Enterococcus faecalis virulence regulator FsrA binding to target promoters

Abstract

The FsrABDC signal transduction system is a major virulence regulator in Enterococcus faecalis. The FsrC sensor histidine kinase, upon activation by the gelatinase biosynthesis-activating pheromone (GBAP) peptide encoded by the fsrBD genes, phosphorylates the FsrA response regulator required for the transcription of the fsrBDC and the gelE-sprE genes from the fsrB promoter and the gelE promoter, respectively. FsrA belongs to the LytTR family of proteins, which includes other virulence regulators, such as AgrA of Staphylococcus aureus, AlgR of Pseudomonas aeruginosa, and VirR of Clostridium perfringens. The LytTR DNA-binding domain that characterizes these proteins generally binds to two imperfect direct repeats separated by a number of bases that place the repeats on the same face of the DNA helix. In this study, we demonstrated that FsrA also binds to two imperfect direct repeats separated by 13 bp, based on the consensus sequence of FsrA, T/AT/CAA/GGGAA/G, which is consistent with the binding characteristics of LytTR domains.

FIG. 1.

Phosphorylation assay of FsrA-His6. MBP-FsrC was incubated with [γ-³²P]ATP in the absence and presence of FsrA-His6 for the indicated times. The samples were separated by 15% SDS-PAGE and exposed to a PhosphorImager screen.

FIG. 2.

Gel mobility shift assays. The 297-bp (positions −283 to +14) end-labeled fsrB promoter fragment (A) or the 359-bp (positions −307 to + 52) end-labeled gelE fragment (B) was incubated with FsrA-His6 at the concentrations indicated in the figure. C, complex; FP, free probe; NS, nonspecific DNA; S, specific DNA.

FIG. 3.

DNase I footprinting analysis of the promoter-regulatory regions of fsrB (A) and gelE (B). The labeled DNA fragments were obtained as described in Materials and Methods. The fragments were incubated with different amounts of FsrA-His6 before DNase I treatment. Protein concentrations used in each reaction mixture were 0 (lanes 1 and 2), 0.66 μM (lane 3), 1.3 μM (lane 4), 2.6 μM (lane 5), and 5.3 μM (lane 6). Dideoxy sequencing reactions of pTOPO-fsrB (A) and pTOPO-gelE (B) are also shown. (C). Alignment of the nucleotide sequence of the fsrB and gelE promoters identifying the regions protected by FsrA and the −10 and −35 promoter elements (26). Regions protected from DNase I digestion are indicated by gray boxes. Arrows show nucleotides hypersensitive to DNase I digestion. The direct repeats shown are based on the consensus sequence identified in Fig. 4.

FIG. 4.

Sequence logo of the repeats of 8 nucleotides that form the FsrA consensus site. (A) Sequence alignment of the regions protected by FsrA in the fsrB and gelE promoters obtained with Clustal W. The sequences in the box were used to generate the logo. An asterisk indicates identical bases, a colon indicates a different nucleotide base group, and a period indicates the same nucleotide base group. (B) The height of each letter is proportional to the frequency of the base, and the height of the letter stack is the conservation in bits at that position. Error bars are shown at the tops of the stacks. The logo was obtained with the WebLogo server (http://weblogo.berkeley.edu) (5).