Studies On Gene Cloning,expression And Biological Characterization Of The Resuscitation-promoting Factor Of Rhodococcus Erythropolis

Viable but non-culturable state is a dormancy state that bacteria formed under unadapted environments.With the improvement of the surrounding environment,the non culturable cells can be recovered to the culturable state,but the mechanism of bacteria recovery is not clear.Resuscitation promoting factor(Rpf)of Micrococcus luteus was first reported to recover the dormancy bacterial cells to the culturable state,and stimulate the growth of normal cells.Rpfs were found to exist widely in gram-positive bacteria with high G+C contents,which were related to the resistance to adverse environments.The numbers and types of Rpf vary in different bacteria.Rhodococcus erythropolis KB1 was isolated from the oil contaminated soil,and showed high efficient oil degradation and environmental adaptabilities.In order to explore the role of Rpfs in the environmental adaptation process of KB1,This article has carried on the rpf gene cloning of KB1,analysis of genetic structure,expression and biological properties.The main results are as follows:The primers of rpf genes were designed,and four rpf genes were amplified from the genome DNA of R.erythropolis KB1.The 4 genes were consisted of 564?1125?1128 and 555 bp and coded for 187?353?375 and 184 amino acid residue peptides respectively.It was found that the sequences of the four genes showed high similarities with those of the rpf gene of Micrococcus luteus rpf B,rpf C genes of Mycobacterium tuberculosi and Streptomyces coelicolor rpf B genes respectively.The proteins encoded by those four rpf genes had promote recovery factor Rpf domains,which were similar with that of the cells promote recovery factor Rpf domain of Micrococcus luteus,and consists of 70 amino acids.Rpf-1 had only one Rpf domain,which was similar to RpfC of Mycobacterium tuberculosis and Streptomyces coelicolor;Rpf-2 had one Rpf domain,one G5 domain and two DUF348,which was similar with Rpf B of Mycobacterium tuberculosis.Rpf-3 had one Rpf domain,one G5 domain and three DUF348,which was similar with Rpf B of Streptomyces coelicolor.Rpf-4 had one Rpf domain and one Lys M domain,which was similar with Rpf of Micrococcus luteus.Rpf-2 and Rpf-3 contain one signal peptide respectively,while Rpf-1and Rpf-4 did not contain any signal peptide.The four proteins were hydrophilic,containing multiple serine and threonine and tyrosine loci and no histidine loci.The specific primers of rpf-1 gene was designed and the rpf-1 gene was subcloned to p ET-32a(+)and expressed in BL21(DE3).The expressed protein was purified by Ni2+-affinity chromatography.SDS-PAGE analysis showed that the molecular weight of the purified protein was 36 k Da.The purified Rpf-1 was found to have lysozyme activity by using synthetic substrate4-Methylumbelliferyl-?-D-N-N'-N'-triacetylchitotriose,with the a specific activity of 2.07 U/mg.The proteinase activity of Rpf-1 was also detected by using azocasein as substrate,and the purified Rpf-1 can hydrolysis azocasein,with a specific activity of 235 U/mg.The recombinant protein Rpf-1 showed distinct recovery and promoting effects on the Rhodococcus erythropolis cells in different states(normal state,VBNC state and preservated at low temperature).Addition of the purified Rpf-1(10%)promoted the growth of Rhodococcus erythropolis cells preservated at low temperature to 4.8-6 times,when compared with that of the control group.The Rhodococcus erythropolis cells were getting into logarithmic phase in 12 h,and the cell growth increased 3 times more than that of the control group in 48 h with addition of the Rpf-1.The Rpf-1 also showed obvious recovery effect on the VBNC state,addion 10% of the pufrified Rpf-1 promoted growth of the VBNCcells by 1.44 times in 48 h when compared with that of the control group.