Resuscitation Promoting Factor (Rpf) from Tomitella biformata AHU 1821^T Promotes Growth and Resuscitates Non-Dividing Cells

Abstract

Functional variation of Rpf, a growth factor found exclusively in Actinobacteria, is differentiated by its source and amino acid sequences. Only purified Rpf proteins from three species have been studied so far. To seek new Rpfs for use in future studies to understand their role in Actinobacteria, the objective of this study was to identify rpf gene homologs in Tomitella biformata AHU 1821^T, a novel Actinobacteria isolated from permafrost ice wedge. Amplification using degenerate primers targeting the essential Rpf domain led to the discovery of a new rpf gene in T. biformata. Gene structure and the deduced Rpf domain amino acid sequence indicated that this rpf gene was not identical to previously studied Rpf. Phylogenetic analysis placed T. biformata Rpf in a monophyletic branch in the RpfB subfamily. The deduced amino acid sequence was 44.9% identical to RpfB in Mycobacterium tuberculosis, the closest functionally tested Rpf. The gene was cloned and expressed in Escherichia coli; the recombinant Rpf protein (rRpf) promoted the growth of dividing cells and resuscitated non-dividing cells of T. biformata. Compared to other studies, this Rpf was required at higher concentrations to promote its growth and to resuscitate itself from a non-dividing state. The resuscitation function was likely due to the highly conserved Rpf domain. This study provides evidence that a genetically unique but functional Rpf can be found in novel members of Actinobacteria and can lead to a better understanding of bacterial cytokines in this phylum.