Isolation And Characterization Of A Bdellovibrio-and Like Organism Strain BDE-1 And Promotion Of Its Bdelloplast Formation

As a relatively new member of predatory marine probiotics,Bdellovibro-and like organisms?BALOs?have a wide application prospect because of its wide spectrum of cracking and strong ability to lyse pathogenic bacteria.However,because of BALOs'special reproductive mode and cycle,the pratical application of BALOs is greatly affected by their parasitic host characteristics and biological activities.Therefore,selecting appropriate parasitic hosts and maintaining the activity of BALOs preparation are very important for BALOs'application.To isolate a strain of BALOs which can use gram-positive probiotics Bacillus subtilis as host,and to increase its bdelloplast concentration in the culture for storage,Bacillus subtilis,which is non-pathogenic to humans and animals,was used for the first time as a host to isolate BALOs from sea mud samples from Hainan by DNB?Dilute nutrient broth?double plate method.A strain of Bdellovibrio was selected,purified and cultured until the plaques grown on the double-layer plate of the DN B were transparent and the ir sizes uniform.Then the target strain was identified by transmission electron microscopy and 16S rRN A gene sequence analysis.It was confirmed that the isolate was Bdellovibrio and named Bdellovibrio BDE-1.In order to obtain the optimum fermentation conditions for Bdellovibrio BDE-1 so as to obtain a high-density Bdellovibrio fermentation broth,the three major factors?temperature,salinity,and p H?of BDE-1 were optimized,and the effect of the addition density of the host strain and the concentration of sodium glutamate on the Bdellovibrio BDE-1 reproductive density were studied through a series of single-factor experiments.It was found that the Bdellovibrio BDE-1 had highest proliferation density under the condition of a temperature of25oC,a salinity of 20‰,a p H of 7.0,a host strain with a density of 1×10¹¹ CFU/mL,and a sodium glutamate concentration of 5 mmol/L.In order to uncover the lysis spectrum and lysis ability of Bdellovibrio BDE-1,the lysis of 28 host strains by BDE-1 and the elimination effect of 2 strains of marine vibrio were investigated,and the effect of the survival of the host strain on the growth and development of BDE-1 was studied.The results showed that BDE-1 could lyse 24 strains of bacteria,which accounted for 85.7%of the total number of strains?28 strains?.Among them,the lysis rate of vibrio?13 strains?was 92.3%;Bdellovibrio BDE-1 had a certain elimination effect on two marine vibrio species,and there was a significant difference in the elimination effect of yellow vibrio,green vibrio,and their mixture?p<0.05?.The elimination ability of the marine vibrio species was yellow-green vibrio mixture>yellow vibrio>green vibrio;the live or death status of the host strain had no influence on the growth and reproduction of BDE-1,and it could utilize the dead host to develop and reproduce.In order to study the density and stability of bdelloplast during the growth and development of Bdellovibrio BDE-1,the single-factor experiments were taken to study the effects of five factors,(including indole,ampicillin,actinomycone,Ca²⁺and Mg²⁺)on the formation and stability of BDE-1.It was found that indole,ampicillin,Ca²⁺,and Mg²⁺played an important role in promoting the formation and stability of BDE-1.The optimal concentrations of these four factors were:3 mmol/L,150?g/mL,20 mmol/L,and 2 mmol/L;actinomycone not only had no promoting effect on the formation of BDE-1,but also had an inhibitory effect on the growth and development of BDE-1 High concentration actinomycone could even cause the death of Bdellovibrio BDE-1 cells.The above results not only provide a feasible solution for the optimal selection of parasitic hosts of the Bdellovibrio BDE-1,but also provide a theoretical basis for maintaining or improving the application activity of the microecological preparations of the Bdellovibrio BDE-1,which is of great significance and value.