Resuscitation-promoting factor diversity of <i>Rhodococcus erythropolis</i> KB1 and their promoting effect on bacterial growth and viable but non-culturable cell resuscitation

Resuscitation-promoting factors (Rpfs) are proteins essential for reactivating microorganisms from the viable but non-culturable (VBNC) state and exhibit considerable diversity across bacterial species. Rhodococcus erythropolis KB1 is an efficient petroleum-degrading actinomycete with strong environmental adaptability. Its genome encodes a variety of Rpf proteins. In this study, we characterized these Rpfs through multiple sequence alignments, domain analysis, tertiary structure modeling, and biological activity assays. Each protein harbors a conserved Rpf domain of ∼70 amino acids and auxiliary domains such as DUF348, G5, and LysM. Reverse Transcription Quantitative Polymerase Chain (RT-qPCR) analysis revealed that rpf1 was highly expressed during the logarithmic phase in R. erythropolis KB1, while rpf4 and rpf5 exhibited lower expression levels. All recombinant proteins, expressed and purified from Escherichia coli, demonstrated muralytic activity. Notably, the muralytic activity was independent of auxiliary domain complexity, with Rpf5 showing the highest activity (18.24 ± 1.06 U nmol-1). Furthermore, all Rpfs promoted the growth and resuscitation of VBNC R. erythropolis KB1 cells, with Rpf5 providing the greatest effect. The efficiencies of the recombinant proteins in promoting bacterial growth and VBNC cell resuscitation varied significantly, and these variations were strongly correlated with their muralytic activities.