Resuscitation Promoting Factor (Rpf) from <i>Tomitella biformata</i> 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 1821T, 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.

Current Perspectives on Viable but Non-Culturable (VBNC) Pathogenic Bacteria

“…Resuscitation-promoting factor or its homologs have also been reported in other bacterial species including Mycobacterium spp., Corynebacterium spp., Streptomyces spp., L. monocytogenes, Tomitella biformata , and S. enterica serovar Typhimurium (1, 92–94). Rpf-mediated resuscitation of M. tuberculosis has been demonstrated in several studies (95, 96).…”

Survivability of Microbes in Natural Environments and Their Ecological Impacts

“…For example, pyruvate worked in the resuscitation process of Salmonella ( 26 ). In the phylum Actinobacteria , the resuscitation promoting factor (Rpf) protein demonstrated both a resuscitating ability and a growth promoting activity ( 27 , 32 , 33 ). However, it is challenging to clarify environmental cues related to microbial activity, and how microbes in non-dividing but metabolically active states sense these cues to decide whether or not initiate active growth.…”

Promoting resuscitation of viable but nonculturable cells ofVibrio harveyiby a resuscitation-promoting factor-like protein YeaZ

“…RPF-like proteins have been found in a number of Gram-positive bacteria including M. luteus, Myco. tuberculosis, Listeria monocytogenes, Corynebacterium and Streptomyces (Panutdaporn et al 2006;Gupta and Srivastava 2012;Dewi Puspita et al 2013;Pinto et al 2013). The structure of RPF proteins is similar to C-type lysozyme and soluble lytic transglycosylases (Cohen-Gonsaud et al 2005), which digest the peptidoglycan in the cell wall by breaking the b (1, 4) linkage between N-acetyl glucosamine and N-acetyl muramic acid (Mukamolova et al 2006).…”