The incongruent gelatinase genotype and phenotype in Enterococcus faecalis are due to shutting off the ability to respond to the gelatinase biosynthesis-activating pheromone (GBAP) quorum-sensing signal

Abstract

The concomitant presence of a complete fsr quorum-sensing system and gelE-sprE operons in Enterococcus faecalis is known to be essential for the detection of gelatinase activity. However, there are reports of the absence of gelatinase activity despite the presence of complete fsr and gelE loci. In order to understand this incongruence between genotype and phenotype we sequenced fsr and gelE loci of the E. faecalis LN68 strain, which was previously found to carry both operons but to lack gelatinase activity. Of the 59 nucleotide differences detected compared with the gelatinase-positive V583 strain, we found a nonsense mutation (a premature STOP codon) predicted to truncate the ATPase sensor domain of the FsrC protein, responsible for sensing and transducing the signal from the quorum-sensing molecule. Strain LN68 was highly affected in the expression of the gelE and sprE genes, further supporting the lack of Fsr-dependent gelE induction. When we constructed a V583 mutant with the same premature stop mutation in the fsrC gene the resulting strain was no longer able to degrade gelatin. We conclude that the reduced ability to transduce the quorum-sensing signal of the prematurely truncated FsrC protein is sufficient to explain the negative gelatinase phenotype. As the incongruent genotype and phenotype is detected in natural isolates, we believe that the silencing of the quorum-sensing system Fsr may be beneficial for some E. faecalis strains.

Fig. 1.

Schematic representation of the fsr and gelE loci in V583 and LN68, and the mutants derived from point mutations, EF_SAVE3 and EF_SAVE4, respectively. The nonsense codon W403STOP is also indicated.

Fig. 2.

Intramembrane structures of FsrC proteins from V583 and LN68, predicted using TopPred software. Mutations found in the fsrC gene of LN68 are shown: tyrosine (Y) 54 to cysteine (C); leucine (L) 124 to isoleucine (I); tyrosine 252 to asparagine (N); lysine (K) 385 to arginine (R); tryptophan (W) 403 to STOP. ES, extracellular space; IS, intracellular space; ECL, extracellular loop.

Fig. 3.

Images from gelatin agar plates inoculated with the V583, EF_SAVE3, LN68 and EF_SAVE5 strains. A transparent halo, indicative of gelatin degradation, is clearly seen around V583 and EF_SAVE5 growth, demonstrating the presence of gelatinase activity in these two strains.

Fig. 4.

LN68 and EF_SAVE3 gelatinase activity on skimmed milk plates. (a) Demonstration of the inability of LN68 and EF_SAVE3 to induce gelatinase upon addition of exogenous GBAP. Strain VI13 was used as a proof of concept of this assay. This strain is able to sense GBAP, and induce gelatinase activity upon GBAP addition, but it is not able to produce GBAP. (b) Demonstration of the ability of both LN68 and EF_SAVE3 to produce GBAP. Proof came from the observation of a transparent halo around growth of the VI13 strain, which is not able to produce GBAP and depends on exogenous GBAP to produce visible gelatinase activity.

Fig. 5.

Enterococcal strains with an incongruent gelatinase genotype and phenotype. The fsrC gene of these strains was sequenced and was found to carry the W403STOP mutation, indicated by a star.