Study On The Lysis-lysogeny Regulation Of Transposable Bacteriophage SHP3

Transposable bacteriophages are a special kind of temperate phages which integrate and replicate their genome through transposition.Due to their unique transposition characteristics,transposable phages not only have great potential in the development of genetic manipulation tools,but also effectively promote gene horizontal transfer and affect host evolution strategies.According to the genomic organization,transposable phages can be divided into Mu type and B3 type,represented by Escherichia coli phage Mu and Pseudomonas aeruginosa phage B3 respectively.Phage SHP3 is a temperate phage released by Stenotrophomonas maltophilia c31.According to the genomic organization,SHP3 is speculated as a B3-like transposable phage,and the predicted lysis-lysogeny regulatory region is quite different from that of Mu.Since the lysis-lysogeny regulation mechanism of B3 type transposable phages has not been reported yet,this study proposed to analyze the biological characteristics of the phage SHP3 first,and then to explore its lysis-lysogeny regulation mechanism.Firstly,the sensitity to temperature of phage SHP3 were tested and it was found that the phage was completely inactivated when the temperature was over 55?;one-step growth curve suggested that SHP3 had a long latent period of about 80 min after infected sensitive bacteria,then entered into lysis period;Tail-PCR detected that the direct repeat sequence on both sides of the SHP3 prophage on the host chromosome was 5'-CGCAGC-3';the product encoded by gene orf3 which is specific to SHP3exhibited exonuclease activity in vitro.In addition,the phylogenetic analysis also showed that SHP3 belongs to B3 type transposable phage.Subsequently,the lysis-lysogeny regulation mechanism of phage SHP3 were studied.According to bioinformatics analysis,genes orf18?orf21 were speculated to encode regulatory proteins,and the region between orf19 and orf20 is the key region for lysis-lysogeny regulation of SHP3.Genes orf18?orf21 were overexpressed in S.maltophilia c31?s and the phage titer was detected.The results showed that the phage titer increased about 100 times when orf19 was overexpressed;in contrast the overexpressing of orf20 resulted in the phage titer decreased about 100 times,while the expression of orf18 and orf21 didn't affect the phage titer.At the same time,experiments proved that the expression of orf20 can confer host bacteria immunity to SHP3 super infection.These results indicated that the lysis-lysogeny switch of phage SHP3 was cpmposed of gp19 and gp20.Among them,gp20 is the lysogeny repressor,which can not only maintain the lysogenic state of phage SHP3,but also can confer superinfection immunity to the host.RLM-RACE and RT-PCR experiments found three promoters that initiate the transcription of the SHP3 genes:P₁₉ located upstream of orf19,P₂₀ located upstream of orf20,and P₂₂ located upstream of orf22.The interactions between promoters and regulatory proteins were veryfied using the plasmid p BCZI.The results showed that the activity of P₁₉ was repressed by gp19 and gp20,the activity of P₂₀ was repressed only by gp20,and the activity of P₂₂ could be activated by gp1.Then the electrophoretic mobility shift assays further proved that both gp19 and gp20 proteins can bind to the promoter regions,indicating that gp19 and gp20 were transcriptional regulators.Finally,this study explored how phage SHP3 responds to MMC induction.The promoter activity of P₁₉ and P₂₀ and the expression of gp20 were tested under MMC-induced and non MMC-induced conditions,but no significant changes were found.Thus,it is still not known how phage SHP3 responds to MMC induction.In summary,based on the previous work,this study further explored the basic biological characteristics of phage SHP3.Besides that,the lysis-lysogeny switch of phage SHP3 was identified to be composed of two novel regulatory proteins,gp19 and gp20,and the regulation mode of gene transcription is completely different from that of Mu phage.The study of the basic biological characteristics and the lysis-lysogeny regulation mechanism of bacteriophage SHP3 expanded the understanding of B3 type transposable phages and provided the theory basis for further application of transposable phages.