Revised Model for Enterococcus faecalis fsr Quorum-Sensing System: the Small Open Reading Frame fsrD Encodes the Gelatinase Biosynthesis-Activating Pheromone Propeptide Corresponding to Staphylococcal AgrD

Abstract

Gelatinase biosynthesis-activating pheromone (GBAP) is an autoinducing peptide involved in Enterococcus faecalis fsr quorum sensing, and its 11-amino-acid sequence has been identified in the C-terminal region of the 242-residue deduced fsrB product (J. Nakayama et al., Mol. Microbiol. 41:145-154, 2001). In this study, however, we demonstrated the existence of fsrD, encoding the GBAP propeptide, which is in frame with fsrB but is translated independently of fsrB. It was also demonstrated that FsrB, an FsrD segment-truncated FsrB, functions as a cysteine protease-like processing enzyme to generate GBAP from FsrD. This revised model is consistent with the staphylococcal agr system.The staphylococcal agr system is the best-understood cyclic peptide-mediated quorum-sensing system among gram-positive bacteria. In the agr system, an autoinducing peptide (AIP) is generated from its propeptide, AgrD, by its processing enzyme, AgrB, and is then sensed by a two-component regulatory system comprising a membrane histidine kinase, AgrC, and a response regulator, AgrA (10,14,18,22,23). The cognate gene cluster consisting of these four components has been identified in the genome databases of Listeria, Clostridium, Lactobacillus, and Bacillus spp., suggesting that cyclic peptide-mediated quorum sensing is widespread among gram-positive bacteria (14,18,21).The fsr system in Enterococcus faecalis controls the expression of pathogenicity-related extracellular proteases, gelatinase, and a serine protease via a quorum-sensing mechanism (11,16,17), and recent studies have suggested that it also regulates biofilm formation (7, 15) and other genes important for virulence (2). The fsr quorum-sensing system also mediates a cyclic peptide named gelatinase biosynthesis-activating pheromone (GBAP), although the ring is formed by lactone instead of the thiolactone found in other gram-positive AIPs (10, 11, 21). However, a small open reading frame corresponding to staphylococcal agrD has not been identified in the nucleotide sequence of the fsr gene cluster (16). The 11-amino-acid sequence of GBAP was identified in the C-terminal part of the 242-residue deduced fsrB product (11). As shown in Fig. 1, the N-terminal part of FsrB (189 residues) shows sequence similarity to staphylococcal AgrB, and the remaining C-terminal part of FsrB (53 residues) appears to be a GBAP propeptide like AgrD. Based on these observations, we have suspected that FsrB autoprocesses its C-terminal part to generate GBAP, which is a unique biosynthetic mechanism compared with those of other cyclic AIPs.In the present study, we demonstrate the existence of a small open reading frame, fsrD, corresponding to agrD, which is carried in frame with fsrB but is translated independently of fsrB. Here we propose a revised fsr system model sharing a common mechanism of AIP biosynthesis with the thiolactonemediated quorum-sensing systems of staphylococci and probably other gram-positive bacteria.Construction of a nisin-inducible expression system for wild-type and mutant fsrBD stra...

Structure-Activity Relationship of Gelatinase Biosynthesis-Activating Pheromone ofEnterococcus faecalis

“…E. faecalis OU510 has an amber mutation in fsrB, resulting in a lack of GBAP biosynthesis. This strain was used as the responder strain for the GBAP agonist activity assay because gelatinase production in this strain completely depends on exogenously added GBAP (28). All E. faecalis strains were cultured in 36.4 g/liter Todd-Hewitt broth (THB) (Oxoid, Basingstoke, Hampshire, United Kingdom) at 37°C with gentle agitation.…”

Whole-genome mapping of 5′ RNA ends in bacteria by tagged sequencing: a comprehensive view in Enterococcus faecalis

“…Several TSSs mapped previously by other methods were retrieved by tagRNA-seq at near-identical locations (±2 bp) attesting to the reliability of the method. For example, we find the TSSs of sodA (ef0463), coding for the superoxide dismutase, ptb (ef1663), coding for the phosphotransbutyrylase, fsrB/D (ef1821), coding for the cysteine protease-like processing enzyme FsrB and the autoinducing propeptide FsrD of the fsr system, a homolog of the accessory gene regulator (agr) of Staphylococcus aureus, and gelE (ef1818), coding for a gelatinase (Qin et al 2000(Qin et al , 2001Ward et al 2000;Nakayama et al 2006;Verneuil et al 2006) (see below; Supplemental Table SB). …”

Making sense of quorum sensing in lactobacilli: a special focus on Lactobacillus plantarum WCFS1

“…In addition, some QS-TCS genes are genetically linked to genes encoding AIP transport and/or modification proteins, bacteriocins and bacteriocin-immunity proteins . For double-glycinetype AIPs the cognate transporters are ABC transporters that contain a characteristic N-terminal peptidase C39 domain (COG2274) (Håvarstein et al, 1995), while in the case of agr-like systems AgrB-type cysteine proteases are involved in transport and modification of AIPs (Nakayama et al, 2006;Qiu et al, 2005;Zhang & Ji, 2004).…”