Article Figures & Data
Figures
- Fig 1
Influence of calcium, magnesium, and the chelator EGTA on biofilm formation by P. putida KT2440. (A to C) Attached biomass was determined by crystal violet staining, as described in Materials and Methods. Data are averages and standard deviations from four experiments. Asterisks indicate statistically significant differences with respect to the control. (A) Biofilm formation on microtiter plates during growth in LB with increasing concentrations of CaCl2 (white bars, control without addition; hatched bars, 10 mM; light gray bars, 20 mM; dark gray bars, 50 mM). (B) Effect of EGTA on biofilm formation, determined as described above (white bars, control; black bars, LB with 0.1 mM EGTA; gray bars, 0.1 mM EGTA and 10 mM CaCl2). (C) Biofilm formation in LB (white bars) and in the presence of 0.1 mM EGTA (hatched bars), 10 mM MgCl2 (black bars), or Mg plus EGTA (gray bars). (D) Biofilm formation on glass coverslips during growth in 1:10 strength LB. Coverslips were directly photographed at 24 h postinoculation.
- Fig 2
Biofilm formation on glass coverslips by KT2440 in the absence (control) or presence of EGTA, compared to that by the lapF mutant strain mus-20. Biofilms were grown in 1:10 strength LB as described in Materials and Methods and observed after 24 h by fluorescence microscopy. Two different fields are shown in each case. Magnification, ×400.
- Fig 3
Bioinformatic analysis of the structure of the 300 C-terminal amino acids of LapF (A) compared to the Serratia marcescens metalloprotease (1SAT in the RCSB PDB database, which shows bound calcium as green balls) (B). The calcium binding motifs are shown in green in the sequence, and the corresponding region is boxed in the structural prediction. Model prediction was obtained using HHPred (24), and the Protein Workshop toolkit (16) was used for visualization.
- Fig 4
Isothermal titration of the C-terminal domain of LapF in the absence and presence of Ca2+. An injection volume of 10 μl and a total number of 25 injections were used. (A) Injection of the ligand CaCl2 into buffer. (B) Injection of the ligand into a 8.5 μM protein solution. Ligand and protein were in 0.1 M Tris–0.5 M NaCl, pH 7.5. Experiments were carried out at 30°C. Upper panel, raw titration data. Lower panel, integrated and dilution-corrected peak areas of raw data. Data were fitted using the “one-binding-site model” of the MicroCal version of ORIGIN.
- Fig 5
(A to C) Transmission electron microscopy of CLapF incubated for 3 days at room temperature without CaCl2 (A), with 10 mM CaCl2 (B), or with CaCl2 and EGTA (C). (D) A control with 10 mM CaCl2 without protein, to ensure that the aggregates in panel B are not artifacts due to calcium precipitation. Scale bar, 500 nm.
- Fig 6
Size distribution histograms for purified CLapF treated with 10 mM EGTA and dialyzed overnight against 10 mM Tris-HCl–50 mM NaCl, pH 7.5 (A), protein from panel A with 10 mM CaCl2 (B), and protein from panel B treated with EGTA to a final concentration of 20 mM (C).
- Fig 7
Study of the molecular mass of CLapF by analytical FPLC. Purified CLapF after treatment with EGTA (gray line) and CLapF incubated with 10 mM CaCl2 (black line) are shown, as well as the molecular masses (open circles) and linear trend (broken line) of the standards. The ∼77-kDa and ∼630-kDa peaks are indicated.
- Fig 8
Transmission electron microscopy and immunodetection of LapF. (A and B) Wild-type cells after 8 h growth and immunogold labeling with anti-LapF antibody; (C) thin sections of resin-embedded wild-type cells after 8 h growth in 6-well microtiter plates; (D) mutant strain mus-20 (lapF). Arrows point to the accumulation of gold particles in the cell surface and in the cytoplasm.
Tables
- Table 1
Binding parameters derived from isothermal titration calorimetry experimentsa
Parameter (unit) Value (mean ± SD) KA (M−1) 2.82 (± 0.16) × 105 KD (μM) 3.54 ± 0.016 ΔH (kcal/mol) −9.8 ± 0.4 ΔS (cal/mol/°) −7.35 N (sites) 0.603 ± 0.0206 ↵a All experiments were conducted at 30°C. Ligands were placed in the syringe and CLapF in the sample cell. The buffer was 10 mM Tris-HCl–50 mM NaCl2 adjusted to a pH 7.5 by the addition of concentrated HCl.
- Table 2
Dynamic light scattering results for CLapF
Peak Control With CaCl2 With CaCl2 + EGTA Hydrodynamic radius (nm) Polydispersity (%) Mass (%) Hydrodynamic radius (nm) Polydispersity (%) Mass (%) Hydrodynamic radius (nm) Polydispersity (%) Mass (%) 1 24.313 11.2 7.8 25.233 17 3 16.183 8.8 0.6 2 148.346 18.7 58.4 136.969 12.8 9.9 153.293 5.7 1.3 3 917.719 4.7 33.8 251.616 20.5 19 913.999 18.5 98.1 4 5262 23.7 68
Supplemental material
Files in this Data Supplement:
- Supplemental file 1 -
Fig. S1, purification of the C-terminal domain of LapF, Western blot with anti-His-tag antibodies, and protein integrity before and after EGTA treatment
PDF, 32K
- Supplemental file 1 -
