity for VP3 RNAP:P1,P2 and P12 showed high specificity,and P7 and P11 showed weak specificity.

To investigate the function for receptor-binding of the tail fiber protein gp44 of Vibrio cholerae typing phage VP3 and to test whether gp44 is the site of interaction with its receptor on V.cholerae cell surface,gp44 was cloned and expressed with 6-His tag added to its N-terminus. The purified His-gp44 in solution was then incubated with the strain susceptible or resistant to the lytic action of VP3, its whether it wuld bind the susceptible or resistant strains was observed by fluorescent microscopy and laser scanning co-focal microscopy. Meanwhile,the sequence of the wav gene cluster wav the wild type V.cholerae with natural resistant to VP3 was DCR,amplified,and sequcnced.It was found that His-gp44 located on the surface of both susceptible and resistant stains,but not on the surface of Escherichiawti. When one of the genes in gene cluster wav was deleted,the susceptible mutant showed no His-gp44 around its cell wall. The sequence of the gene cluster wav showed only one single base difference in susceptible and resistant strains to VP3. It is concluded from the above mentioned observations that the tail fiber protein gp44 of V. cholerae typing phage VP3 can bind specifically with the surface receptors on V. cholerae.