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.

FIG. 1.

Deduced amino acid sequences and alignments of FsrB and FsrD with AgrBs, AgrDs, and their homologs. Ef, Enterococcus faecalis (GenBank accession no. AAF14219); Sa, Staphylococcus aureus (accession no. CAA36781 and CAA36782); Se, Staphylococcus epidermidis (accession no. AAC38295 and ACC38294); Li, Listeria innocua (accession no. CAC95274 and CAC95275); Lm, Listeria monocytogenes (accession no. CAC98263 and CAC98264); Bc, Bacillus cereus (accession no. ZP_00237848 and ZP_00237849); Ca, Clostridium acetobutylicum (accession no. AAK78063 and AAK78064); Lp, Lactobacillus plantarum (accession no. NP_786783 and NP_786782). Dashes indicate gaps in the alignment. Conserved residues are indicated by black shading. Dotted and solid underlines indicate the predicted transmembrane segments showing scores higher than 1.7 and 2.2, respectively, using the dense alignment surface method (http://www.sbc.su.se/∼miklos/DAS/) (4). Inverted triangles indicate the positions where site-directed mutagenesis was performed. Sequences of AIPs (for E. faecalis, S. aureus, S. epidermidis, and L. plantarum) and putative AIPs (for the corresponding positions for L. innocua, L. monocytogenes, and C. acetobutylicum) are enclosed in boxes. The fsrB ⁵⁶⁸ATG codon for Met-190 (also for Met-1 in fsrD) is directly connected in frame to the fsrB codon for Phe-189.

FIG. 2.

(A) Genetic map of the fsr gene cluster and the downstream regions in gelatinase-positive wild-type E. faecalis and in two gelatinase-negative isolates, OU510 and OU598. White and black flags represent constitutive and GBAP-inducible promoters, respectively. Black arrows indicate fsrD. (B) Genetic map of the plasmids used in this study. Double flags indicate the nisin-inducible promoter.

FIG. 3.

Site-directed mutagenesis of pQU2100 in E. faecalis OU598. The nucleotide sequences around the mutagenesis site are indicated by vertical arrows, and the GBAP activities of each transformant are shown. Black shading indicates the putative start codon of fsrD. Open boxes indicate the putative ribosomal binding site of fsrD. The GBAP activity of each transformant was measured as described previously (12), except that E. faecalis OU510 was used as a responder strain instead of OG1SP and gelatinase was induced for 5 h instead of 3 h. The GBAP activity is represented by the induced gelatinase activity (change in A₅₄₀) determined by two independent experiments.

FIG. 4.

LC-MS analysis of culture supernatants of OU598(pQU2100) and OU598(pQU2101). After the strains were cultured in a chemically defined medium (200 ml) (21) for 2 h, nisin was added to a final concentration of 25 ng/ml, and the strains were then cultured for another 5 h. The culture supernatants were partially purified by using a Spe-pak octyldecyl silane cartridge column (720 mg; Waters Co., Milford, Mass.) according to a procedure previously described (12, 21) and were then injected into a LC-MS device (Accutof T100LC; JEOL, Tokyo, Japan; LC column, Agilent Zorbax Eclipse XDB-C8 [2.1 by 150 mm]). The column was eluted with a linear gradient of acetonitrile (24% to 50% in 26 min) in an 0.05% trifluoroacetic acid aqueous solution, and the eluates at a protonated molecular mass of GBAP (m/z,1303.7) were monitored. The mass spectra of the peaks indicated by an arrow are shown in insets. As a standard, the synthetic GBAP was monitored by using the same LC-MS system.

FIG. 5.

Gelatinase activity of nisin-induced E. faecalis OU510 carrying each plasmid. (A) Gelatinase activity in liquid culture. Each strain was cultured in medium with (black bars) or without (white bars) nisin for 5 h, and the culture supernatant was subjected to an Azocoll assay, as described previously (11). The experiment was performed in duplicate, and average values were plotted. (B) Gelatinase activity on solid agar medium. Overnight liquid cultures (0.5 μl) of each strain were spotted and grown on agar medium containing 3% gelatin with (+) or without (−) nisin overnight, cooled at 4°C for 1 h, and photographed.