Preliminary Study On The Role Of Two-component System In The Protocooperation Relationship Of Fermented Yoghurt Strains

Lactobacillus delbrueckii subsp.bulgaricus and Streptococcus thermophilus are necessary for starter cultures for yogurt manufacturing,and widely used in yogurt production in the world.The two species are synergistic,and they stimulate each other's growth and acid production during the milk formation.The mechanism of protocooperation between L.delbrueckii subsp.bulgaricus and S.thermophilus is complex and not very clear at present.The two component systems(TCSs)are one of the most important mechanisms for external environmental sensing and signal transduction for the majority of Gram-positive and Gram-negative bacteria.In our previous study,L.delbrueckii subsp.bulgaricus CH3 and S.thermophilus CS5 were isolated from yogurt in traditional pasturing area,and showed potential used as yogurt starter culture.They were used in this study.The aim of this study is to identify the function of TCSs in the protocooperation between two species;clarify the signal regulation mechanism of protocooperation;lay a theoretical basis for consummating the protocooperation mechanism,and improving the protocooperation effect of two species;and accelerate the development of high-efficiency yogurt starter cultures.The primers were designed for the detection of TCS distribution in CH3 and CS5,according to the nucleotides information of known TCS gene in S.thermophilus LMG18311 and L.delbrueckii subsp.bulgaricus BAA-365.The results showed that L.delbrueckii subsp.bulgaricus CH3 contained 7 TCSs named HPK1/RR1-HPK7/RR7;S.thermophilus CS5 contained 8 TCSs named HPK1/RR1-HPK2/RR2,HPK4/RR4-HPK9/RR9.The growth ability and acid productin ability of L.delbrueckii subsp.bulgaricus CH3 and S.thermophilus CS5 strains in monoculture and mixed culture were analyzed.The key fermented periods of strains were determined.The TCSs of L.delbrueckii subsp.bulgaricus CH3 and S.thermophilus CS5 strains in monoculture and mixed culture were analyzed by means of real-time PCR technology.The differentially expressed TCSs were identified.The TCSs of L.delbrueckii subsp.bulgaricus CH3 and S.thermophilus CS5 strains in monoculture and mixed culture were analyzed by means of real-time PCR technology.The differentially expressed TCSs were identified.Thesignificant differences in TCSs included hpk2/rr2,hpk4/rr4,hpk7/rr7 in S.thermophilus CS5,hpk1/rr1,hpk2/rr2,hpk3/rr3,hpk7/rr7 in L.delbrueckii subsp.bulgaricus CH3.Gene inactivation and functional identification were used to determine the role of key TCSs in the protocooperation relationship between the two bacteria.First of all,the role of key TCS gene hpk1/rr1 of CH3 in the protocooperation relationship was studied.In our previous study,the mutants CH3-H and CH3-R were constructed by means of inactivation of gene hpk1/rr1.The mutants CH3-H,CH3-R and wild type CH3 respectively with the CS5 strains mixed fermentation yoghurt.The growth ability,the production of acetaldehyde and viscosity capacity were analyzed.The results showed that the three indexes of mutant CH3-H and CH3-R were lower than those of wild-type strain CH3,which indicated that hpk1/rr1 had significantly decreased on the growth ability,acetaldehyde production ability and viscosity ability of strain CH3.Primers were designed according to the key TCS gene of S.thermophilus CS5,hpk2-u,rr5-u were amplificated by PCR,The fragments were ligated into the lactic acid bacteria suicide vector pRV300,and two suicide recombinant plasmids were successfully obtained.And it has established for preparing S.thermophilus CS5 competent method,clarifying the erythromycin sensitivity,and determining the screening concentration for subsequent experiments to screen for the key gene inactivation of the CS5 mutant strain to lay the foundation.