Promoting Resuscitation of the VBNC Bacteria and Enrichment of Naphthalene-degrading Bacteria from Oil-contaminated Soils with the Rpf4 from Rhodococcus erythropolis KB1

Nowadays, more than 90% of bacteria in nature are in a viable but not culturable (VBNC) state, and they cannot be separated by traditional plate culture methods. However, resuscitation-promoting factors (Rpf) participate in recovery of dormant cells and promoting bacterial growth. The Rpf4 of Rhodococcus erythropolis KB1 is a Rpf like protein of Micrococcus luteus and contained the Rpf domain and LysM domain. In this study, an expression vector for the rpf4 gene was constructed and expressed in Escherichia coli. The recombinant protein Rpf4 was purified by Ni2+-affinity chromatography. The recombinant protein showed a muralytic activity of 549 +/- 28.77 U/mg. The purified Rpf4 could increase R. erythropolis KB1 cell proliferation and improve the resuscitation of R. erythropolis cells that were VBNC. The Rpf4 altered the diversity of naphthalene-degrading bacteria enriched from oil-contaminated soils. Based on the high-throughput sequencing, we found that Proteobacteria and Actinobacteria phyla increased from 76.10% and 0.029% to 98.23% and 0.401% after the Rpf4 treatment, while the Bacteroidetes phylum decreased from 22.70% to 0.76%. The genera Enterobacter, Citrobacter, and Acinetobacter increased from 0.0054%, 0.001%, and 0.745% to 19.02%, 6.34% and 2.49%. Some new genera such as Bifidobacterium, Limnobacter, Alloprevotella, Hydrogenophaga, and Nevskia were founded in the treatment group. Meanwhile, 23 naphthalene-degrading bacteria were isolated by adding the Rpf4 protein to the oil-contaminated soil samples. Study on the naphthalene-degrading bacteria will provide a basis for the microbial degradation of naphthalene and other polycyclic aromatic hydrocarbons and discover the wider application value of Rpf.