Characterization of two resuscitation promoting factors of Listeria monocytogenes

In actinobacteria, resuscitation promoting factor (Rpf) proteins have been described as having the ability to increase the viable count of dormant cultures and stimulate growth of vegetative cells through lag phase reduction. Recently, it was suggested that proteins Lmo0186 and Lmo2522 of Listeria monocytogenes are equivalent to Rpf proteins based on their genomic context and conserved domain architecture. It was proposed that they have evolved through non-orthologous displacement of the Rpf domain found in actinobacteria. Here we present biological and biochemical data supporting a function of Lmo0186 and Lmo2522 as Rpfs. These proteins are collectively dispensable for growth but a lmo0186 lmo2522 double mutant exhibits an extended lag phase when diluted in minimal medium. This phenotype could be partially complemented by medium supplementation with fM to nM concentrations of purified hexahistidine-tagged versions of Lmo0186 and Lmo2522, showing that these proteins can stimulate growth. Gel filtration analysis and cross-linking experiments suggest that the recombinant proteins in solution are elongated monomers. Both proteins display muralytic activity against crude cell wall preparations and are active against an artificial lysozyme substrate. Our study thus supports the hypothesis that Lmo0186 and Lmo2522 are functional equivalents of actinobacteria Rpf proteins and represents the first characterization of two Rpf homologues from firmicutes.