Expression And Bioactivity Of Rpf2 Gene In Rhodococcus Erythropolis And Its Promotion Effect On Isolation Of Petroleum Degrading Bacteria

There are abundant microbial resources in the natural environments,and the number of microorganisms that can be obtained by artificial culture is currently limited.Most of the microorganisms are difficult-to-culture or in the viable but nonculturable state(VBNC).Resuscitation promoting factor(Rpf)is a cytokine secreted by some bacteria,which can promote the resuscitation of dormant bacteria and promote the growth of normal cells.Rhodococcus erythropolis KB1 is an efficient petroleum-degrading bacterium isolated from the desert soil,which showed strong environmental adaptabilities and high petroleum-degrading ability at low temperature.Multiple rpf genes had been found in this bacterium.In this paper,a prokaryotic expression vector of Rpf2 gene of Rhodococcus erythropolis KB1,was constructed and highly expressed in Escherichia coli.The expressed protein was purified by Ni-agarose affinity chromatography,and the enzyme activity and biological activity of the purified recombinant protein were analyzed.The effect of recombinant protein on microbial degradation ability and microbial diversities of the contaminated soil was studied by adding purified recombinant Rpf2 to petroleum-contaminated soil samples,the recombinant Rpf2 protein was used to isolate efficient petroleum-degrading bacteria from the contaminated soil,and the main findings were as follows:The recombinant plasmid p ET28a(+)-rpf2 was constructed by linking the rpf2 gene of Rhodococcus erythropolis KB1 with prokaryotic expression vector and highly expressed in E.coli,and then the recombinant rpf2 protein was purified by Ni-agarose affinity chromatography.The molecular weight of the expressed protein was about 45 KDa by polyacrylamide gel electrophoresis(SDS-PAGE)analysis,the lysozyme activity of the recombinant Rpf2 protein was 1503 U/mg,the protease activity was1528 U/mg when measured by azocasein substrate.The amidase and esterase activities were 1395 U/mg and 1026 U/mg,respectively when measured by BAPAN and BTEE as the substrates.The purified recombinant Rpf2 was added to R.erythropolis KB1 cells,and it was found that the recombinant Rpf2 significantly promoted the growth of R.erythropolis cells,and the growth promoting effect was directly proportional to the concentration of the added recombinant protein.The effects of recombinant proteins Rpf1 and Rpf2 on the degradation abilities of indigenous microorganisms in oil-polluted soil were studied.It was found that the purified recombinant Rpf2 could improve the oil degradation ability of indigenous microorganisms,and the oil degradation rates of the recombinant Rpf2 treatment groups increased from 30.86% of the control group to 43.45% and 45.20%,respectively.The petroleum degradation rates were related with concentrations of the added recombinant protein.The effect of the purified recombinant Rpf2 on the community of the culturable bacteria in oil-contaminated soil was studied by using high-throughput sequencing technology.The results showed that addition of the purified recombinant Rpf2 had a greater effect on the communities of the culturable bacteria in the oil-contaminated soil.The bacterial community with the control group of 9 phyla 82 genera increased to13 phyla 116 genera and 16 phyla 138 genera after the addition of different concentrations of the purified Rpf2.Some genera significantly changed in abundance,Flavobacterium was increased from 0.20% to 11.09% and Ochrophatrum increased from 0.94% to 15.37% in the high concentration group of the Rpf2.Some new petroleum degrading bacterial genera were founded,including Oceanisera,Streptococcus,Pseudohongiella,Vagococcus,Geodermatophilus and Sphingobium.Recombinant Rpf1 and Rpf2 were added to the oil-contaminated soil samples in to study their effects on the isolation of oil-degrading bacteria,and it was found that the addition of the purified recombinant Rpf1 and Rpf2 could promote the isolation effect of the oil-degrading microorganisms from the oil-contaminated soil.A total of25 oil-degrading bacteria were isolated,including six bacteria strains isolated from the Rpf1 treatment group,which belonged to Alcanivorax sp.CQ2,Acinetobacter sp.CQ4,Pseudomonas sp.CQ7,Citromonas sp.CQ15,Brevundimonas sp.CQ16 and Sphingobacterium sp.CQ21 as identified by 16 S r DNA sequencing.Eight new petroleum-degrading bacteria were isolated from Rpf2 treatment groups,including two strains(CQ2 and CQ21),which were belonged to the same species as those isolated in Rpf1.The other six strains were Pseudomonas sp.CQ9,Pseudomonas sp.CQ10,Alcaligenes sp.CQ17,Exiguobacterium sp.CQ19,Martelella sp.CQ22,and Idiomarina sp.CQ25,of which CQ25 may be potential new species.Analysis of the degradation performance of the isolated strains revealed that the degradation rates of the seven strains was more than 30.00%.The degradation rates of Serratia sp CQ18,Acinetobacter sp.CQ4,Providencia sp.CQ13,Pseudomonas sp.CQ8,Alcanivorax sp.CQ1,Idiomarina sp.CQ25 and Aeromonas sp.CQ11 were32.24%,33.88%,31.77% and 32.81%,36.12%,31.84%,30.77%,respectively.