Construction Of Lactobacillus Delbrueckii Subsp.bulgaricus Mutants By ISS1Transposon Insertion

The lactic acid bacteria (LAB) is a kind of gram-positive bacteria which can produce lactic acid asthe major metabolic end-product of carbohydrate fermentation，and they are generally regarded as safefood microorganism．The Lactobacillus delbrueckii subsp.bulgaricus is one of the most important of thelactic acid bacteria, and is widely used in the medicine,health products,food industies and animalfeeding. It is significative of using modern molecular biology technology to change the characteristic ofLactobacillus delbrueckii subsp.bulgaricus.We use the plasmid pGhost9:ISS1to generate an efficienttransposon mutagenesis,and screening the mutants.The plasmid pGhost9:ISS1with the insertion sequence ISS1replicates at28℃but losts above37℃.In this study, pGhost9:ISS1was used to test for ISS1transposition at the nonpermissivetemperature.Replicative transposition of ISS1into the chromosome of Lactobacillus delbrueckiisubsp.bulgaricus leads to the integration of the plasmid vector (pGh) between duplicated ISS1sequences.Transposition of pGhost9:ISS1is random and efficient.The frequency of transposition ishigher than10-2. Every cell have only one transposition.To excise the transposed vector was incubatingat the permissive temperature. Recombination between the duplicated sequences would give rise to astable structure in which a single ISS1element remains in the chromosome.In the study,transposants were successfully incubated at42℃on MRS agar containing2μg·mL-1Em,50μg·mL-1Deoxythymidine and some concentration trimethoprim (TMP).Then samples werediluted and plated at42℃on MRS agar containing Erythromycin (Em), Deoxythymidine andTMP.Colonies were transferred with toothpicks to containing Deoxythymidine and non containingDeoxythymidine SA plates.Then the thyA-null mutants of Lactobacillus delbrueckii subsp.bulgaricuswere obtained after33generations of cultivation in the presence of500μg·mL-1TMP.In this study,in order to abtain wesk-postacidification mutants of Lactobacillus delbrueckiisubsp.bulgaricus,transposants,which was treatment for5h on MRS containing2000μg·mL-1penicillinand pH4.3conditions.The mortality rate of cell is50.6%,low pH conditions were still growing cellswere killed.The optimum enrichment effect was abtained.Then samples were diluted and plated at42℃on MRS agar containing2μg·mL-1Em. Colonies were transferred with toothpicks to low pH andhigh pH MRS plates.The obtained44strains were poor growth or no growth under the low pHconditions.After further testing, I screened out3weak postacidification mutants.Efficient excision ofthe replicon by a temperature shift gives rise to a stable food-grade mutant strain.