Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Mon, 07 Jan 2019 19:52:57 Characterization of two resuscitation promoting factors of Listeria monocytogenes Daniela Pinto, 1 Carlos Sa ˜ o-Jose ´, 2 Ma ´ rio A. Santos 1 and Le ´ lia Chambel 1 Correspondence Le ´ lia Chambel lmchambel@fc.ul.pt Received 19 March 2013 Accepted 9 May 2013 1 Center for Biodiversity, Functional and Integrative Genomics (BioFIG), Faculty of Sciences, University of Lisbon, Lisbon, Portugal 2 Center of Molecular Pathogenesis, Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal 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. INTRODUCTION Since the 1980s, it has been known that bacteria, when exposed to stressful environmental conditions, can become non-culturable and yet remain viable (Xu et al., 1982) retaining the possibility of regaining culturability (resus- citation) later (Roszak et al., 1984). This viable but non- culturable (VBNC) state has been described in more than 70 bacterial species (Oliver, 2005, 2010), including the foodborne pathogen Listeria monocytogenes (Besnard et al., 2000a, b, 2002), but little is known about resuscitation. Resuscitation promoting factor (Rpf) proteins were first described by Mukamolova et al. (1998a) in Micrococcus luteus as having the ability to increase the viable count of dormant (not metabolically active) cultures of this micro- organism and stimulate growth of vegetative cells through lag phase reduction (Mukamolova et al., 1998a, b). These Rpf proteins can be isolated from supernatants of actively growing bacteria and are active at picomolar concentra- tions (Mukamolova et al., 1998a). While M. luteus possesses one essential Rpf, Mycobacterium tuberculosis has five non-essential ones (Mukamolova et al., 2002b) that are only partially redundant as shown through growth, survival and resuscitation in vitro as well as in mouse infection models (Biketov et al., 2007; Downing et al., 2005; Kana et al., 2008; Tufariello et al., 2004). RpfB of Myc. tuberculosis is a small protein with a lysozyme-like globular catalytic domain and an elongated G5 domain, which gives the protein a comma-like shape (Ruggiero et al., 2009). Muralytic activity of Rpf proteins has been demonstrated in vitro against crude cell wall preparations and using an artificial lysozyme substrate; this activity has been proved responsible for resuscitation of dormant cells and growth promotion (Mukamolova et al., 1998a; Telkov et al., 2006). Currently three models are proposed for the action of Rpf proteins. (i) According to the original hypothesis, Rpf can act as a bacterial cytokine (Mukamolova et al., 1998a) that through binding to an extracellular receptor stimulates growth, although to date, such receptor has not been found. (ii) Since Rpfs are muralytic enzymes (Mukamolova et al., 2002a) they could, through degradation of the non- culturable state peptidoglycan (Signoretto et al., 2000), promote the physical changes in the cell wall necessary for cell division. (iii) Rpf degradation of peptidoglycan releases Abbreviations: (NAG) 3 -MUF, 4-methylumbelliferyl-b-D-N,N9,N9- triacetylchitotrioside; Rpf, resuscitation promoting factors; Sps, stationary phase survival. One supplementary table is available with the online version of this paper. Microbiology (2013), 159, 1390–1401 DOI 10.1099/mic.0.067850-0 1390 067850 G 2013 SGM Printed in Great Britain