Showing papers on "Bacillus thuringiensis published in 1991"

Crystal structure of insecticidal delta-endotoxin from Bacillus thuringiensis at 2.5 A resolution.

Abstract: The structure of the delta-endotoxin from Bacillus thuringiensis subsp. tenebrionis that is specifically toxic to Coleoptera insects (beetle toxin) has been determined at 2.5 A resolution. It comprises three domains which are, from the N- to C-termini, a seven-helix bundle, a three-sheet domain, and a beta sandwich. The core of the molecule encompassing all the domain interfaces is built from conserved sequence segments of the active delta-endotoxins. Therefore the structure represents the general fold of this family of insecticidal proteins. The bundle of long, hydrophobic and amphipathic helices is equipped for pore formation in the insect membrane, and regions of the three-sheet domain are probably responsible for receptor binding.

Comparative analysis of Bacillus anthracis, Bacillus cereus, and related species on the basis of reverse transcriptase sequencing of 16S rRNA

Abstract: The primary structures of the 16S rRNAs of Bacillus anthracis, Bacillus cereus, Bacillus mycoides, and Bacillus thuringiensis were determined by using the reverse transcription-dideoxy sequencing method. All of the strains exhibited very high levels of sequence similarity T (T = type strain) by only a single nucleotide. The 16S rRNA sequences of B. mycoides and B. thuringiensis differed from each other and from the sequences of B. anthracis and B. cereus by four to nine nucleotides.

Resistance to the Bacillus thuringiensis bioinsecticide in a field population of Plutella xylostella is due to a change in a midgut membrane receptor.

Abstract: The biochemical mechanism for resistance to Bacillus thuringiensis crystal proteins was studied in a field population of diamondback moths (Plutella xylostella) with a reduced susceptibility to the bioinsecticidal spray. The toxicity and binding characteristics of three crystal proteins [CryIA(b), CryIB, and CryIC] were compared between the field population and a laboratory strain. The field population proved resistant (greater than 200-fold compared with the laboratory strain) to CryIA(b), one of the crystal proteins in the insecticidal formulation. Binding studies showed that the two strains differ in a membrane receptor that recognizes CryIA(b). This crystal protein did not bind to the brush-border membrane of the midgut epithelial cells of the field population, either because of strongly reduced binding affinity or because of the complete absence of the receptor molecule. Both strains proved fully susceptible to the CryIB and CryIC crystal proteins, which were not present in the B. thuringiensis formulation used in the field. Characteristics of CryIB and CryIC binding to brush-border membranes of midgut epithelial cells were virtually identical in the laboratory and the field population.

Prediction of insecticidal activity of Bacillus thuringiensis strains by polymerase chain reaction product profiles.

Abstract: A rapid analysis of Bacillus thuringiensis strains predictive of insecticidal activity was established by using polymerase chain reaction (PCR) technology. Primers specific to regions of high homology within genes encoding three major classes of B. thuringiensis crystal proteins were used to generate a PCR product profile characteristic of each insecticidal class. Predictions of insecticidal activity were made on the basis of the electrophoretic patterns of the PCR products. Included in the screen were PCR primers specific for cryI, cryIII, and cryIV genes, which are insecticidal for lepidopterans, coleopterans, and dipterans, respectively. Known B. thuringiensis strains as well as unidentified strains isolated from soil and insect cadavers were analyzed by PCR. Small amounts of crude sample lysates were assayed in a single PCR reaction containing 12 to 20 primers capable of distinguishing between the different insecticidal genes. Insecticidal activity predicted by the PCR screen was found to correspond with the insecticidal activity of insect bioassays. In addition to identifying strains with known insecticidal genes, the PCR screen can identify strains with altered electrophoretic patterns containing potentially novel genes. Images

The phylloplane as a source of bacillus thuringiensis variants

Abstract: Novel variants of Bacillus thuringiensis were isolated from the phylloplane of deciduous and conifer trees as well as of other plants. These isolates displayed a range of toxicity towards Trichoplusia ni. Immunoblot and toxin protein analysis indicate that these strains included representatives of the three principal B. thuringiensis pathotypes active against larvae of the orders Lepidoptera, Diptera, and Coleoptera. We propose that B. thuringiensis be considered part of the common leaf microflora of many plants.

Identification of putative insect brush border membrane-binding molecules specific to Bacillus thuringiensis delta-endotoxin by protein blot analysis.

Abstract: Binding sites for insecticidal toxins of Bacillus thuringiensis are located in the brush border membranes of insect midguts Two approaches were used to investigate the interactions of B thuringiensis subsp kurstaki HD-73 CryIA(c) toxin with brush border membrane vesicles from sensitive and naturally resistant insects: 125I-toxin-vesicle binding assays and protein blots probed with 125I-CryIA(c) toxin In bioassays, Manduca sexta and Heliothis virescens larvae were highly sensitive, Helicoverpa zea larvae were moderately sensitive, and Spodoptera frugiperda larvae were resistant to CryIA(c) toxin Studies of binding of 125I-CryIA(c) toxin to brush border membrane vesicles from the larval midguts revealed that all insects tested had high-affinity, saturable binding sites Significantly, S frugiperda larvae bind but are not killed by CryIA(c) toxin Labeled CryIA(c) toxin incubated with protein blots identifies a major binding molecule of 120 kDa for M sexta and 148 kDa for S frugiperda H virescens and H zea are more complex, containing 155-, 120-, 103-, 90-, and 63-kDa proteins as putative toxin-binding molecules H virescens also contains a minor toxin-binding protein of 81 kDa These experiments provide information that can be applied toward a more detailed characterization of B thuringiensis toxin-binding proteins

Bacillus thuringiensis strains and their genes encoding insecticidal toxins

Abstract: Two new Bacillus thuringiensis strains, which are deposited at the DSM under accession numbers 5870 and 5871, produce new crystal proteins during sporulation that are toxic to Coleoptera and that are encoded by new genes. The crystal proteins contain protoxins, which can yield toxins as trypsin-digestion products. A plant, the genome of which is transformed with a DNA sequence that comes from either one of the strains and encodes an insecticidally effective portion of its respective protoxin or encodes its respective toxin, is resistant to Coleoptera. Each strain, itself, or its crystals, crystal proteins, protoxin, toxin and/or insecticidally effective protoxin portion can be used as the active ingredient in an insecticidal composition for combatting Coleoptera.

Analysis of unstable RNA transcripts of insecticidal crystal protein genes of Bacillus thuringiensis in transgenic plants and electroporated protoplasts.

Abstract: We have examined expression of several insecticidal crystal protein (ICP) genes of Bacillus thuringiensis in transgenic tobacco plants and electroporated carrot protoplasts. We determined that low levels of lepidopteran toxin cryIA(b) ICP gene expression in plants and electroporated carrot cells is due to RNA instability. We used a series of 3′ deleted cryIA(b) constructs directed by the cauliflower mosaic virus 35S promoter to demonstrate that this instability is minimally contained in the first 579 bases of the gene in both systems. This instability may result from 5′ → 3′ as well as 3′ → 5′ RNA metabolism. The coleopteran toxic cryIIIA gene was also examined in electroporated carrot cells, and found to be poorly expressed. A model for improvement of ICP RNA stability in plants is presented.

Isolation and characterization of a novel insecticidal crystal protein gene from Bacillus thuringiensis subsp. aizawai.

Abstract: Bacillus thuringiensis subsp. aizawai EG6346, a novel grain dust isolate, was analyzed by Southern blot hybridization for its insecticidal crystal protein (ICP) gene profile. Strain EG6346 lacks previously characterized cryIA ICP genes yet does possess novel cryI-related gene sequences. A recombinant genomic plasmid library was constructed for strain EG6346 in Escherichia coli. One recombinant plasmid, pEG640, isolated from the library contained a novel ICP gene on a 5.7-kb Sau3A insert. The sequence of this gene, designated cryIF, was related to, but distinct from, the published sequences for other cryI genes. A second novel cryI-related sequence was also located on pEG640, approximately 500 bp downstream from cryIF. Introduction of cryIF into a Cry- B. thuringiensis recipient strain via electroporation enabled sufficient production of CryIF protein for quantitative bioassay analyses of insecticidal specificity. The CryIF crystal protein was selectively toxic to a subset of lepidopteran insects tested, including the larvae of Ostrinia nubilalis and Spodoptera exigua.

Managing Resistance to Bacillus thuringiensis: Lessons from the Diamondback Moth (Lepidoptera: Plutellidae

Abstract: Laboratory selection increased resistance to B. thuringiensis in three strains established from a moderately resistant field population of diamondback moth, Plutella xylostella (L.). Five generations of laboratory selection caused 5- to 7-fold increases in LC50, resulting in 150- to 190-fold resistance compared with a susceptible laboratory colony. Nine generations of selection produced 430- to 820-fold resistance. In contrast, five foliar applications of B. thuringiensis in the field did not increase the LC50 of a moderately resistant population. Field-selected resistance to B. thuringiensis declined slowly in the absence of treatments. The rapid response to laboratory selection shows intrapopulation genetic variation in susceptibility to B. thuringiensis and suggests that intense selection may produce much higher levels of resistance to B. thuringiensis than those previously reported from the field. We hypothesize that resistance increased faster in the laboratory than in the field because selection intensity was lower in the field. Because susceptibility was not restored quickly when treatments were discontinued, rotations may not be especially effective for managing resistance to B. thuringiensis in diamondback moth. Field populations of diamondback moth developed resistance to a commercial formulation containing a mixture of B. thuringiensis toxins, an event that raises doubts about the ability of mixtures to retard resistance development. Extensive and intensive exposure of pests to B. thuringiensis toxins through transgenic crop plants or other tactics may cause widespread pest resistance. We urge judicious use of B. thuringiensis to conserve its efficacy.

The solubility of inclusion proteins from Bacillus thuringiensis is dependent upon protoxin composition and is a factor in toxicity to insects.

Abstract: Bacillus thuringiensis subsp. aizawai HD133 is one of several strains particularly effective against Plodia interpunctella selected for resistance to B. thuringiensis subsp. kurstaki HD1 (Dipel). B. thuringiensis subsp. aizawai HD133 produces inclusions containing three protoxins, CryIA(b), CryIC, and CryID, and the CryIC protoxin has been shown to be active on resistant P. interpunctella as well as on Spodoptera larvae. The CryIA(b) protoxin is very similar to the major one in B. thuringiensis subsp. kurstaki HD1, and as expected, this protoxin was inactive on resistant P. interpunctella. A derivative of B. thuringiensis subsp. aizawai HD133 which had been cured of a 68-kb plasmid containing the cryIA(b) gene produced inclusions comprising only the CryIC and CryID protoxins. Surprisingly, these inclusions were much less toxic for resistant P. interpunctella and two other Lepidoptera than those produced by the parental strain, whereas the soluble protoxins from these strains were equally effective. In contrast, inclusions from the two strains were about as active as soluble protoxins for Spodoptera frugiperda larvae, so toxicity differences between inclusions may be due to the solubilizing conditions within particular larval guts. Consistent with this hypothesis, it was found that a higher pH was required to solubilize protoxins from inclusions from the plasmid-cured strain than from B. thuringiensis subsp. aizawai HD133, a difference which is probably attributable to the absence of the CryIA(b) protoxin in the former. The interactions of structurally related protoxins within an inclusion are probably important for solubility and are thus another factor in the effectiveness of B. thuringiensis isolates for particular insect larvae.

Modified bacillus thuringiensis insecticidal-crystal protein genes and their expression in plant cells

Abstract: A DNA fragment, encoding all or an insecticidally-effective part of a Bt crystal protein, is modified by changing A and T sequences to corresponding G and C sequences encoding the same amino acids.

Deletion by in vivo recombination shows that the 28-kilodalton cytolytic polypeptide from Bacillus thuringiensis subsp. israelensis is not essential for mosquitocidal activity.

Abstract: The cytA gene encoding the 28-kDa polypeptide of Bacillus thuringiensis subsp. israelensis crystals was disrupted in the 72-MDa resident plasmid by in vivo recombination, thus indicating that homologous recombination occurs in B. thuringiensis. The absence of the 28-kDa protein in B. thuringiensis did not affect the crystallization of the other toxic components of the parasporal body (68-, 125-, and 135-kDa polypeptides). The absence of the 28-kDa protein abolished the hemolytic activity of B. thuringiensis subsp. israelensis crystals. However, the mosquitocidal activity of the 28-kDa protein-free crystals did not differ significantly from that of the wild-type crystals when tested on Aedes aegypti and Culex pipiens larvae. The 28-kDa protein contributed slightly to the toxicity to Anopheles stephensi larvae. This indicates that the 28-kDa protein is not essential for mosquitocidal activity, at least against the three species tested.

N-acetyl galactosamine is part of the receptor in insect gut epithelia that recognizes an insecticidal protein from Bacillus thuringiensis.

Abstract: Proteins synthesized by the bacterium Bacillus thuringiensis are potent insecticides. When ingested by susceptible larvae they rapidly lyse epithelial cells lining the midgut. In vitro the toxins lyse certain insect cell lines and show saturable, high-affinity binding to brush-border membrane vesicles (BBMVs) prepared from insect midguts. We observed that the sugar N-acetyl galactosamine (GalNAc) specifically decreased the cytolytic activity of a CryIA(c) toxin towards Choristoneura fumiferana CF1 cells, completely abolished toxin binding to Manduca sexia BBMVs, partially inhibited binding to Heliothis virescens BBMVs and had no apparent effect on binding to Pieris brassicae BBMVs. In ligand blotting experiments the toxin bound proteins of 120 kDa in M. sexta, 125 kDa in P. brassicae and numerous proteins in H. zea. Toxin binding to these proteins was specifically inhibited by GalNAc. The toxin binding proteins of M. sexta and H. zea also bound the lectin soybean agglutinin. Taken together these findings suggest that N-acetyl galactosamine might be a component of a CryIA(c) toxin receptor of CF1 cells and of at least two of the insects tested.

Binding of Bacillus thuringiensis proteins to a laboratory-selected line of Heliothis virescens.

Abstract: A laboratory-selected colony of Heliothis virescens displaying a 20- to 70-fold level of resistance to Bacillus thuringiensis proteins was evaluated to identify mechanism(s) of resistance. Brush-border membrane vesicles were isolated from larval midgut epithelium from the susceptible and resistant strains of H. virescens. Two B. thuringiensis proteins, CryIA(b) and CryIA(c), were iodinated and shown to specifically bind to brush-border membrane vesicles of both insect strains. Multiple changes in the receptor-binding parameters were seen in the resistant strain as compared with the susceptible strain. A 2- to 4-fold reduction in binding affinity was accompanied by a 4- to 6-fold increase in binding-site concentration for both proteins. Although these two B. thuringiensis proteins competed for the same high-affinity binding site, competition experiments revealed different receptor specificity toward these proteins in the resistant H. virescens line. The H. virescens strains were not sensitive to a coleopteran-active protein, CryIIIA, nor did these proteins compete with the CryIA proteins for binding. Complexity of the mechanism of resistance is consistent with the complex mode of action of B. thuringiensis proteins.

Efficient transformation of Bacillus thuringiensis requires nonmethylated plasmid DNA.

Abstract: The transformation efficiency of Bacillus thuringiensis depends upon the source of plasmid DNA. DNA isolated from B. thuringiensis, Bacillus megaterium, or a Dam- Dcm- Escherichia coli strain efficiently transformed several B. thuringiensis strains, B. thuringiensis strains were grouped according to which B. thuringiensis backgrounds were suitable sources of DNA for transformation of other B. thuringiensis strains, suggesting that B. thuringiensis strains differ in DNA modification and restriction. Efficient transformation allowed the demonstration of developmental regulation of cloned crystal protein genes in B. thuringiensis.

Effects of Bacillus thuringiensis and HD-73 delta-endotoxin on growth, behavior, and fitness of susceptible and toxin-adapted strains of Heliothis virescens (Lepidoptera : Noctuidae)

Abstract: A commercial formulation of Bacillus thuringiensis var. kurstaki Berliner (Dipel 2X) and purified HD-73 delta-endotoxin were evaluated for effects on the growth, behavior, and fitness of susceptible and toxin-adapted strains of Heliothis virescens (F.) by incorporation of the materials into artificial diets at varying concentrations. Neonate larvae were placed in experimental arenas where they could choose a diet containing toxin and a control diet, or were placed in arenas where the only diet available contained a toxin. The difference between the strains in growth and survival was much more pronounced when larvae could not choose to feed on the control diet. Both strains avoided moderate and high concentrations of the toxins. At the lowest concentration of HD-73, neither strain avoided the diet that contained the endotoxin. At the two lowest concentrations of Dipel 2X, only the susceptible strain avoided the diet containing Dipel. Data on growth rate, survival, and fecundity of the two strains reared on HD-73 delta-endotoxin were used to set parameters of a Single-locus genetic model to predict the rate at which H. virescens populations would adapt to B. thuringiensis var. kurstaki or its endotoxin when exposed to it in choice and no-choice situations. Results from the model generally predict much more rapid adaptation in no-choice situations.

Phosphatidylinositol-specific phospholipases C from Bacillus cereus and Bacillus thuringiensis

Abstract: Publisher Summary Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus and Bacillus thuringiensis catalyzes the cleavage of the sn -3 phosphodiester bond of phosphatidylinositol (PI). Bacillus cereus and B. thuringiensis are closely related rod-shaped gram-positive bacteria that are nonpathogenic to man. The enzymes are excreted in relatively large quantities across the single cell membrane into the growth medium. This facilitates the purification of PI-PLC as intracellular proteins are removed with the cells during an initial centrifugation step. PI-PLC is isolated from the supernatants of B. cereus and B. thuringiensis cultures in milligram quantities. The final product is homogeneous on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), exhibits high specific activity, and is used to raise monoclonal antibodies and provide polypeptide sequence information for the cloning and DNA sequencing of PI-PLC from B. cereus . The B. cereus or B. thuringiensis culture is cooled to 4°, and the cells are removed by centrifugation for 30 min at 13,000 g. Solid ammonium sulfate is added slowly to the supernatant to 90% saturation (576 g/liter), and the solution is kept overnight at 4°. The purity of the final enzyme preparations is generally better than 95%, with similar specific activities for the B. cereus and B. thuringiensis enzymes in the range of 1200 to 1500 units/mg. During the early steps of the purification, the specific enzyme activities are affected by the presence of colored contaminants that contribute strongly in the protein determinations. These contaminants are completely removed by the final Phenyl-Sepharose step.

Toxicity of Bacillus thuringiensis to laboratory populations of the olive fruit fly (Dacus oleae).

Abstract: A survey of Bacillus thuringiensis recovered from the environments of olive groves in Greece was carried out. Of 80 soil samples, 24 were found to contain B. thuringiensis with parasporal crystal inclusions; these were tested for toxicity against the olive fruit fly (Dacus oleae). Mortality levels of larvae caused by the different isolates varied from 7 to 87%. Higher levels of mortality were observed if a mixture of relatively pure crystals and spores was used compared with the mortality resulting from either fraction alone. We were able to show that the toxicity of the most active isolate is likely to be specific for D. oleae.

Plant defenses: Chlorogenic acid and polyphenol oxidase enhance toxicity ofBacillus thuringiensis subsp.kurstaki toHeliothis zea.

Abstract: Two chemicals implicated in resistance of the tomato plant, chlorogenic acid and polyphenol oxidase, are known to form orthoquinones in damaged plant tissue. Orthoquinones have been reported to alkylate -NH2 and -SH groups of proteins and amino acids, altering solubility, digestibility, and, for some pathogenic viruses, infectivity. Here we explore effects of quinone alkylation on toxicity of an important microbial insecticide,Bacillus thuringiensis subsp.kurstaki (BTk), to larvalHeliothis zea. BTk incubated with these phytochemicals and fed to larvae was more toxic than untreated BTk. Similar but less dramatic results arose when BTk was incubated with polyphenol oxidase alone. Digestibility experiments suggest that alkylation enhanced the solubilization and/or proteolysis of crystal protein in vivo. Implications of our results for compatibility of BTk with host-plant resistance and biological control are discussed.

Bacillus thuringiensis isolate active against dipteran pests

Abstract: Disclosed and claimed is a novel Bacillus thuringiensis isolate designated B.t. PS192N1 which has dipteran activity. Thus, this isolate, or mutants thereof, can be used to control such insect pests. Further, genes encoding novel δ-endotoxins can be removed from the isolate and transferred to other host microbes, or plants. Expression of the δ-endotoxins in such hosts results in the control of susceptible insect pests in the environment of such hosts.

Bacillus thuringiensis Berliner and Beauveria bassiana (Balsamo) Vuillimen for European corn borer control: program for immediate and season-long suppression.

Abstract: Combinations of Bacillus thuringiensis Berliner subspecies kurstaki and Beauveria bassiana (Balsamo) Vuillimen formulated on granules were applied separately and in combination to field corn to suppress larval populations of the European corn borer, Ostrinia nubilalis (Hubner). Research was conducted in 2 separate years. Laboratory-reared O. nubilalis eggs or larvae were placed on the plant during either the whorl stage (V6) or the pollen-shedding stage (R1) to simulate first- and second-generation O. nubilalis oviposition periods, respectively. Efficacy was determined by measuring the length of European corn borer tunnels in the corn stalk at harvest time. In the 1st year, first generation, and second generation (of 2nd year) B. thuringiensis and B. bassiana alone and in combination caused significant reductions in tunneling compared with that in the check populations. There were no significant differences in tunneling between any treatments in the second-generation study of year 1. Bacillus thuringiensis and B. bassiana were independent of each other in their suppression of insects. Tunneling by the naturally occurring second-generation larvae (year 2) was recorded to determine if B. thuringiensis and B. bassiana applied in the V6 stage persisted in the plant. Pith samples were excised from nodal plates 7–10 of the corn stalk to determine the incidence of B. bassiana. There was a significant correlation (r = −0.376) (P≤0.05) between the occurrence of B. bassiana in the corn plant and tunneling by second-generation larvae. These data indicate that B. bassiana placed in the whorl of the corn plant may provide season-long suppression of O. nubilalis.

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

Abstract: 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.

Sequence of an operon containing a novel δ-endotoxin gene from Bacillus thuringiensis

Abstract: An insect larval toxin designated CryII is produced by several subspecies of Bacillus thuringiensis and differs from the other major δ-endotoxins in these bacteria in its size, toxicity profile and presence as part of an operon with three open reading frames (ORF). Such an operon from a novel B. thuringiensis isolate has been cloned and differs from one previously characterized in the following ways: (a) the size and number of amino acid repeats in one of the ORFs; (b) the smaller size of the CryII protoxin and the presence of a unique 110-kDa CryII-related antigen; and (c) high larvicidal activity for a particular Lepidopteran but low activity for a Dipteran. Various subclones of this operon were introduced into a plasmid-free B. thuringiensis strain and only the cryII gene was found to be necessary for protoxin accumulation.

Effect of a 20-kilodalton protein from Bacillus thuringiensis subsp. israelensis on production of the CytA protein by Escherichia coli.

Abstract: CytA, a 27-kDa cytolytic crystal protein of Bacillus thuringiensis subsp. israelensis, is produced only at very low levels by recombinant Escherichia coli cells unless a 20-kDa B. thuringiensis subsp. israelensis protein is also present (K. M. McLean and H. R. Whiteley, J. Bacteriol. 169:1017-1023, 1987; L. F. Adams, J. E. Visick, and H. R. Whiteley, J. Bacteriol. 171:521-530, 1989). However, the data reported here demonstrate that the 20-kDa protein is not required for high-level CytA production in E. coli strains carrying mutations in rpoH, groEL, or dnaK, all of which affect the proteolytic ability of the cells. The 20-kDa protein also increases the amount of CryIVD (another B. thuringiensis subsp. israelensis crystal protein) and LacZX90 (a mutant of beta-galactosidase) made by E. coli. The latter phenomenon is attributable to an increase in the half-life of LacZX90, suggesting that the 20-kDa protein may stabilize this protein. The effect of the 20-kDa protein was also examined in vitro and in a T7 RNA polymerase expression system, and the possible significance of these results for the timing of proteolysis and of 20-kDa protein activity is discussed. Finally, the ability of a single antibody to coimmunoprecipitate CytA and the 20-kDa protein from E. coli extracts provides evidence for a protein-protein interaction that may be related to the mechanism of action of the 20-kDa protein.