Table 1.
Preys selected by the PhoN2 bait.
Figure 1.
PhoN2 is a periplasmic protein.
Exponentially-growing bacteria were harvested by centrifugation and supernatant, periplasmic and membrane fractions were prepared as described in Materials and Methods. Fractions were solubilised in Laemmli buffer and analyzed by Western blot analysis using polyclonal anti-PhoN2 [21], monoclonal anti-HA, polyclonal anti-SurA and anti-OmpA antibodies, as indicated. Panel A, bacterial fractions of wild-type S. flexneri strain M90T and of its mutant derivative strain HNDHA10 carrying the phoN2::HA fusion under the control of its natural promoter (Table S1). Panel B, bacterial fractions of the ΔphoN2 mutant derivative strain HND115 complemented with plasmids pHND10, 19, 11, 23, 14, 15, 16 and 21 encoding the different HA-fused recombinant proteins under the control of an L-arabinose inducible promoter (Table S1). In these cases, 0.016% of L-arabinose was used to induce phoN2::HA expression. No specific signals of PhoN2-HA were evidenced when bacteria were grown in the absence of the inducer. Experiments were repeated at least three times and images are representative.
Figure 2.
PhoN2-HA localizes at the poles of both exponentially-growing S. flexneri and of E. coli K-12 strains.
Images are representative of phase-contrast, DAPI, anti-HA, anti-IcsA, and the merged fields. Panels A–D, HNDHA10 phoN2::HA. Panel A, phase-contrast; Panel B, bacteria stained with anti-HA (green); Panel C, bacteria stained with anti-IcsA (red); Panel D, overlay of fluorescence of panels A–C. Panels E–F, semi-confluent monolayers of HeLa cells were infected with exponentially-growing HNDHA10 phoN2::HA. After 2 h of infection, HeLa cells and bacteria inside cells were stained with DAPI (panel E) and with DAPI (blue) and anti-HA (red) (panel F, overlay of fluorescence). Squares indicate subpopulation of bacteria displaying polarly localized PhoN2-HA. Panels G–L, immunofluorescent staining of the ΔphoN2 strain HND115, of the ΔicsA strain SC560 and of the E. coli K-12 strain DH10b (Table S1) complemented with plasmid pHND10 (panels G, I and K, anti-HA staining, green; panels H, J and L, overlay of the immunofluorescent images with the corresponding phase-contrast images). Bacteria shown in panels G–L were incubated in the presence of 0.016% of L-arabinose to induce phoN2::HA expression. No specific fluorescence signals were evidenced when bacteria were grown in the absence of the inducer (data not shown). Experiments were repeated at least three times and images are representative. Bars = 2 µm.
Figure 3.
Determination of domains in PhoN2 required for polar localization and catalytic activity.
Panel A, in-frame deletion and amino acid substitution mutants of the HA-tagged phoN2 gene. Mutants were generated by PCR, as described in Materials and Methods, using pHND10 (Table S1) DNA as template and the primer pairs reported in Table S2. Amino acid residue 1 represents the first residue of the leader peptide (dotted box), while numbers on the right indicate the last residue of the tagged HA epitope (white box). The positions (not in scale) of the N-terminal polyproline PPPP motif, of the different P to S and of the Y to A amino acid substitutions are indicated. The conserved D1, D2 and D3 domains corresponding to the putative catalytic site of PhoN2 [24], [44] and the R192P substitution are indicated. Panel B, fluorescence microscopy and overlay of fluorescence with phase-contrast images of HND115 complemented with each of the PhoN2-HA recombinant plasmids listed in Panel A and grown in the presence of 0.016% of L-arabinose. Bacteria were labeled with anti-HA monoclonal antibody (green). Arrows indicate polar foci of PhoN2-HA. Panel C, units of ATP-hydrolyzing activity; one unit corresponds to 1 μmol of Pi min−1 mg−1 of protein; means ± standard deviations (error bars) of experiments that were performed more than three times. Images are representative. Bar = 2 µm.
Figure 4.
The polyproline PPPP motif and the Y155 residue are required for PhoN2 stability.
The intracellular stability of recombinant proteins (Table S1) was determined in the ΔphoN2 mutant strain HND115 complemented with the recombinant plasmids listed in Fig. 3 (Panel A). The relative stability of selected PhoN2-HA recombinant proteins (plasmids pHND10, pHND11, 15, 16 and 21) was evaluated in the E. coli K-12 strain DH10b (Panel B) and in BS176, a virulence plasmid-cured derivative of wild-type S. flexneri strain M90T (Table S1) (Panel C). Stability was measured after protein synthesis had been inhibited by the addition of 100 μg/ml of spectinomycin, as described in Materials and Methods. 0.016% of L-arabinose was used to induce phoN2::HA expression. Samples were removed at time points indicated at the top and whole cell extracts were analyzed by Western blot using anti-HA monoclonal antibody. Quantitative measurements were carried out and the calculated half-life of each recombinant protein is shown (right).
Figure 5.
The 43PPPP46 motif is required for proper IcsA exposition.
Bright fields and fluorescence microscopy of IcsA of the wild-type strain M90T, HND115 and HND115 complemented with recombinant plasmids pHND10, 11, 19, 23, 14, 15, 16 and 21 (indicated at the top). 0.016% of L-arabinose was used to induce phoN2::HA expression. IcsA was detected in exponentially-growing bacteria, fixed with paraformaldehyde and labelled with anti-IcsA rabbit polyclonal antibody. Subpopulations of IcsA-labelled bacteria are shown. Arrows, polar foci of IcsA. Experiments were repeated at least three times and typical results are shown. Bar = 2 µm.
Figure 6.
In vivo cross-linking experiments.
The 43PPPP46 motif of PhoN2 is not required for the PhoN2-OmpA interaction. Cross-linking of HND115 (pHND10) was achieved by treating bacteria with formaldehyde to a final concentration of 1%, as described in Materials and Methods. Samples were suspended in Laemmli buffer and either heated at 37°C for 10 min to maintain cross-links or at 95°C for 20 min to break cross-links. Equal amounts of proteins were analyzed by Western blot. A protein molecular weight marker (Pierce) was used to determine the molecular weight of proteins. Immunoblotting was carried out with monoclonal anti-HA (Panels A and B) or polyclonal anti-OmpA antibodies (Panels C and D). Expression of PhoN2-HA was achieved by growing bacteria in the presence of 0.016% of L-arabinose. Panels A and C, bacteria not induced with L-arabinose; Panels B and D, L-arabinose induced bacteria. OmpA (U), unfolded OmpA; OmpA (F), folded OmpA [48]. Experiments were repeated at least three times and typical results are shown.
Figure 7.
OmpA is not required in the process of polar localization of PhoN2-HA.
Fluorescence microscopy of the S. flexneri mutant strain HND93 (ΔphoN2 ΔompA), of the E. coli strain ME9062 (ompA+) and of its ΔompA derivative strain JW0940 (Table S1) complemented with plasmid pHND10. Fluorescence microscopy and overlay of fluorescence with phase contrast images are shown. Bacteria were grown in the presence of 0.016% of L-arabinose to induce phoN2::HA expression and labeled with anti-HA monoclonal antibody. Panel A, HND93 (pHND10); Panel B, ME9062 (pHND10); Panel C, JW0940 (pHND10). Arrows, polar foci of PhoN2-HA. Experiments were repeated at least three times and typical results are shown. Bar = 2 µm.
Figure 8.
Model of the role of periplasmic PhoN2 in S. flexneri in proper IcsA exposition.
Polarly localized PhoN2, by binding the C-terminal periplasmic domain of OmpA only at the old bacterial pole, stabilizes OmpA in the small pore conformation thereby enabling proper IcsA exposition and function.