Screening,Heterologous Expression And Activity Of Actinomycetes Producing Resuscitation Promoting Factors From Mangroves

The environment of mangrove sediment is unique and complex,which is rich in actinomycetes resources.at present,the isolated strains only account for 0.01%-10%of mangrove actinomycetes resources.most of them periodically enter a living but non-culturable(viable but non-culturable,VBNC)state in order to adapt to changes in the environment.Resuscitation promoting factor(Resuscitation-promoting factor,Rpf)has the ability to resuscitate VBNC strains and can revive and promote the growth of Gram-positive strains at the concentration of picomole.Actinomycetes are one of the main sources of rpfs gene.Therefore,the study of rpfs-producing strains from mangroves is beneficial to enrich the rpfs gene pool and provide important guidance for the further application and development of Rpfs protein.In this study,using mangrove sediments as samples,a strain containing rpf B gene and certain growth-promoting ability was obtained by using the method of concentrated fermentation supernatant to promote the growth of bacteria.The expression and enzymatic properties of rpf B gene of Rhodococcus sp.(GX12401)in prokaryotic expression system were studied.The main results are as follows:(1)Through the isolation of strains from the mangrove sediments of Maowei Sea in Beibu Bay,Guangxi,a total of 63 strains of actinomycetes were obtained,belonging to 10 orders,15 families and 22 genera,of which Mycobacteriales,Micrococcales and Streptomycetales accounted for21.88%,20.64%and 15.3%of all actinomycetes,respectively.Through single genome analysis,it was found that 13 of the 63 actinomycetes contained rpf gene,accounting for 20.63%of the actinomycetes.16S r RNA and protein phylogenetic tree analysis of 13 strains with rpfs gene showed that the taxonomic status of the two strains was similar,which may indicate that the rpfs gene evolved with the evolution of the species,not through horizontal gene transfer.Through the study on the growth promotion of new microbial species by concentrated fermentation supernatant,it was found that five strains had growth promoting activity,of which Rhodococcus sp.(GX12401)and Glutamicibacter arilaitensis(GX3430)could promote the growth of multi-genus strains,and Rhodococcus,Glutamicibacter and Brevibacteriales were bacteria with both growth-promoting function and rpfs gene.(2)based on the whole genome analysis of Rhodococcus sp.(GX12401),the primers of rpf B gene were designed,and the p ET-30a(+)-rpf B recombinant was constructed by enzyme digestion and ligation with p ET30a(+)as cloning vector.The rpf B gene was cloned with E.coli TOP10 as host cell.Bioinformatics analysis showed that the RpfB coding frame carried by Rhodococcus sp.(GX12401)consists of 1128 bases and the encoded protein consists of 375 amino acids and one stop codon.(3)The recombinant p ET-30a(+)-rpf B was introduced into the host cell E.coil BL21.The RpfB protein was heterogeneously expressed and the soluble protein was obtained.The theoretical value of RpfB protein size is46.6 k Da.After 12%SDS-PAGE detection,the protein size is about 55 k Da,which is larger than the predicted 8.4 k Da..The optimal expression conditions were determined and the crude enzyme solution of RpfB was collected.The heterologous expressed RpfB was isolated and purified by nickel column and gradient elution.Through MALDI-TOF MS detection and analysis,the purified protein band was the target protein.The enzymatic characteristics of RpfB showed that RpfB had low lysozyme activity of 4.74 U.The optimum enzyme reaction temperature of RpfB is45°C;when it reacts with the substrate for 7 h at 50°C,the relative enzyme activity is still more than 50%,indicating that the enzyme has good thermal stability to some extent.The enzyme maintained more than 50%relative enzyme activity under the condition of 25°C?45°C(incubation time 7 h).Metal ions Mg²⁺,Na⁺,Al³⁺and organic matter DMSO can promote the enzyme activity of RpfB.On the contrary,Ni²⁺,Co²⁺,K⁺,Tris,glycerol,acetic acid and Tween80 could inhibit the activity of RpfB.(4)Rhodococcus sp.(GX12401)was successfully induced into VBNC state by ciprofloxacin.Rhodococcus sp.(GX12401)in VBNC state was resuscitated with recombinant protein RpfB.The highest dosage of recombinant protein RpfB was 1000 pmol/L and the optimum dosage was 1pmol/L.In addition,the addition of recombinant protein RpfB to mangrove sediment samples could promote the rapid growth of Gram-positive(G⁺)strains at pmol/L level.In this study,a stable potential strain Rhodococcus sp.(GX12401)was screened from mangrove sediments,and its resuscitation promoting factor(Rpf)was heterogeneously expressed.The experiment of enzymatic properties showed that the recombinant protein RpfB had good stability,enzyme activity and low lysozyme activity of 4.74 U,in which Mg²⁺,Na⁺,Al³⁺and DMSO could significantly increase the activity of RpfB,recover the VBNC state of Rhodococcus sp.(GX12401)and promote the rapid growth of Gram-positive strains.The above analysis results lay a solid foundation for the practical application of Rhodococcus sp.(GX12401)and its source resuscitation promoting factors.