| Lactobacillus delbrueckii subsp.bulgaricus is a kind of lactic acid bacteria which is widely used in the world food fermentation industry.Lactic acid bacteria often encounter acid environment during their growth and application,which has adverse effects on cell physiology,including membrane damage,inhibition of enzyme expression and transport system.Therefore,it is very important to clarify the mechanisms of acid resistance regulation and establish acid resistance regulation network for the survival and industrial application of bacteria.In this study,two-dimensional electrophoresis and real-time quantitative PCR were used to screen the genes related to acid tolerance regulated by two-component system HPK1/RR1 in histidine protein kinase HPK1 mutant CH3-H and response regulator protein RR1 mutant CH3-R compared with wild-type CH3 strain under the conditions of natural fermentation and acid induction.Lactococcus lactis NZ9000 was used for heterogenous expression and functional identification of key differentially expressed genes.The interaction between HPK1 or RR1 in two-component signal transduction system TCS1 and key differential proteins was preliminarily validated by yeast two-hybrid technique,and the key interaction sites were initially identified.The research contents and results are as follows:Acid-induced treatment can better simulate a series of genes that may be involved in the improvement of viability of strains when encounter ed with acid.Therefore,differentially expressed proteins in L.bulgaricus CH3 under normal fermentation and acid stress conditions were isolated,identified and the expression of transcriptional level was analyzed.Two-dimensional electrophoresis and mass spectrometry were used to identify the different proteins under normal fermentation and acid stress.Under natural fermentation,there were 22 differentially expressed proteins in mutant CH3-H and 24 differentially expressed proteins in mutant CH3-R compared with wild type CH3.Under acid induction,there were 6 differentially expressed proteins in mutant CH3-H and 20 differentially expressed proteins in mutant CH3-R compared with wild type CH3.Real-time quantitative PCR was used to study the expression of differentially expressed proteins related to acid stress at the transcriptional level.The above results indicate that the acid tolerance regulation of CH3 strain by two-component signal transduction system HPK1/RR1 is achieved through comprehensive regulation of multiple metabolic pathways.Heterogenous expression and functional validation of differentially expressed genes related to acid tolerance were carried out.The differentially expressed proteins Aprt,Ddl,Opp DII and Tsf regulated by HPK1 or RR1 i n the two-component signal transduction system of L.bulgaricus CH3 were successfully expressed in L.lactis NZ9000.The tolerance of the recombinant strains to acid stress and other environmental stresses were also studied compared with the control strain NZCK.Expression of Aprt and Ddl proteins had significant effects on acid tolerance of the strain,which increased 75 times and 114 times,respectively.The expression of Opp DII and Tsf had no significant effect on the acid resistance of the recombinant strains.Moreover,the bile salt tolerance of the recombinant strain carrying Aprt increased by 5 times,while that of the recombinant strain carrying Ddl increased by 12 times,and the tolerance to cold stress increased by 8 times.Thus,Aprt and Ddl are one of the reasons for the increased tolerance of strains in acidic environment.At the same time,acid-tolerance regulation in strains is a complex regulation process involving multiple metabolic pathways,and various metabolic processes play a synergistic role to achieve self-protection of strains.The interactions between two-component signal transduction proteins and acid-tolerance regulatory proteins were studied.In this project,yeast two-hybrid system was used to screen the interactions between two-component signal transduction system HPK1 or RR1 and Aprt or Ddl related to acid tolerance of CH3 strain,which were screened by two-dimensional electrophoresis,and the key interaction sites between the interacting proteins were verified.The results of this study preliminarily clarify the interaction between HPK1 and Ddl,and initially identify that HPK1-C was the key interaction site with Ddl.This study laid a theoretical foundation for understanding the acid tolerance regulatory network and mechanism regulated by the two-component signal transduction system HPK1/RR1 in L.bulgaricus CH3,and provided some new ideas and directions for further study of the functions of HPK1 and Ddl. |
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