Establishment of a Protein Concentration Gradient in the Outer Membrane Requires Two Diffusion-Limiting Mechanisms

Abstract

OmpA-like proteins are involved in the stabilization of the outer membrane, resistance to osmotic stress, and pathogenesis. In Caulobacter crescentus, OmpA2 forms a physiologically relevant concentration gradient that forms by an uncharacterized mechanism, in which the gradient orientation depends on the position of the gene locus. This suggests that OmpA2 is synthesized and translocated to the periplasm close to the position of the gene and that the gradient forms by diffusion of the protein from this point. To further understand how the OmpA2 gradient is established, we determined the localization and mobility of the full protein and of its two structural domains. We show that OmpA2 does not diffuse and that both domains are required for gradient formation. The C-terminal domain binds tightly to the cell wall and the immobility of the full protein depends on the binding of this domain to the peptidoglycan; in contrast, the N-terminal membrane β-barrel diffuses slowly. Our results support a model in which once OmpA2 is translocated to the periplasm, the N-terminal membrane β-barrel is required for an initial fast restriction of diffusion until the position of the protein is stabilized by the binding of the C-terminal domain to the cell wall. The implications of these results on outer membrane protein diffusion and organization are discussed.IMPORTANCE Protein concentration gradients play a relevant role in the organization of the bacterial cell. The Caulobacter crescentus protein OmpA2 forms an outer membrane polar concentration gradient. To understand the molecular mechanism that determines the formation of this gradient, we characterized the mobility and localization of the full protein and of its two structural domains an integral outer membrane β-barrel and a periplasmic peptidoglycan binding domain. Each domain has a different role in the formation of the OmpA2 gradient, which occurs in two steps. We also show that the OmpA2 outer membrane β-barrel can diffuse, which is in contrast to what has been reported previously for several integral outer membrane proteins in Escherichia coli, suggesting a different organization of the outer membrane proteins.

Keywords: Caulobacter crescentus; FRAP; cell wall; limited diffusion; outer membrane; protein concentration gradient.

FIG 1

OmpA2 requires both structural domains to form its concentration gradient. The localization, stability, subcellular localization, and folding of the full OmpA2 fluorescent fusion and of the truncated version are shown. (A) Schematic representation of the fluorescent fusions. The length (in residues) is indicated. Periplasmic mCherry was expressed from the vanA promoter. SP, signal peptide; P-rich, proline rich linker. (B) Western blot of the soluble (S) and insoluble (I) fractions obtained from cell cultures expressing the proteins indicated in the figure. The following indicated molecular weights correspond to mature proteins: OmpA2-mCh (67 kDa), PBD-mCh (45 kDa), β-barrel–mCh (57 kDa), and periplasmic mCherry (30 kDa). (C) Western blot showing the unfolding pattern of OmpA2 and the β-barrel at the indicated temperatures. Both Western blots were revealed with anti-mCherry antibody. (D) Florescence images of cells expressing the indicated fluorescent fusion (OmpA2, OmpA2-mCh, strain LDG1; PBD, PBD-mCh, strain LDG4; β-barrel, β-barrel–mCh, strain LDG3; mCh, periplasmic mCherry, strain LDG5). The arrow in the PBD panel indicates an area with a reduced amount of the protein around the cell division site, and the arrowhead indicates outer membrane vesicles. Cells were grown in PYE to a 0.3 OD₆₆₀. Scale bar indicates 1 μm.

FIG 2

Mobility of OmpA2 and its truncated or point mutant versions. The mobility of the full OmpA2-mCh protein, the two domains fused to mCherry, and the single amino acid variants in the PBD domain of the full protein (OmpA2_D336A-mCh and OmpA2_R351A-mCh) was determined in FRAP experiments. (A) FRAP assays in filamented cells obtained by FtsZ depletion and treated to inhibit protein synthesis (OmpA2, OmpA2-mCh, strain LDG7; β-barrel, β-barrel–mCh, strain LDG8; PBD, PBD-mCh, strain LDG9; OmpA2 D336A, OmpA2_D336A, strain LDG22; and OmpA2 R351A, OmpA2_R351A, strain LDG23). Representative cells in different times after photobleaching are shown. Arrows in the first column indicate the selected area for photobleaching. The scale bar indicates 1 μm. (B) Quantification of the fluorescence recovery after photobleaching. The scattered symbols show the recovery of the cells in (A), and the continuous lines show the fitting of the signal of the cells that show recovery (see Materials and Methods for more details). The fluorescence level in the bleached area after the photobleaching (0 minutes) was used as basal level. The t_1/2 values for β-barrel–mCh and OmpA2_R351A-mCh are indicated in the plot and correspond to the mean for at least 10 cells from independent experiments.

FIG 3

Diffusion of the OmpA2 β-barrel is not caused by FtsZ depletion. The mobility of the complete OmpA2 protein and of the β-barrel were determined in unfilamented cells and for the β-barrel in cells filamented by FtsI depletion or cephalexin treatment. (A) FRAP assays of OmpA2 (OmpA2-mCh) and the β-barrel (β-barrel–mCh) in unfilamented cells. (B) FRAP of the β-barrel–mCh in filaments obtained by the FtsI depletion or the cephalexin treatment. Protein synthesis was inhibited as in the experiments in Fig. 2. Arrows in the first column indicate the selected area for photobleaching. The t_1/2 values for the β-barrel in FtsI and cephalexin filaments are indicated in the text.

FIG 4

Binding of OmpA2 to PG is essential for gradient formation. The localization and stability of two OmpA2 point mutants were tested in wild-type and chaperone-overexpressing cells (A) Cells expressing OmpA2-mCh or the point mutants OmpA2_D336A or OmpA2_R351A fused to mCherry in a wild-type background (first column, strains LDG1, LDG11, and LDG12), in cells overexpressing SurA (second column, strains LDG13, LDG16, and LDG19), in cells overexpressing DegP (third column, strains LDG14, LDG17, and LDG20), and in cells overexpressing Skp (fourth column, strains LDG15, LDG18, and LDG21). Arrows indicate cells with a weak gradient in the cells expressing OmpA2_D336A. (B) Quantification of the number of cells showing a gradient. The percentage of cells showing a gradient was determined for the strains shown in panel A. The percentages were obtained from at least 100 cells. (C) Western blot α-mCherry showing the stability of OmpA2 point mutants in wild-type and chaperone-overexpressing cells. The arrow indicates the degradation product of the fluorescent fusions (free mCherry). Scale bar = 1 μm.

FIG 5

Pattern of appearance of newly synthesized OmpA2 and PBD proteins. The fluorescence pattern of synchronized single cells expressing OmpA2-mCh or PBD-mCh was recorded after complete bleaching. (A) Time lapse of cells expressing OmpA2 (strain LDG24) or PBD-mCh (strain LDG4). Fluorescence was bleached in the whole cell body, and the appearance of newly synthesized protein was followed. Arrows in the prebleach image of the strain expressing OmpA2-mCh indicate the old cell poles assigned by the localization of MipZ-YFP (not shown). The scale bar represents 1 μm. (B and C) Fluorescence intensity profiles of representative cells (marked with asterisks in panel A) expressing OmpA2-mCh or the PBD-mCh, respectively. Cell length was normalized, fluorescence levels were plotted, the fluorescence of the cell before bleaching was scaled down and correspond to one-third of the real one. The time in minutes is indicated.