Comparison of OG1RF and an isogenic fsrB deletion mutant by transcriptional analysis: the Fsr system of Enterococcus faecalis is more than the activator of gelatinase and serine protease

Abstract

The FsrABC system of Enterococcus faecalis controls the expression of gelatinase and a serine protease via a quorum-sensing mechanism, and recent studies suggest that the Fsr system may also regulate other genes important for virulence. To investigate the possibility that Fsr influences the expression of additional genes, we used transcriptional profiling, with microarrays based on the E. faecalis strain V583 sequence, to compare the E. faecalis strain OG1RF with its isogenic mutant, TX5266, an fsrB deletion mutant. We found that the presence of an intact fsrB influences expression of numerous genes throughout the growth phases tested, namely, late log to early stationary phase. In addition, the Fsr regulon is independent of the activity of the proteases, GelE and SprE, whose expression was confirmed to be activated at all three time points tested. While expression of some genes (i.e., ef1097 and ef0750 to -757, encoding hypothetical proteins) was activated in late log phase in OG1RF versus the fsrB deletion mutant, expression of ef1617 to -1634 (eut-pdu orthologues) was highly repressed by the presence of an intact Fsr at entry into stationary phase. This is the first time that Fsr has been characterized as a negative regulator. The newly recognized Fsr-regulated targets include other factors, besides gelatinase, described as important for biofilms (BopD), and genes predicted to encode the surface proteins EF0750 to -0757 and EF1097, along with proteins implicated in several metabolic pathways, indicating that the FsrABC system may be an important regulator in strain OG1RF, with both positive and negative effects.

FIG. 1.

Characterization of the strains used in this study. (A) Genetic organization of the fsr and gelE loci in V583 and locations of the PCR products of these genes used to construct the microarray. (B) Genotype and phenotype of the OG1RF parental and isogenic strains used in this study. TX5266 and TX5264 are both in-frame deletion mutants in which the deleted sequence contains the portion used for the microarray (fsrB and gelE, respectively). TX5128 is a gelE disruption mutant in which the minitransposon, mγδ, is inserted in the 3′ end of the gene. (a) Expression pattern using the V583 array. (b) Gelatinase activity. (c) Although the gene is truncated and the protein inactive, due to the location of the PCR primers, gelE transcript was detected in TX5128 by microarray.

FIG. 2.

Growth profile of E. faecalis in BHI and semiquantitative RT-PCR. (A) The means and standard deviations were calculated from three independent cultures grown in parallel. OG1RF is the parental strain, while TX5266 is the in-frame fsrB deletion mutant, and TX5128 is a gelE disruption mutant. The arrows indicate when samples were taken for microarray analysis. Hour 3 corresponds to late log phase, hour 4 to ENT-stat, and hour 5 to EAR-stat. (B) Semiquantitative RT-PCR analysis of gyrB, fsrC, gelE, ef1097, ef1561, ef0750, ef754 to -755, ef1632, efaA, efaB, and efaC showing RT-PCR products from RNA extracted from OG1RF or TX5266 at hour 3 (late log phase), and hour 5 (EAR-stat). The three lanes for each RNA represent undiluted cDNA and two 10-fold dilutions of cDNA before the PCR. The gDNA used as a control for the PCR was extracted from OG1RF.

FIG. 3.

Alteration in gene expression between OG1RF and TX5266 (fsrB in-frame deletion) and between OG1RF and TX5128 (gelE disruption mutant). Each panel represents an average of microarray results obtained with RNA preparations from at least three different cultures. The genes are represented if at least one average ratio per RNA preparation was available for statistical analysis. For all panels, the x axis represents the gene identification number as annotated by TIGR (NCBI ID, NC_004668). The y axis indicates the change (n-fold); the change was considered positive when the expression was higher in OG1RF than in the mutant. The gray circles correspond to nonsignificantly affected genes. The black triangles (A, B, and C) correspond to genes that were regulated at least twofold with a P value of <0.05 for OG1RF versus TX5266, while the black diamonds (D and E) represent genes that were regulated at least twofold with a P value of <0.05 for OG1RF versus TX5128. The arrows indicate specific genes of interest (“ef” has been omitted). In the boxes in the right lower corners of panels A to C are shown the numbers of regulated genes and their classification into four categories: >10-fold repression, between 2- and 10-fold repression, between 2- and 10-fold activation, and >10-fold activation.

FIG. 4.

ef1097 locus and transcriptional start site. (A) Genetic organization of the ef1097 chromosomal region. (B) Sequence alignment between ef1097, gelE, and fsrB promoter regions. Putative −10 and −35 sequences are underlined, and transcription start sites (+1) are boldface and underlined. The two boxes characterized by Qin et al. (36) as essential for expression are shaded. Common nucleotides between ef1097, fsrB, and gelE promoters are boldface. The stem loops indicate a putative transcriptional terminator.