Response of Zymomonas mobilis levansucrase activity to sodium chloride

An activation of levansucrase-catalysed levan formation by NaCl, KCl and Na2 SO4 (0.03–0.7 M) was observed using cell-free extract of Zymomonas mobilis. A sigmoidal response of the rate of levansucrase-catalysed reaction to the sucrose concentration was significantly reduced in the presence of salts the Hill coefficient 2.10 and 1.0–1.2 respectively), possibly, due to the heterotropic activation of levansucrase as an allosteric enzyme. © Rapid Science Ltd. 1998     

Towards the characterization of squalene synthase activity in extracts of Zymomonas mobilis

Abstract Extracts of Zymomonas mobilis in the presence of NADPH converted tritium-labelled farnesyl diphosphate (FPP) into squalene, resulting from the activity of squalene synthase, as well as diploptene and diplopterol, derived from further squalene cyclisation. An unidentified isoprenoid representing up to 70% of the conversion products of FPP and different from presqualene alcohol was also formed, even in the absence of NADPH. Addition of squalestatin 1, an inhibitor of squalene synthase, blocked biosynthesis from FPP of the three former triterpenes, in accordance with the role of squalene synthase in their formation, as well as that of the unknown compound.     

The progress in insect cross‐resistance among Bacillus thuringiensis toxins

Bt crop pyramids produce two or more Bt proteins active to broaden the spectrum of action and to delay the development of resistance in exposed insect populations. The cross‐resistance between Bt toxins is a vital restriction factor for Bt crop pyramids, which may reduce the effect of pyramid strategy. In this review, the status of the cross‐resistance among more than 20 Bt toxins that are most commonly used against 13 insect pests was analyzed. The potential mechanisms of cross‐resistance are discussed. The corresponding measures, including pyramid RNA interference and Bt toxin, “high dose/refuge,” and so on are advised to be taken for adopting the pyramided strategy to delay the Bt evolution of resistance and control the target pest insect.     

Lectin activity of Bacillus thuringiensis parasporal inclusion proteins

Parasporal inclusion proteins from a total of 151 Bacillus thuringiensis strains, consisting of 139 Japanese isolates and the type strains of 12 H serovars, were screened for haemagglutination (HA) activity against sheep erythrocytes. Of 58 B. thuringiensis strains with HA activity, nine strains exhibited high activity and the remaining 49 strains were moderately active. The strains with high HA activity were derived from phylloplanes and soils of five geographically different localities, and belonged to H serovars kurstaki and other undefined serotype(s). The HA activities in the four selected strains were generated only when alkali-solubilised parasporal inclusion proteins were proteolytically processed. Furthermore, the lectin activity of the four strains was strongly inhibited by preincubation with N-acetylgalactosamine. The lectin-producing B. thuringiensis strains were heterogeneous in other biological activities of parasporal inclusions: insecticidal activity and cytocidal action on human leukaemia T cells.     

Effect of insect larval midgut proteases on the activity of Bacillus thuringiensis Cry toxins

To test the possibility that proteolytic cleavage by midgut juice enzymes could enhance or inhibit the activity of Bacillus thuringiensis insecticidal toxins, once activated, the effects of different toxins on the membrane potential of the epithelial cells of isolated Manduca sexta midguts in the presence and absence of midgut juice were measured. While midgut juice had little effect on the activity of Cry1Aa, Cry1Ac, Cry1Ca, Cry1Ea, and R233A, a mutant of Cry1Aa from which one of the four salt bridges linking domains I and II of the toxin was eliminated, it greatly increased the activity of Cry1Ab. In addition, when tested in the presence of a cocktail of protease inhibitors or when boiled, midgut juice retained almost completely its capacity to enhance Cry1Ab activity, suggesting that proteases were not responsible for the stimulation. On the other hand, in the absence of midgut juice, the cocktail of protease inhibitors also enhanced the activity of Cry1Ab, suggesting that proteolytic cleavage by membrane proteases could render the toxin less effective. The lower toxicity of R233A, despite a similar in vitro pore-forming ability, compared with Cry1Aa, cannot be accounted for by an increased susceptibility to midgut proteases. Although these assays were performed under conditions approaching those found in the larval midgut, the depolarizing activities of the toxins correlated only partially with their toxicities.     

Immunological properties of entomocidal protein of Bacillus thuringiensis and its insecticidal activity

The immunological properties of the proteinaceous component of the parasporal crystal (δ-endotoxin) of Bacillus thuringiensis var. kurstaki were analyzed by rocket immunoelectrophoresis. Two antisera, one against the k-l-type crystal containing two components, and the other against the k-73-type crystal containing one component, were made in rabbits. The antigens consisting of purified and dissociated crystals were run in electrophoresis with these two antisera. The ratio between the two peak heights of precipitin lines, which were formed by the dissociated crystal of one B. thuringiensis isolate in two antisera, was compared with the ratios of other isolates under identical conditions. The difference in the ratio reflected a difference in the structure of the crystal component and correlated closely with the insecticidal activity spectrum. This method can be used to evaluate a newly isolated B. thuringiensis, and it can further differentiate the isolates which have been classified as one serotype.     

Overexpression of extracellular sucrase (SacC) of Zymomonas mobilis in Escherichia coli

Abstract The extracellular sucrase (SacC) gene of Zymomonas mobilis was overexpressed in Escherichia coli BL21 using the T7 polymerase expression system. A low cell density induction method was designed to have maximum expression, and the conditions (IPTG concentration, ampicillin addition) were optimised to overexpress to the level of more than 60% of the total cellular protein representing SacC protein.     

Ultrastructure of the bacterial spores and crystals of various Bacillus thuringiensis serotypes

Spores and crystals of various Bacillus thuringiensis serotypes were studied by electron microscopy. The serotypes were shown to differ in the fine structure of the exosporium and crystals. The packing parameters of morphological subunits were determined by analysing electron photomicrographs by the technique of optical diffraction and filtration. Some of the strains are characterised by hypersynthesis of surface spore structures. The individual morphological properties of crystals can be used for their differentiation.     

Characterization of novel Brazilian Bacillus thuringiensis strains active against Spodoptera frugiperda and other insect pests

Brazilian strains of Bacillus thuringiensis, namely S701, S764 and S1265 were analysed regarding their cry gene and protein contents, crystal type, and activity against larvae of the lepidopteran fall armyworm (Spodoptera frugiperda Smith), the velvet caterpillar (Anticarsia gemmatalis), the dipterans (Culex quinquefasciatus and Aedes aegypti) and the coleopteran (Tenebrio molitor). The LC₅₀ of the strains against second instar larvae of S. frugiperda or A. gemmatalis revealed a high potency against those insect species. The spore–crystal mixtures of the isolates were analysed by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and showed similar protein pattern as the B. thuringiensis subsp. kurstaki strain HD‐1 (proteins approximately 130 and 65 kDa) for isolates S701 and S764, respectively, and only one major protein of approximately 130 kDa for isolate S1265. The polymerase chain reaction (PCR) using total DNA of the isolates and general and specific primers showed the presence of cry1Aa, cry1Ac, cry1Ia and cry2Ab genes in the two isolates serotyped as B. thuringiensis kurstaki (S701 and S764) and the presence of cry1D and cry2Ad in B. thuringiensis morrisoni S1265 strain. Scanning electron microscopy of strains S701 and S764, showed the presence of bipyramidal, cuboidal and round crystals, like in strain HD‐1 and bipyramidal and round crystals like in strain S1265.     

苏云金杆菌杀虫蛋白的多样性

苏云金杆菌是生物防治中应用最为广泛的一种杀虫剂,其杀虫蛋白具有广泛的多样性。本文就苏云金杆菌杀虫蛋白的基因、基因分布、杀虫蛋白结构以及作用机制的多样性进行了概述。     

Toxicity of Bacillus thuringiensis entomocidal protein toward cultured insect tissue

The entomocidal protein from crystalline inclusion bodies of Bacillus thuringiensis can be bioassayed in vitro using cultured insect tissue. Larval cells of the spruce budworm, Choristoneura fumiferana, are damaged by enzyme-digested (activated) protein isolated from B. thuringiensis crystals. Measurement of toxicity is accomplished by detection of adenosine triphosphate (ATP) in treated cultures using firefly bioluminescence. The ATP content of toxin-treated tissue is inversely proportional to the amount of toxin added. Tissue cells from the spruce budworm exhibited maximum susceptibility to activated δ-endotoxin after 120 hr incubation. Probit analysis of tissue ATP response to toxin dose indicated 50% of the cells were damaged by 14.6 μg or less of toxin protein per 2 × 10⁵ insect tissue cells. Activated δ-endotoxin was entomocidal to insects as well, as detemined by mortality studies with second-instar larvae of the European corn borer. Electron microscopic observations of insect tissue treated with activated δ-endotoxin protein for 60 min revealed massive outer membrane disruption and subsequent loss of cytoplasmic constituents, accompanied by swelling of the nuclear membrane.     

Batch fermentation kinetics of sugar beet molasses by Zymomonas mobilis

A new osmotolerant mutant strain of Zymomonas mobilis was successfully used for ethanol production from beet molasses. Addition of magnesium sulfate to hydrolyzed molasses allowed repeated growth without the need of yeast extract addition. The kinetics and yields parameters of fermentation on media with different molasses concentrations were calculated. The anabolic parameters (specific growth rate, mu, and biomass yield, Y(X/S)) were inhibited at elevated molasses concentrations while the catabolic parameters (specific ethanol productivity, q(p), and ethanol yield, Y(p/s)) were not significantly affected. In addition to ethanol and substrate inhibition, osmotic pressure effects can explain the observed results.     

Purification of the mosquitocidal and cytolytic proteins of Bacillus thuringiensis subsp. israelensis

Two proteins from parasporal crystals of Bacillus thuringiensis subsp. israelensis were purified to electrophoretic homogeneity by gel filtration and anion-exchange chromatography. The larger of the two proteins (molecular weight, 68,000) was not cytolytic, whereas the smaller protein (molecular weight, 28,000) was highly cytolytic when assayed against rat erythrocytes. When these proteins were assayed against larvae of the yellow fever mosquito, Aedes aegypti, the larger protein was at least 100-fold more toxic than the smaller protein. Although proteolytic activity was not detected in solubilized crystals nor in purified protein preparations, the toxin (molecular weight, 68,000) was readily degraded to smaller, nontoxic molecules, even when maintained at 4 degrees C. Mixtures of the two purified proteins were significantly more toxic to mosquito larvae than was either protein alone. Thus, it is likely that both the mosquitocidal and the cytolytic protein play roles in the overall insecticidal action of the parasporal crystal produced by this bacterium.     

Bacillus thuringiensis Vip3 mutant proteins: Insecticidal activity and trypsin sensitivity

Vip3 is a novel insect toxin isolated from Bacillus thuringiensis (Bt), and could be used as an alternative toxin for Bt δ-endotoxins for transgenic insect control. Vip3 mutants with deletion, addition or mutations at the very end of the C-terminus were generated. The deletion and addition of a few amino acid residues at the C-terminus totally abolished the insecticidal activity. The mutation of the last two residues from IK to LG also resulted in the total loss of its insecticidal activity; however, the mutation from IK to LR increased its activity substantially against beet armyworm. Interestingly, all the inactive mutants were found to be highly sensitive to trypsin digestion, while the active mutant generated a trypsin-resistant polypeptide of 62-kDa upon trypsin digestion. However, this 62-kDa polypeptide expressed in E. coli from the 5' end truncated vip3 gene was biologically inactive and sensitive to trypsin digestion. Thus, the N-terminal part of the protein is required to form the 62-kDa trypsin resistant core. This study suggested that the 62-kDa peptide core could be a hallmark of active insecticidal Vip3 proteins.