Study On The Role And Mechanism Of Autolysins In Lactic Acid Bacteria Autolysis

Lactic acid bacteria （LAB）are generallly recognized as safe and the most typical representativeof probiotics. LAB occcupies a central role in fermentation process and has a long and safe history ofapplication and consumption in the worldwide production of fermented food processing because oftheir physiological functions in maintaining the balance of intestinal flora of host body.Autolysis of lactic acid bacteria is a common phenomenon in fermented dairy production and playsan important role of regulating the quality, flavors and production cycle of fermenteddairy products. Therefore, the study of the influential factors and mechanism of autolysis of lactic acidbacteria would supply the better guide for the application of lactic acid bacteria inindustrial production and provide a theoretical basis for starter breeding to suit the demand offermented dairy industry.In this study, LABs with various autolysis rate were screened from the Chinese traditionalfermented dairy products. The highest and lowest autolysis rate strains were selected as the mainresearch object. First, the effects of environmental factors on the autolysis and various autolysinsexpression of lactic acid bacteria were studied. Second, low autolysis strains was induced by N+implantation, positive mutants and negative mutants were selected on the basis of autolysis rate. Thenanalyzing expression variation of selected autolysin genes between wild type strain and mutant byfluorescence quantitative real-time PCR. Furthermore, Sequencing the selected autolysin genes to findout probable base mutant site. Third, the key autolysin of the high autolysis strain was knocked out forverifing its effect on the autolysis of LAB. Finally, the autolysis rate，morphological characteristics,and other biological characteristics was investigated between wild type strain and gene knockout strain.According to the above results, to illuminate the affect of autolysin on autolysis and reveal theinteraction among different autolysins in different LAB.The main achievements of this study are as follows:1. Through analyzing the autolysis rate of7lactic acid bacteria strains isolated from the Chinesetraditional fermented dairy products by the autolysis rate test method based on detecting nucleic acid/protein dissolution, we achieved the order of autolysis rate of7strains from high to low is: LJJ>SY15>LB1>AST18>LGG>LB2>116-a and screened high autolysis strain Lactobacillus bulgaricusLJJ and low autolysis strain Lactobacillus casei116-a. There are significant differences betweenautolysis rate of LJJ and116-a (P<0.01). Under the same autolysis conditions, the LJJ cell showedvarious degrees of atrophy and sunken and holes appeared on the cell wall surface. Intracellular contentsalso began to gradually be released to the surrounding environment as a result of the damage of cellintegrity during the autolysis process. In contrast, the low autolysis strain116-a showed slight cellautolysis. Although cell displayed atrophy and intracellular contents of little cell was released to thesurrounding environment, most of the cells still remained the original integrity of cell wall, cell surface did not appear obvious holes and damage.2. The environment temperature, pH value, SDS concentration, Triton X-100concentration anddifferent growth stages can influence autolysis rate and extent of Lactobacillus bulgaricus LJJ througheffectting on autolysis enzyme yield and activity. Lactobacillus bulgaricus LJJ cells from differentgrowth stages displayed various autolysis rate. The cells from logarithmic growth phase showed themaximum autolysis rate. The cell from stability growth phase diplayed the lower autolysis rate than thatof the cell from logarithmic growth phase. Then the cells from recession phase owned the minimumautolysis rate and cells from the mid to late of recession phase no longer occurred autolysis because ofcell death. Autolysis of Lactobacillus bulgaricus LJJ had the optimum environment pH range of6.0-8.0and when environment pH≤4or≥10, autolysis can be almost inhibited completely. In thetemperature range of0-50℃, the autolysis rate increased with temperature increase. However, when thetemperature was higher than60℃, its autolysis was partially inhibited and autolysis rate began todecrease. The presence of SDS can suppress autolysis, when the concentration of SDS increased to0.05g/100mL, autolysis of lactic acid bacteria was almost completely inhibited. SDS can also suppress theactivity of lysozyme and suppression curve was consistent with the autolysis supperssion trend. TritonX-100can enhance lysozyme activity and autolysis rate and extend of LJJ increased with content ofTriton of X-100increase.3. Autolysis of LABs was regulated by different key autolysins. Among AcmA, AmiA, GlcNAcaseand Dac, the expression trend of AcmA and AmiA was consistent with trend of auolysis rate inLactobacillus bulgaricus LJJ. The relative expression range of AcmA of LJJ from several times tohundred times, depending on different conditions.So the most likely key autolysin of Lactobacillusbulgaricus LJJ is AcmA. While The expression trend of GlcNAcase and AmiA was consistent withtrend of auolysis rate in Lactobacillus casei116-a. The relative expression range of GlcNAcase of116-afrom several times to hundred times, depending on different conditions. So the most likely key autolysinof Lactobacillus casei116-a is GlcNAcase. For Lactobacillus bulgaricus LJJ, The expression of fourautolysins were almost not influenced by environment pH value variration. The changes of cell autolysisrate with environment pH value variation were due to high autolysin activity under the optimum pHconditon rather than autolysins expression increase. Dac expression was up-regulated under low pH anddownregulated under high pH, which suggested that the expression of Dac will be affected by theenvironment pH in Lactobacillus casei116-a. Triton X-100can accelerated autolysis of LAB throughthe upregulation of AcmA and AmiA expression.4. Lactobacillus casei116-a was induced by N+implantation with energy of30Kev. Twomaximum positive mutants and two negative mutants with good genetic stability were screened underN+implantation dose2.5×1015ions/cm2and2×1015ions/cm2respectively. Real time fluorescencequantitative PCR analyzed the relative expression of aotulysins between wild type strain and the mutantstrain.The results showed only the expression of N-acetylglucosaminidase was significantlyup-regulated or down-regulated among four selected autolysins. N-acetylglucosaminidase was up-regulated exceeded30times in positive mutant while that was down-regulated6times in negaitivemutant. Sequencing four autolysin of mutants showed several base site of N-acetylglucosaminidase andN-acetylmuramidase were change and leaded to the amino acid change.5. Through suicide plasmid vector gene knockout strategies, we successfully constructed theinactivation of N-acetylmuramidase Lactobacillus bulgaricus mutant containing erythromycinresistance gene. The erythromycin resistance gene (Emr) as a selective marker, was successfully insertedinto the N-acetylmuramidase gene. Then it was connected with the suicide plasmid vector pUC19forlactic acid bacteria successfully. So N-acetylmuramidase gene knockout component was constructed forLactobacillus bulgaricus LJJ. Optimizing electroporation conditions of Lactobacillus bulgaricus LJJ byresponse surface method and the optimal electroporation conditions electric voltage, resistence andglycine content of1.5kV,400and0.5g/100mL culture medium. Under the optimal electroporationconditions, the N-acetylmuramidase gene knockout component was transformed into Lactobacillusbulgaria LJJ cell. Then N-acetylmuramidase insertion inactivation Lactobacillus bulgaricus mutant wasscreened by MRS plate containing erythromycin sucessfully and identified by PCR verification.6. N-acetylmuramidase of Lactobacillus bulgaricus LJJ not only affects the autolysis but alsoplays an vital role of other physiological and biochemical characteristics, such as the growth rate ofbacteria, acid producing capacity and storage. Compared with the wild type strain, N-acetylmuramidasegene deletion strains produced the following changes: the autolysis rate was decreased significantly(P<0.01). The autolysis rate of24h decreased to about1/2than that of wild type strain. The result ofscanning electron microscope showed the single cell length of the gene deletion mutant increased fromthe previous ten micrometers up to tens micrometers, which is several times longer than the length ofwild type strain single cell. For gene deletion strain, the growth rate decelerated, the highest cellconcentration reduced from3.5to around2.5(OD600value), the maximum viable count of bacteriawas decreased to7.0.-8.0×107cfu/mL, compared to the wild type strain the value decreased by almostone order of magnitude and biomass was reduced by about10%. Besides, althrough fermentation ratedescended, the achievable minimum pH decreased from3.89to3.78. Storage stability of gene deletionstrains were greatly improved, the14day survival rate was increased from0.5%to50%under4℃temperature.