| Vibrio parahaemolyticus is a gram-negative halophilic bacterium and often exists in seawater, seafood and salted food. V. parahaemolyticus is an important huaman seafood-borne pathogen, which contributed most to the food safety issues caused by microorganisms in China.In this study, we investigated the mutagenesis condition to eliminate the plasmidsin V. parahaemolyticus Chn25 using chemical curing agent(sodium dodecyl sulfonate, SDS) combined with increased growth temperatures. Our results showed that the optimal mutagenesis condition was the supplement of SDS at a final concentration of 0.2%(w/v) in Tryptic Soy Broth(TSB) medium and a sublethal growing temperature of 42℃. Under this condition, elimination rate of plasmids was more than 60%. In this study, for the first time, we found that this method can also be applied to the elimination of SXT/R391-family of the integrative and conjugative element(ICE, ICEVpa Chn1) of V. parahaemolyticus Chn25. The ICEVpa Chn1 and or plasmid deletion mutants were obtained, namely ICEs-eliminated strain(K), plasmid A and ICE together eliminated strain(PA), plasmids AC and ICEs together eliminated strain(PAC), which displayed no antibiotic resistance phenotypes. The morphological analysis revealed that the bacterial flagella and other major subsidiary structures appeared not affected by the elimination condition, indicating that this method was a relatively mild elimination method. Bacterial phenotypic analysis also showed that the eliminated ICE element was not involved in the regulation of heavy metal tolerance and bacterial motility. In addition, conjugation experiments revealed that the method established in this study did not damaged the original genetic insertion site of the ICE, as the mutant strains could obtain the original ICE element under appropriate conditions. The obtained mutant strains in this study have successfully been applied in our laboratory in gene knock out research of V. parahaemolyticus Chn25We further investigated the transcriptomes of the wild-type and three mutant strains using the high-throughput paired-end Solexa sequencing technique. When compared to the wild type strain, the differentially expressed genes derived from the mutants were analyzed against the GO(Gene Ontology), COG(Clusters of Orthologous Group) and KEGG(Kyoto Encyclopedia of Genes and Genomes) databases. This analysis revealed that the mutants had different numbers of the differentially expressed genes, among which the up-regulated genes become preponderant. When compared with wild type strain, 11.4% of the differentially expressed genes of the mutant K was significantly up-regulated, mainly involved in carbohydrate metabolism, energy metabolism, amino acid metabolism and signal transmission system; whereas 16.3% of the genes was significantly down-regulated, including those in fat metabolism, membrane system and peptidoglycan synthesis, cofactors and vitamin, nucleic acid replication and repair, exogenous material degradation and metabolism, as well as the translation function. For the PA mutant,11.3% of the differentially expressed genes was significantly up-regulated, mainly involved in carbohydrate metabolism, energy metabolism, amino acid metabolism, fatmetabolism, signal transmission system, membrane transport system and peptidoglycan synthesis, metabolism of cofactors and vitamins, exogenous material degradation and metabolism; whereas 5% of the genes was significantly down-regulated, mainly involved in nucleic acid replication and repair, and the translation function. When compared to the wild type strain, 6.5% of the differentially expressed genes in the PAC mutant was significantly up-regulated, mainly involved in energy metabolism, fat metabolism, signal transmission system, membrane transportsystem, auxiliary factors and sustenance, exogenous material degradation and metabolism, translation function; whereas 6.6% of the genes was significantly down-regulated, including those in carbohydrate metabolism, amino acid metabolism, nucleic acid replication and repair, nucleotide metabolism. The preliminary analysis of the transcriptome data showed that some biological functions such as nucleotide repaire were enhanced in different degrees in the three mutants. Our data in this study will facilitate future research in the stability mechanism of the ICE in V. parahaemolyticus Chn25... |
PDF Full Text Request