Specificity of Activated CryIA Proteins from Bacillus thuringiensis subsp. kurstaki HD-1 for Defoliating Forest Lepidoptera.

The insecticidal activity of the CryIA(a), CryIA(b), and CryIA(c) toxins from Bacillus thuringiensis subsp. kurstaki HD-1 was determined in force-feeding experiments with larvae of Choristoneura fumiferana, C. occidentalis, C. pinus, Lymantria dispar, Orgyia leucostigma, Malacosoma disstria, and Actebia fennica. The toxins were obtained from cloned protoxin genes expressed in Escherichia coli. The protoxins were activated with gut juice from Bombyx mori larvae. Biological activity of the individual gene products as well as the native HD-1 toxin was assessed as the dose which prevented 50% of the insects from producing frass within 3 days (frass failure dose [FFD(50)]). The three toxins were about equally active against M. disstria. In the Choristoneura species, CryIA(a) and CryIA(b) were up to fivefold more toxic than CryIA(c). In the lymantriid species, CryIA(a) and CryIA(b) were up to 100-fold more toxic than CryIA(c). The toxicity of HD-1 was similar to that of the individual CryIA(a) or CryIA(b) toxins in all of these species. None of the CryIA toxins or HD-1 exhibited and toxicity towards A. fennica. Comparison of the observed FFD(50) of HD-1 with the FFD(50) expected on the basis of its crystal composition suggested a possible synergistic effect of the toxins in the two lymantriid species. Our results further illustrate the diversity of activity spectra of these highly related proteins and provide a data base for studies with forest insects to elucidate the molecular basis of toxin specificity.

The mechanism of sunlight-mediated inactivation of Bacillus thuringiensis crystals.

Detailed photostability studies were carried out using purified delta-endotoxin crystals from Bacillus thuringiensis subspecies HD-1 and HD-73. The mechanism and time course of sunlight inactivation was investigated by: (a) monitoring the tryptophan damage in the intact crystals by Raman spectroscopy, (b) amino acid analysis and (c) biological assays using insects. The results demonstrate that, for purified HD-1 or HD-73 crystals, the 300-380 nm range of the solar spectrum is largely responsible for bringing about crystal damage and consequent loss of toxicity. Purified Bacillus thuringiensis crystals that were exposed to fermentation liquor after cell lysis were more quickly degraded by sunlight than were crystals from cells that were lysed in water. This effect is attributed to adsorption of chromophores by crystals exposed to the fermenter liquor and the subsequent ability of these chromophores to act as photosensitizers. The importance of a photosensitization mechanism in crystal degradation was further emphasized by irradiating Bacillus thuringiensis crystals in vacuo. The latter crystals were found to be less damaged (20% tryptophan loss after 24 h irradiation by the solar spectrum) compared with crystals from the same sample irradiated in air (60% (60% tryptophan loss). Other methods of decreasing exposure of the crystals to oxygen, e.g. by using glycerol as a humectant, were also found to be successful in controlling photodamage. The results concerning photodegradation support a photosensitization mechanism involving the presence of exogenous (and possibly endogenous) chromophores which create singlet oxygen species upon irradiation by light.

Resonance Raman and Fourier transform infrared spectroscopic studies of the acyl carbonyl group in [3-(5-methyl-2-thienyl)acryloyl]chymotrypsin: evidence for artifacts in the spectra obtained by both techniques.

The acyl carbonyl group of [3-(5-methyl-2-thienyl)acryloyl]chymotrypsin (5MeTA-chymotrypsin) has been investigated by using both resonance Raman (RR) and Fourier transform infrared (FTIR) spectroscopies. The spectrum of the acyl-enzyme carbonyl group has been obtained as a function of pH over the range 3.0-10.0 in the RR experiments and over the range 3.4-7.6 (p2H) in the FTIR experiments. The carbonyl spectral profiles obtained by using FTIR spectroscopy are substantially different from the carbonyl profiles obtained by using RR spectroscopy. The FTIR spectra were obtained by subtracting the spectrum of the free enzyme from that of the acyl-enzyme. Use of the active-site inhibitor phenylmethanesulfonyl fluoride demonstrates that part of the intensity observed in the FTIR spectra of 5MeTA-chymotrypsin is due to a subtraction artifact giving rise to enzyme-associated bands, probably from peptide groups perturbed by substrate binding. The enzyme bands can be removed by subtracting the FTIR spectrum of 13C=O acyl-enzyme from that of 12C=O acyl-enzyme. Additionally, this procedure reveals that one of the acyl-enzyme carbonyl bands observed at 1727 cm-1 using RR spectroscopy is absent in the FTIR acyl-enzyme spectrum. However, a feature near 1720 cm-1 can be induced in the FTIR spectrum by actinic light in the near-UV region. Thus, it is proposed that the 1727 cm-1 RR carbonyl band results from a population of acyl-enzymes which is generated by exposure to the laser beam during RR data collection. When both the RR and FTIR data are adjusted to remove artifacts, they provide essentially identical carbonyl stretching profiles.