Oral toxicity of Photorhabdus luminescens W14 toxin complexes in Escherichia coli

Abstract

Previous attempts to express the toxin complex genes of Photorhabdus luminescens W14 in Escherichia coli have failed to reconstitute their oral toxicity to the model insect Manduca sexta. Here we show that the combination of three genes, tcdA, tcdB, and tccC, is essential for oral toxicity to M. sexta when expression in E. coli is used. Further, when transcription from native toxin complex gene promoters is used, maximal toxicity in E. coli cultures is associated with the addition of mitomycin C to the growth medium. In contrast, the expression of tcdAB (or the homologous tcaABC operon) with no recombinant tccC homolog in a different P. luminescens strain, K122, is sufficient to confer oral toxicity on this strain, which is otherwise not orally toxic. We therefore infer that P. luminescens K122 carries a functional tccC-like homolog within its own genome, a hypothesis supported by Southern analysis. Recombinant toxins from both P. luminescens K122 and E. coli were purified as high-molecular-weight particulate preparations. Transmission electron micrograph (TEM) images of these particulate preparations showed that the expression of tcdAB (either with or without tccC) in E. coli produces visible approximately 25-nm-long complexes with a head and tail-like substructure. These data are consistent with a model whereby TcdAB constitutes the majority of the complex visible under TEM and TccC either is a toxin itself or is an activator of the complex. The implications for the potential mode of action of the toxin complex genes are discussed.

FIG. 1

Diagram of toxin complex genes present in the recombinant plasmids used in this study. These are derived from either the tca (pA) or the tcd (pD) locus of P. luminescens W14. Plasmids pA1 and pD1 are Sau3A restriction fragments cloned into the BamHI site of Bluescript pBCKS(+). Plasmid pD2 is an SphI-BamHI fragment of pD1 cloned into pBR322. Plasmids pD3 and pD4 represent the pD2 backbone with a PCR-generated copy of tccC cloned into the BamHI site. In pD4, tccC is transcribed by the tetracycline resistance gene promoter on pBR322.

FIG. 2

Effect of the presence of TccC and mitomycin C induction on the oral toxicity of recombinant P. luminescens K122 and E. coli. (a) Histogram showing relative weight gain (mean and standard error for six larvae per treatment) of M. sexta fed on a diet treated with whole cultures of strains containing plasmids with or without tccC. Note that plasmids containing tccC alone [E. coli(pBADtccC)] do not inhibit weight gain, whereas plasmids carrying tcdAB in P. luminescens K122 [K122(pD2)] or tcdAB and tccC in E. coli [E. coli(pD3) and E. coli(pD4)] inhibit growth to 10% or less of the control weight. (b) Relative weight gain of larvae fed on supernatants grown in the presence (dark shading) or absence (light shading) of mitomycin C. Note that although some toxicity can be observed without mitomycin C [e.g., the 20% reduction seen with E. coli(pD4)], toxicity is substantially increased (∼90% weight reduction) by the addition of mitomycin C to cultures containing plasmids coexpressing both tcdAB and tccC [E. coli(pD3) and E. coli(pD4)]. The dagger represents a sample in which the mortality of the insects was 100%.

FIG. 3

Relative weight gain of M. sexta larvae fed on different dilutions (1× to 0.002×) of P. luminescens supernatants. Strains used were orally toxic P. luminescens W14 and P. luminescens K122 (lacking oral toxicity) expressing recombinant Tca or Tcd from the W14-derived recombinant plasmid pA1 or pD1 (Fig. 1), respectively. Note that the expression of Tcd alone in strain K122 produces a more orally toxic supernatant than does the expression of strain W14 (which contains a mixture of Tca and Tcd). Weights (mean and standard error for nine larvae) are relative to the mean weight for larvae fed on a supernatant from nonrecombinant K122 (which is set at 1.0). The means and standard errors for three independent experiments are shown. The estimated EC₅₀s are as follows: W14, 0.05×; Tca, 0.23×; and Tcd, 0.003× (see Discussion).

FIG. 4

Southern analysis supports the hypothesis that P. luminescens K122 carries homologs of W14 tccC-like and tcdA-like genes. The blots were probed with the highly conserved core region of the tccC gene (10) (a) and the P. luminescens W14 tcdAB operon (b). Probes were derived by PCR from the W14 genome. The DNA was digested and electrophoresed on 0.7% agarose gels and blotted using standard techniques. Probe hybridization was visualized using the Boehringer digoxigenin system. The locations of 1-kb marker DNA fragments are shown between the two gels (from top to bottom, 10, 8, 6, 5, 4, 3, and 2 kb). Digests were as follows: E, EcoRI; P, PstI; and H, HindIII.

FIG. 5

(a) Denaturing gel electrophoresis (SDS-PAGE) of particulate preparations from P. luminescens wild-type K122 and K122 expressing recombinant Tca (pA1) and Tcd (pD1). Note that expression from the pA1 and pD1 plasmids leads to the production of additional protein species, presumably toxin components. (b) Western analysis of P. luminescens K122 expressing recombinant Tca with an anti-Tca antibody. Note the detection of an ∼60-kDa cross-reacting species (arrowhead), putatively TcaBii. Note also that the antibody recognizes the same species in a preparation from P. luminescens W14.

FIG. 6

(a) Native gel of particulate preparations from P. luminescens K122 and K122 expressing recombinant Tca (pA1) and Tcd (pD1). Note that preparations from the acceptor strain P. luminescens K122 migrate as two clearly separable complexes, whereas coexpression of W14 Tca or Tcd results in a single native complex being produced by recombinant P. luminescens K122. The asterisk represents the recombinant Tca complex excised from the gel and reanalyzed by SDS-PAGE. (b) Denaturing gel electrophoresis (SDS-PAGE) of the same K122 and K122-Tca preparations as those analyzed in panel a. Note that the native Tca complex excised from the gel in panel a contains three predominant polypeptides (arrowheads). Note also that this native complex forms visible particles under TEM examination (see Fig. 8d).

FIG. 7

Denaturing gel electrophoresis (SDS-PAGE) of particulate preparations from E. coli expressing recombinant TcdAB from three plasmids, pD2, pD3, and pD4. Species corresponding to TcdA and TcdB are indicated by arrowheads.

FIG. 8

Transmission electron micrographs of particulate preparations from wild-type and recombinant strains of E. coli and P. luminescens showing the supramolecular structure of the toxin complexes. (a) E. coli expressing TcdAB (pD2). Note the presence of visible complexes (circled); however, these preparations are not orally toxic (Fig. 2a). (b) E. coli coexpressing TcdAB and TccC. Note the presence of the same visible particles, which are now orally toxic (Fig. 2a). (c) Preparation from wild-type P. luminescens W14 showing visible toxin complexes. (d) Native gel preparation (asterisk in Fig. 6a) from K122 expressing TcaABC, again showing the same particles. Bar, 50 nm. These data show that TcdA and TcdB together are sufficient to produce visible particles but that coexpression of TccC is essential for oral toxicity to M. sexta (see Discussion).