Abstract
Quorum‐sensing mechanisms regulate gene expression in response to changing cell‐population density detected through pheromones. In Enterococcus faecalis, Fsr quorum sensing produces and responds to the gelatinase biosynthesis‐activating pheromone (GBAP). Here we establish that the enterococcal FsrB membrane protein has a direct role connected with GBAP by showing that GBAP binds to purified FsrB. Far‐UV CD measurements demonstrated a predominantly α‐helical protein exhibiting a small level of conformational flexibility. Fivefold (400 μm) GBAP stabilised FsrB (80 μm) secondary structure. FsrB thermal denaturation in the presence and absence of GBAP revealed melting temperatures of 70.1 and 60.8 °C, respectively, demonstrating GBAP interactions and increased thermal stability conferred by GBAP. Addition of GBAP also resulted in tertiary structural changes, confirming GBAP binding.
Highlights
The Gelatinase Biosynthesis‐Activating Pheromone binds and stabilizes the FsrB membrane protein in Enterococcus faecalis quorum sensing
Plasmid was transformed into the expression host E. coli BL21 [DE3] which was cultured in selective Luria–Bertani (LB) medium as described in Materials and Methods and induced for FsrB production using 0.2 mM isopropyl thiogalactoside (IPTG) followed by 3-h incubation at 33 °C prior to harvesting
IPTG induction resulted in an almost immediate cessation in growth compared with cultures lacking IPTG addition (Fig. 2), indicative of successful FsrB expression due to FsrB insertion into E. coli membranes resulting in deleterious effects on E. coli growth, as commonly observed during membrane protein expression [30,31]
Summary
The Gelatinase Biosynthesis‐Activating Pheromone binds and stabilizes the FsrB membrane protein in Enterococcus faecalis quorum sensing. It is advisable to refer to the publisher’s version if you intend to cite from the work. All outputs in CLoK are protected by Intellectual Property Rights law, including Copyright law. IPR and Moral Rights for the works on this site are retained by the individual authors and/or other copyright owners. Terms and conditions for use of this material are defined in the policies page. The gelatinase biosynthesis-activating pheromone binds and stabilises the FsrB membrane protein in Enterococcus faecalis quorum sensing.
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