Studies On Rpf Gene Mutation And Biological Characterization Of Rhodococcus Erythropolis And Its Application

There exist abundant microbial resources in the natural environments,but only0.01%-1%of the total microorganisms has been isolated by the laboratory methods.Resuscitation promoting factor?Rpf?is found to be a cytokine to promote the recovery and growth of the dormant bacterial cells.Petroleum pollution has long jeopardized the natural environments and human health.Microbial remediation has become one of the main techniques for oil-contaminated soil remediation.However,many of the petroleum-degrading microorganisms in the natural environments are difficult to be isolated and culturated.In this paper,the biological properties of a resuscitation promoting factor from Rhodococcus erythropolis KB1 and its promoting effect on the recovery of dormant cells were studied by mutation technique.The promoting mechanism of the Rpf was explored.The purified recombinant protein was also used for isolation of high-efficiency oil degrading bacteria in natural environments.The recombinant plasmid BL21-pET-32a-rpf containing the rpf gene of Rhodococcus erythropinus KB1 was expressed in Escherichia coli,and the recombinant Rpf protein was purified by Ni-Sepharose affinity chromatography.The molecular weight of the expressed protein was 37 kDa on SDS-PAGE gel.The muralytic activity of the Rpf recombinant protein was 1760 U/mg and the protease activity was 1634 U/mg.0.1 mM of Zn²⁺could increase the muralytic activity,but Ca²⁺,Mg²⁺,Co²⁺,DTT,EDTA,PMSF had no obvious effect on the enzyme activity.The purified recombinant Rpf protein can promote the growth of Rhodococcus erythropois KB1 and recovery of the VBNC KB1 cells.The resuscitation effect was related with the concentrations of the recombinant protein.Site-directed mutagenesis was used to study the muralytic activity related amino acids such as Asp⁴⁵,Cys⁵⁰,Glu⁵¹,Thr⁶⁰,Gln⁶⁹,Thr⁷⁴,Trp⁷55 and Cys¹¹⁴.Several recombinant plasmids with different amino acid replcemrnts were constructed and expressed in Escherichia coli.The mutant proteins were also expressed and purified with Ni²⁺-affinity chromatography.The muralytic activities of the mutant proteins decreased at different degree when compared with that of the wild type Rpf.The muralytic activity of the C50G+C114T mutant lost 86.68%,the enzyme activity of the D45A+E51K mutant lost 84.43%,the activity with the Q69K mutant lost 86.11%,and the E51A mutant lost 11.14%of the enzyme actvity,but the muralytic activity of the W75V mutant increased 94.45%.The growth-promoting and resuscitation effects of the Rpf were closely related to its muralytic activity.The growth-promoting and resuscitation activities of the C50G+C114T mutant were lost completely.Addition of the recombinant Rpf mutant with W75V increases the growth and recovery of the VBNC Rhodococcus erythropolis cells more effectively when compared with that of the equal amount of the wild type Rpf.The recombinant Rpf protein of Rhodococcus erythropolis was added to the oil-contaminated soil samples to study the effect on isolation of oil degrading bacteria in the soil.It was found that addition of the Rpf could promote the isolation of oil-degrading bacteria from the soil samples.Nine additional oil-degrading bacteria strains were isolated from the oil-contaminated soil samples.The 16S rDNA sequence analysis confirmed that the isolated bacteria strains belonged to Bacillus,Ochrobactrum,Burkholderia,Leifsonia,pseudomonocytes,Pseudomonasand Microbacterium.The oil degradation rates of the strains were determined.It was found that the degradation rates of Bacillus sp.JC1,Bacillus sp.JD5,Bacillus sp.FD1,Bacillus sp.JP3 and Bacillus sp.FP3 were 43.94%,41.58%,28.96%,44.38%and 29.87%respectively.The degradation rates of Ochrobactrum sp.JP1 and Ochrobactrum sp.FP1 were 36.12%and 34.27%respectively.The degradation rates of Microbacterium sp.JD2 and Microbacterium sp.FD2 were 26.01%and 20.46%respectively.