Purification and characterization of a high-molecular-weight insecticidal protein complex produced by the entomopathogenic bacterium photorhabdus luminescens

Abstract

Photorhabdus luminescens is a gram-negative enteric bacterium that is found in association with entomopathogenic nematodes of the family Heterorhabditidae. The nematodes infect a variety of soil-dwelling insects. Upon entering an insect host, the nematode releases P. luminescens cells from its intestinal tract, and the bacteria quickly establish a lethal septicemia. When grown in peptone broth, in the absence of the nematodes, the bacteria produce a protein toxin complex that is lethal when fed to, or injected into the hemolymph of, Manduca sexta larvae and several other insect species. The toxin purified as a protein complex which has an estimated molecular weight of 1,000,000 and contains no protease, phospholipase, or hemolytic activity and only a trace of lipase activity. The purified toxin possesses insecticidal activity whether injected or given orally. Analyses of the denatured complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed it to be composed of several protein subunits ranging in size from 30 to 200 kDa. The complex was further separated by native gel electrophoresis into three components, two of which retained insecticidal activity. The purified native toxin complex was found to be active in nanogram concentrations against insects representing four orders of the class Insecta.

FIG. 1

HPLC chromatograms, from the HPLC 4000 SW molecular sieve column, of the toxic fraction following each step of the purification. (A) Crude culture broth; (B) DEAE-Sephacel; (C) Sephacryl S-400HR; (D) HPLC 4000 SW molecular sieve column; (E) standard proteins (37.07 min, thyroglobin, 669 kDa; 47.74 min, aldolase, 158 kDa; 51.40 min, ovalbumin, 42.7 kDa). The arrows below the chromatograms indicate fractions containing the insecticidal activity. The numbers on the chromatograms indicate the elution times in minutes. Absorbance was monitored at 280 nm.

FIG. 2

Analysis of protein components of the insecticidal toxin complex of P. luminescens. (A) SDS–10% PAGE analysis of HPLC-purified P. luminescens insecticidal toxin preparations. The gel was stained with Coomassie brilliant blue. Sta, size standard; tox, purified toxin sample. (B) HPLC-purified toxin separated by native agarose gel electrophoresis; 5 μg of total protein was loaded. The gel was stained with Coomassie brilliant blue. (C) SDS–18% PAGE analysis of protein bands separated by native agarose gel electrophoresis. Lanes: 1, band 1; 2, band 2; 3, band 3. The gel was silver stained. (D) HPLC-purified toxin separated by agarose gel electrophoresis in the presence of 0.1% Triton X-100; 4 μg of total protein was loaded. The gel was stained with Coomassie brilliant blue. (E) SDS–10% PAGE analysis of protein bands separated in native agarose gels in the presence of 0.1% Triton X-100. Lanes: 1, band 1; 2, band 2; 3, band 3. The positions of molecular mass markers are indicated.