PrgB promotes aggregation, biofilm formation, and conjugation through DNA binding and compaction

Abstract

Gram-positive bacteria deploy type IV secretion systems (T4SSs) to facilitate horizontal gene transfer. The T4SSs of Gram-positive bacteria rely on surface adhesins as opposed to conjugative pili to facilitate mating. Enterococcus faecalis PrgB is a surface adhesin that promotes mating pair formation and robust biofilm development in an extracellular DNA (eDNA) dependent manner. Here, we report the structure of the adhesin domain of PrgB. The adhesin domain binds and compacts DNA in vitro. In vivo PrgB deleted of its adhesin domain does not support cellular aggregation, biofilm development and conjugative DNA transfer. PrgB also binds lipoteichoic acid (LTA), which competes with DNA binding. We propose that PrgB binding and compaction of eDNA facilitates cell aggregation and plays an important role in establishment of early biofilms in mono- or polyspecies settings. Within these biofilms, PrgB mediates formation and stabilization of direct cell-cell contacts through alternative binding of cell-bound LTA, which in turn promotes establishment of productive mating junctions and efficient intra- or inter-species T4SS-mediated gene transfer.

Figure 1

Schematic overview of the different domains of PrgB. PrgB contains a signal sequence (ss, cyan) at the N-terminus that targets it to the outside of the cell. It contains a C-proximal LPXTG motif that allows PrgB to be anchored to the cell wall (cw, green). Gray areas indicate areas of the protein predicted to be unstructured. Amino acid numbers are shown.

Figure 2

Crystal structure of the adhesion domain PrgB_246-558 at 1.6 Å resolution. A: Ribbon representation, coloured in rainbow. The numbers of individual β-strands important for protein function are indicated. B: Surface charge distribution calculated by APBS, scaled from -5 (red) to 5 (blue) k_bT. A bound Tris-molecule within the negatively charged cleft is shown in spheres. The top and back side of the protein is dominated by positively charged surface areas.

Figure 3

DNA interaction studies. A: SPR measurements of PrgB_188-1233 with DNA-1 and DNA-2, the normalized response is plotted against the concentration of PrgB. The determined K_d values are 3.4 ± 0.3 μM and 3.2 ± 0.3 μM for DNA-1 and DNA-2, respectively. B: Binding of 250 bp long DNA to PrgB_188-1233 monomer and dimer determined by EMSAs. Determined K_D values are 1.0 ± 0.02 μM and 0.55 ± 0.02 μM, respectively. C: Binding of 100bp and 250bp long DNA to PrgB_188-1233 monomer determined by EMSAs. Determined K_D values are 1.6 ± 0.1 μM and 1.0 ± 0.01 μM, respectively. D: Binding of 100bp and 250bp long DNA to PrgB_188-1233 dimer determined by EMSAs. Determined K_D values are 0.2 ± 0.01 μM and 0.05 ± 0.02 μM, respectively.

Figure 4

Crystal structure of the adhesion domain PrgB_246-558 with bound DNA. A: Surface charge distribution calculated by APBS, scaled from -5 (red) to 5 (blue) k_bT. The bound DNA molecule is shown in cartoon representation. Crystal symmetry related molecules are shown in grey for protein and light orange for DNA B: Crystal packing of the PrgB-DNA complex. Contents of the asymmetric unit are shown as colored surface and cartoon, symmetry related molecules are represented in grey for protein and light orange for DNA.

Figure 5

Nanochannel experiments with DNA and dimeric PrgB. A: A sketch showing a DNA molecule confined inside a nanochannel. DNA will be partially stretched out in the nanochannel, with an extension R, shorter than its contour length, Lc. B: Snapshots and kymographs of λ-DNA with PrgB bound confined in 800×150 nm² channels. i) non-compacted DNA. ii) partly compacted DNA. iii) fully compacted DNA. C: Snapshots and kymographs of λ-DNA molecules confined in 800×150 nm² channels. Left: Partially compacted DNA with two ends condensed. Right: DNA dimer with three condensed points. D: Boxplot of extension of λ-DNA inside 800×150 nm² channels. The box is determined by the 25^th and 75^th percentiles and the whiskers are determined by the 5^th and 95^th percentiles. The line in the box is the median value and the square symbol in the box is the mean value. The cross symbols are the maximum and minimum values, respectively. The DNA concentration is 5 μM base pairs. The scale bar is 1μm in all figures.

Figure 6

LTA competition to PrgB bound DNA, demonstrated by EMSA. A: LTA binding to either dimeric or monomeric PrgB_188-1233 pre-incubated with 250 bp long DNA. B: Relative intensity of PrgB-DNA bands plotted against LTA concentration for PrgB_188-1233 monomer and dimer. The determined IC₅₀ values are 0.39 ± 0.09 and 0.69 ± 0.03 mg/mL, respectively.

Figure 7

Contributions of the PrgB adhesin domain to aggregation, biofilm formation, and conjugation. In all panels, strains analysed were OG1RF alone (denoted ‘no plasmid’) or with pCF10 or pCF10ΔprgB (ΔprgB); these strains also were engineered to produce native PrgB or PrgBΔ246-558 from the constitutive P₂₃ promoter. A: Steady-state levels of Prg proteins in strains induced for 1 h with cCF10 pheromone (10 ng ml⁻¹). Immunoblots were developed with anti-PrgB antibodies. Protein sizes (in kilodaltons, kDa) are listed at the right. Protein extracts were loaded on a per-cell equivalent basis. B: Photographs of strains grown in microtiter plates showing the effects of DNase and LTA on PrgB-mediated clumping. C: Biofilm formation on polystyrene microtiterplates. The biofilm biomass was assayed as a function of crystal violet stain retained. DNase or LTA treatment was initiated at the onset of cCF10 induction and biofilm formation and continued for the full incubation time of 24 h. Results are expressed as OD₅₅₀ values and values represent the average of at least three independent experiments; the error bars represent standard deviations. D: Effect of the PrgB adhesin domain on plasmid transfer. Transfer frequencies are presented as the number of transconjugants per donor cell. Experiments were repeated at least three times in duplicate, and results from a representative experiment are shown.