Monoclonal antibodies (MAbs) raised against Escherichia coli O6:H16 were screened against: 15 strains of E. coli and 19 non-E. coli bacteria. A MAb-luminescence assay using MAb-5.8, which shows no cross-reactions with...
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Monoclonal antibodies (MAbs) raised against Escherichia coli O6:H16 were screened against: 15 strains of E. coli and 19 non-E. coli bacteria. A MAb-luminescence assay using MAb-5.8, which shows no cross-reactions with non-E, coli bacteria, and a photon-counting television camera were developed for rapid enumeration of E. coli O16:H16 in water. The membrane filter that retained bacteria was boiled for 5 min in a buffer and incubated with biotinylated MAb5.8. Alter incubation with streptavidin-peroxidase conjugate, it was reacted with luminol-based reaction mixture. Luminous image and light intensity of the filter was recorded with a Biocell Counter. Levels of E. coli O6 higher than 7 x 10(3) CFU were detected by the MAb-luminescence assay when E. coli O6 was spotted onto the membrane filter. The sample that contained E, coli O6:H16 was filtered through a membrane filter, and the filter that retained bacteria was incubated on a filter paper soaked with nutrient: broth supplemented with 0.5% NaCl at 37'% for 6 h. The number of light emission points on the filter correlated well with initial E. coli O6:H16 counts within the range of 1 to 3 x 10(2) CFU. The correlation coefficient was 0.89.
A new cvy1I-type gene, cry1Id1, was cloned from a B. thuringiensis isolate, and its nucleotide sequence was determined. The deduced amino acid sequence of Cry1Id1 is 89.7%, 87.2%, and 83.4% identical to the Cry1Ia, Cr...
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A new cvy1I-type gene, cry1Id1, was cloned from a B. thuringiensis isolate, and its nucleotide sequence was determined. The deduced amino acid sequence of Cry1Id1 is 89.7%, 87.2%, and 83.4% identical to the Cry1Ia, Cry1Ib, and Cry1Ic proteins, respectively. The upstream sequence of the cry1Id1 structural gene was not functional as promoter in B. subtilis. The Cry1Id1 protein, purified from recombinant E. coli cells, had a toxicity comparable to that of Cry1Ia against Plutella xylostella, but it was significantly less active than Cry1Ia against Bombyx mori. Cry1Id1 was not active against the coleopteran insect, Agelastica coerulea.
Bacillus anthracis lethal factor (LF) is a 90-kDa zinc metalloprotease that plays an important role in the virulence of the organism. LF has previously been purified from Escherichia coli and Bacillus anthracis, The y...
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Bacillus anthracis lethal factor (LF) is a 90-kDa zinc metalloprotease that plays an important role in the virulence of the organism. LF has previously been purified from Escherichia coli and Bacillus anthracis, The yields and purities of these preparations were inadequate for crystal structure determination. In this study, the genes encoding wild-type LF and a mutated, inactive LF (LF-E687C) were placed in an E. coli-Bacillus shuttle vector so that LF was produced with the protective antigen (PA) signal peptide at its N-terminus. The resulting vectors, pSJ115 and pSJ121, express wild-type and mutated LF fusion proteins, respectively. Expression of the LF genes is under the control of the PA promoter and, during secretion, the PA signal peptide is cleaved to release the 90-kDa LF proteins. The wild-type and mutated LF proteins were purified from the culture medium using three chromatographic steps (Phenyl-Sepharose, Q-Sepharose, and hydroxyapatite). The purified proteins were greater than 95% pure and yields (20-30 mg/L) were higher than those obtained in other expression systems (1-5 mg/L). These proteins have been crystallized and are being used to solve the crystal structure of LF. Their potential use in anthrax vaccines is also discussed. (C) 2000 Academic Press.
Both the bacterium Photorhabdus luminescens alone and its symbiotic Photorhabdus-nematode complex are known to be highly pathogenic to insects, The nature of the insecticidal activity of Photorhabdus bacteria was inve...
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Both the bacterium Photorhabdus luminescens alone and its symbiotic Photorhabdus-nematode complex are known to be highly pathogenic to insects, The nature of the insecticidal activity of Photorhabdus bacteria was investigated for its potential application as an insect control agent. It was found that in the fermentation broth of P. luminescens strain W-14, at least two proteins, toxin A and toxin B, independently contributed to the oral insecticidal activity against Southern corn rootworm, Purified toxin A and toxin B exhibited single bands on native polyacrylamide gel electrophoresis and two peptides of 208 and 63 kDa on SDS-polyacrylamide gel electrophoresis. The native molecular weight of both the toxin A and toxin B was determined to be approximately 860 kDa, suggesting that they are tetrameric, NH2-terminal amino acid sequencing and Western analysis using monospecific antibodies to each toxin demonstrated that the two toxins were distinct but homologous, The oral potency (LD,,) of toxin A and toxin B against Southern corn rootworm larvae was determined to be similar to that observed with highly potent Bt toxins against lepidopteran pests, In addition, it was found that the two peptides present in toxin B could be processed in vitro from a 281-kDa protoxin by endogenous P. luminescens proteases. Proteolytic processing was shown to enhance insecticidal activity.
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