Transposon mutagenesis of Psychrobacter cryohalolentis PAMC 21807 by tri-parental conjugation

Random mutagenesis is commonly used to study gene function. The screening of mutants exhibiting specific pheno- types assists in the identification of phenotype-related genes. In the current study, we isolated Antarctic bacteria, and developed a transposon Tn5 mutagenesis system. A total of 26 strains were isolated from seawater and freshwater near Antarctic King Sejong Research Station, King George Island. Six Psychrobacter strains were identified as psychrophilic, with optimal growth tempera- tures of 10~C or 15~C Psychrobacter cryohalolentis PAMC 21807 with a high growth rate at 4~C was selected for transposon mutagenesis. Tri-parental conjugation with a plasmid containing Tn5 produced 13 putative recombinants containing the selectable marker. Genomic Southern hybridization confirmed Tn5 existed as episomes for seven recombinants, and for a single recombinant, Tn5 was integrated into the genome of Psychrobacter cryohalolentis PAMC 21807. The result indicates that the mutagenesis method, although successful, has a relatively low rate. The psychrophilic bacteria isolated in this study may be a useful resource for studying cold adaptation mechanisms, and the mutagenesis method can be applied to genetic analysis.

Dynamic interaction of neutrophils and RFP-labelled Vibrio parahaemolyticus in zebrafish(Danio rerio)

Vibrio parahaemolyticus(Vp)is an aquatic zoonotic pathogen that causes vibriosis in marine animals as well as sepsis,gastroenteritis and wound infection in human.In vertebrates,the innate immune system plays a critical defense mechanism against Vp infection including transmigration of neutrophils.In this study,we have examined the genetic recombination and infectious process of Vp in the zebrafish(Danio rerio),a vertebrate model system extensively used for studying host-pathogen interactions.A pathogenic Vp strain,Vp57,tagged with red fluorescent protein(RFP)(Vp57^(RFP))was introduced into larval zebrafish at 3 days post fertilization(dpf),in which the innate immunity is present whereas the adaptive immunity has not yet developed.Vp57 and Vp57^(RFP)showed similar LD50 and induced similar symptoms and pathological changes in the hosts.We microinjected 579 colony-forming units(CFU)Vp57^(RFP)into the zebrafish caudal vein,and observed that neutrophils were recruited to the injection site and within 3 h post infection(hpi)Vp57^(RFP)were mainly distributed in the tail,eyes,heart and optic vesicle.After 3 hpi,the fish died with slight spine bending.Several Vp57^(RFP)were also detected in somites,the phagocytosis of neutrophils was activated through the progress of bacterial infections.We found that chemotaxis and phagocytosis of neutrophils occurs when zebrafish is infected by Vp57^(RFP),whereas Vp57^(RFP)can escape from neutrophils and colonize other remote regions using the blood circulation system.