Use of Pseudomonas putida EstA as an anchoring motif for display of a periplasmic enzyme on the surface of Escherichia coli

Abstract

The functional expression of proteins on the surface of bacteria has proven important for numerous biotechnological applications. In this report, we investigated the N-terminal fusion display of the periplasmic enzyme beta-lactamase (Bla) on the surface of Escherichia coli by using the translocator domain of the Pseudomonas putida outer membrane esterase (EstA), which is a member of the lipolytic autotransporter enzymes. To find out the transport function of a C-terminal domain of EstA, we generated a set of Bla-EstA fusion proteins containing N-terminally truncated derivatives of the EstA C-terminal domain. The surface exposure of the Bla moiety was verified by whole-cell immunoblots, protease accessibility, and fluorescence-activated cell sorting. The investigation of growth kinetics and host cell viability showed that the presence of the EstA translocator domain in the outer membrane neither inhibits cell growth nor affects cell viability. Furthermore, the surface-exposed Bla moiety was shown to be enzymatically active. These results demonstrate for the first time that the translocator domain of a lipolytic autotransporter enzyme is an effective anchoring motif for the functional display of heterologous passenger protein on the surface of E. coli. This investigation also provides a possible topological model of the EstA translocator domain, which might serve as a basis for the construction of fusion proteins containing heterologous passenger domains.

FIG. 1.

(A) Schematic representation of the structure of the EstA preprotein. The EstA preprotein is predicted to be composed of a 24-amino-acid signal sequence (SP), the α-domain (EstAα) including the catalytic triad, the linking region (LR), and the β-domain (EstAβ). The amino acids indicated in the EstAα region show a putative catalytic triad (serine 14, aspartate 286, and histidine 289). The predicted linking region (S307 to G349) is assumed to contain an α-helical structure. (B) Schematic representation of the construction of the Bla-EstA fusion proteins. The fusion protein is composed of the Bla signal sequence (BP) and the mature portion of Bla, fused to successively truncated C-terminal portions (numbered 1 to 8) of the EstA. Additional amino acids between Bla and the EstA C-terminal domain which were derived from a restriction enzyme site (NheI) for linking bla and each estA C-terminal region are underlined. Recombinant plasmids pABETU1 to pABETU8 encode the fusion proteins FP1 to FP8, respectively. As a control construct, pABla encodes wild-type Bla in the periplasm.

FIG. 2.

(A) Expression of fusion proteins in E. coli as assessed by Western blots. Equal amounts of whole-cell lysates of recombinant E. coli JK321 cells corresponding 10 μl of a suspension with an OD₆₀₀ of 10.0 were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent Western blot analysis with anti-Bla antibody. Lane 1, pACYC184 (vector); lane 2, pABla; lane 3, pABETU1; lane 4, pABETU2; lane 5, pABETU3; lane 6, pABETU4; lane 7, pABETU5; lane 8, pABETU6; lane 9, pABETU7; lane 10, pABETU8. The poorly expressed fusion proteins are indicated by the open arrowheads (lanes 3 to 7), and periplasmic expressed Bla-derived bands are indicated with an arrow (lane 2). (B) Surface exposure of fusion proteins assessed by whole-cell immunoblots. Equal amounts of recombinant E. coli JK321 cell suspensions were applied directly to nitrocellulose membrane and subsequently subjected to immunodetection analysis. Each lane is the same as those described for panel A. (C) Surface exposure of fusion proteins in various E. coli strains assessed by whole-cell immunoblots. Equal amounts of recombinant E. coli cell suspensions were applied directly to nitrocellulose membrane and subsequently subjected to immunodetection analysis.

FIG. 3.

Protease accessibility of fusion proteins assessed by (A) Western blots and (B) whole-cell immunoblots. Samples were prepared and analyzed as described in the legend of Fig. 2. Tryptic digestion of physiologically intact cells was performed as described in Materials and Methods.

FIG. 4.

(A) FACS histograms of recombinant E. coli cells to determine surface localization of Bla. The E. coli cell line harboring pACYC184 (vector) was used as the negative control, and the corresponding histogram (dotted line) was superimposed on results (solid line) with E. coli expressing periplamsic Bla (pABla) or Bla-EstA fusion proteins (indicated recombinant plasmid). (B) Mean fluorescence intensities of E. coli cells expressing periplasmic Bla and Bla-EstA fusion proteins.

FIG. 5.

Growth kinetics and viability of E. coli cells expressing the Bla-EstA fusion proteins and periplasmic Bla. The E. coli cells harboring pACYC184 (vector) (•), pABla (▾), and pABETU8 (▪) were grown in LB broth, and the viability of each recombinant E. coli cell suspension (open symbols) was estimated by using DiBAC₄(3) staining (determined in duplicate without significant variation).

FIG. 6.

Whole-cell Bla activity of E. coli cells expressing the Bla-EstA fusion proteins. The E. coli cell line harboring pABETU8 was grown on an LB agar plate (solid) or in LB broth (liquid) at indicated temperatures with (open bars) and without (filled bars) ampicillin. Whole-cell Bla activities are shown as the mean values, representing the results of five independent experiments, which are calculated for the amounts of bacteria present in 1 ml of a suspension with an OD₅₇₅ of 1.0 (∼2.5 × 10⁹ CFU). One unit of Bla activity is defined as the amount of enzyme that hydrolyzes 1 μmol of penicillin G per min.

FIG. 7.

Possible topological model of the organization of the EstA β-domain in the outer membrane. The C-terminal β-domain of EstA might be composed of 12 amphipathic β-strands (pentagons) and one α-helical structure (cylinder). Eleven amphipathic β-strands (solid lines) are clearly predicted by the 3D-PSSM program within the C-terminal region Y374 to F622 as shown by Wilhelm et al. (49), while one more β-strand and an α-helix are hypothetical (dotted line). The first amino acid residues of the N-terminally truncated EstA β-domains are indicated by arrows.