Construction And Thermal Tolerance Analysis Of Enterococcus Faecium Hdrsef1Dnak Mutant Strain

Probiotics which has been widely used abroad have great commercial value because of its probiotic properties. It will be conducive to the health of animals when a sufficient number of viable cellobiotics were ingestioned by the host, such as antioxidation, immune enhancement, infection prevention and so on. However, the sufficient number of viable cellobiotics decreases in the process of processing and storage, which is due to the changing of environmental conditions, for example, heat, acid, oxygen, etc. This phenomenon appears in lactic acid bacteria most significantly. Our laboratory independently isolated a new heat-resistant probiotic called Enterococcus faecium. In order to study the heat shock response of probiotic Enterococcus faecium. We firstly constructed a gene knock-out molecular platform of the new probiotic Enterococcus faecium and knock out the main heat shock protein Dnak by homologous recombination. The results obtained are as follows:1. Construction of Dnak deleted mutant from Enterococcus faecium suis:Enterococcus faecium suis is sensitive to ampicillin, we repalced coding sequence (CDS sequence) of pSET4S spectinomycin acetyltransferase with β-lactamase coding sequence (CDS sequence) by directional cloning technology. The new transformation plasmid which was successfully constructed named pSET7S. For Dnak gene sequencing and blast analysis, Dnak amplifation product subcloned into pMD18-T. By Sequence alignment, we designed four primers to amplify Dnak gene homologous arm. By the method of overlapping PCR, Ligation product of the Dnak gene homologous left and right arm was subcloned into suicide plasmid pSET7S. This recombinant plasmid called pSET7S-LR which was later electroporated into Enterococcus faecium suis competent. After transconjugation, Dnak gene of Enterococcus faecium suis was knock out by homologous recombination and the result were analyzed by bacterium liquid PCR and southernblot. We successfully obtained Enterococcus faecium heat shock protein Dnak gene mutant strain.2. Heat tolerance analysis about Enterococcus faecium HDRsEfl:(1) The results of growth curve showe that wile type and deletion type of Enterococcus faecium HDRsEfl have similar growth characteristics.(2) The flat dilution and analysis of Enterococcus faecium HDRsEfl wild type and deletion type which hatched15min at the temperature of40℃、45℃、50℃、55℃、60℃、65℃and70℃. The difference of40℃and45℃is not significant. The resuil of50℃、55℃and65℃indicate the significant difference at5%level. The resuil of60℃and70℃indicate the significant difference at1%level.(3) Enterococcus faecium HDRsEfl wild type and deletion type were hatched1h at the temperature of40℃、60℃and70℃, plate count were carried out every10min. The resuil of20min、30min and40min at the temperature of60℃and10min、20min at the temperature of70℃indicate the significant difference at1%level.(4) Enterococcus faecium HDRsEfl wild type and deletion type were hatched12h with MRS culture media which contains different concentration of bile salt. The flat dilution and analysis indicates the significant difference at1%level, while concentration of bile salt is0.15%、0.3%and0.5%.(5) Enterococcus faecium HDRsEf1wild type and deletion type were hatched2h with pH2.0MRS while plate count was carried out every30min.The resuil indicates the significant difference at1%level when treated1.5h. The resuil indicates the significant difference at5%level when treated2h.(6) There is little difference between Enterococcus faecium suis and ADnak Enterococcus faecium HDRsEf1on antibiotics stress.