Degradation of the insecticidal toxin produced by Bacillus thuringiensis var. kurstaki by extracellular proteases produced by Chrysosporium sp.

AIMS: Some Cry proteins produced by the soil bacterium Bacillus thuringiensis (Bt) or by transgenic Bt plants persist in agricultural soils for an extended period of time, which may pose a hazard for nontarget soil organisms. The aims of our study were to screen for soil fungi capable of degrading the Cry1Ac toxin and to identify the mechanisms that lead to the inactivation of this protein. METHODS AND RESULTS: Of the eight fungal strains screened, only one, Chrysosporium sp., was found to produce extracellular proteases capable of degrading the 66-kDa Cry1Ac at the N-terminal end of amino acid 125 (alanine). The proteolytic products of the Cry1Ac toxin did not exhibit any insecticidal activity against Helicoverpa armigera, in contrast to its high toxicity exhibited in the native form. CONCLUSIONS: Proteases elaborated by the Chrysosporium sp. degrade the Cry1Ac toxin in a way that it looses its insecticidal activity against H. armigera. SIGNIFICANCE AND IMPACT OF THE STUDY: Chrysosporium sp., a specific soil micro-organism capable of producing proteases that degrade the Cry1Ac toxin into inactive products under controlled conditions is being reported for the first time. Application of this observation needs to be further tested in field conditions.

Intracellular proteases of Bacillus thuringiensis subsp. kurstaki and a protease-deficient mutant Btk-q.

The commencement of intracellular protease synthesis was studied by gelatin zymography in Bacillus thuringiensis ( Btk) HD1, Btk HD73, and a protease-deficient mutant Btk-q derived from the former strain. By gelatin zymography, a 92-kDa protease was detected first at 3 h of sporulation, which continued until 48 h, whereas two other proteases of mol wt 78 and 69 kDa were detectable from 6 h onwards and continued until 48 h of growth in Btk HD1. Similar studies revealed the presence of two major intracellular proteases in Btk HD73 by gelatin zymography, which first appeared at 6 h of sporulation and continued until 48 h of growth. The quantitative azocasein assay confirmed that the total protease activity increases from 3 to 21 h, thereafter reaching a plateau up to 48 h of growth examined, in HD1 and HD73 strains. Btk-q, a protease-deficient mutant, showed traces of protease activity by azocasein analysis that could not be detected by gelatin zymography. The free amino acid pool content was also increased parallel to the way that the protease activity increased in all three strains. However, this increase was found to be low (16-fold) in Btk-q when compared with Btk HD1 and HD73 strains. The following amino acids were detected by paper chromatography in Btk HD1: DL-alanine, L-glutamic acid, L-aspartic acid, tyrosine, tryptophan/methionine/valine, arginine, leucine/norleucine/isoleucine, and glycine, whereas only DL-alanine, L-glutamic acid, and L-aspartic acid were in Btk-q at 24 and 48 h, when the protease activity was maximum.

In vivo chitin-cadmium complexation in cell wall of Neurospora crassa.

Fungal cell wall, mainly composed of chitin, an N-acetylglucosamine polymer, is known to participate in heavy metal detoxification. In the present study, an effort was made to elucidate the sites involved in complexation of cadmium by the chitin material of cell wall of Neurospora crassa. Based on the results of physical techniques, such as solid-state 13C-NMR, X-ray diffraction, IR and molecular modeling, a structure was proposed for the chitin-cadmium complex. The ring and C-3 hydroxyl oxygens of N-acetylglucosamine were implicated in the complexation of cadmium by the chitin of the fungal cell wall. The studies further revealed that the conformation of chitin did not alter after cadmium complexation.

Bacillus thuringiensis crystal delta-endotoxin: role of proteases in the conversion of protoxin to toxin.

The conversion of delta-endoprotoxins of Bacillus thuringiensis to active toxins is mediated by trypsin, insect gut (exogenous) and bacterial (endogenous) proteases. The biochemical aspects of exogenous and endogenous proteases involved in the conversion of protoxin to toxin are reviewed. Perhaps, these proteases also play a role in influencing the host range of toxin and in the development of resistance to toxin.

Identification and purification of the 69-kDa intracellular protease involved in the proteolytic processing of the crystal delta-endotoxin of Bacillus thuringiensis subsp. tenebrionis.

The dynamics of appearance of intracellular proteases in relation to the synthesis of crystal delta-endotoxin was studied to identify the native intracellular protease(s) involved in the proteolytic processing of the 73-kDa protoxin of Bacillus thuringiensis subsp. tenebrionis. In vitro proteolytic activation of the 73-kDa protoxin indicated the possible role of 69-kDa protease in the proteolytic processing of 73-kDa protoxin. The purified 69-kDa protease was able to cause the proteolytic activation of the 73-kDa protoxin to 68-kDa toxin and this conversion was inhibited by ethylenediamine tetraacetic acid and 1,10-phenanthroline.

The alkaline single cell gel electrophoresis: a new test for assessing DNA single strand breaks in Neurospora crassa.

The single cell gel electrophoresis (comet assay) is a potent technique in testing double and single strand breaks in DNA. In this paper, we present an application of alkaline comet assay to filamentous fungi for genotoxicological assessment of heavy metals for the first time. A wild strain of Neurospora crassa SLA 4200 was grown in presence of cadmium sulfate (CdSO4) (10 microM and 100 microM) for 12 h. Protoplasts from 12-h old mycelia were prepared by using Novozym 234 and DNA damage was evaluated by alkaline comet assay. Hydrogen peroxide (H2O2) (50 microM and 100 microM) was taken as an internal standard for DNA damage. Both CdSO4 and H2O2 induced significant single strand breaks in DNA. The results indicate that alkaline comet assay is a sensitive and rapid method for DNA damage analysis in filamentous fungal systems.

Analysis of 66 kDa toxin from Bacillus thuringiensis subsp. kurstaki reveals differential amino terminal processing of protoxin by endogenous protease(s).

The endogenous protease(s) activated crystal toxin from Bacillus thuringiensis subsp. kurstaki was purified and examined. The purified toxin was homogenous, as demonstrated by two-dimensional polyacrylamide gel electrophoresis and contained 1.38 mumoles neutral sugar and 9 nmoles sialic acid per mg protein amino terminal amino acid sequence data revealed that the toxin is a cleavage product of 132 kDa protoxin with glutamic acid-30 of the deduced amino acid sequence of the crystal protein (Schnepf, H.E., Wong, H.C. and Whiteley, H.R. (1985) J. Biol. Chem. 260: 6264-6272) at the amino terminus.

Endogenous protease-activated 66-kDa toxin from Bacillus thuringiensis subsp. kurstaki active against Spodoptera littoralis.

The anti-lepidopteran toxin from sporulated Bacillus thuringiensis subsp. kurstaki cells, generated by the proteolytic action of endogenous protease(s) on the protoxin, was purified and studied to identify the effect of such proteolysis on the biochemical nature of the toxin. The active toxin was purified employing anion-exchange chromatography to absolute homogeneity, as indicated by SDS-PAGE and Western blotting. Antisera to the purified toxin (66 kDa) crossreacted with the protoxin (132 kDa) confirming its origin from protoxin. The purified toxin with a pI of 7.95 was derived from the N-terminal region of the protoxin (pI 7.6). Circular dichroism data revealed that the toxin has significant secondary structure and it undergoes pH dependent conformational change. Unlike the toxin generated by exogenous proteases such as trypsin, etc., the endogenous protease(s) activated toxin is highly lethal to a tolerant insect variety of the lepidopteran order, Spodoptera littoralis.

Involvement of an endogenous metalloprotease in the activation of protoxin in Bacillus thuringiensis subsp. kurstaki.

Insecticidal crystal proteins harvested from sporulated cultures of Bacillus thuringiensis subsp. kurstaki contain the protoxin (Mr 132 kDa) and minor amounts of toxin (66 kDa). The proteolytic processing of 132 kDa protoxin to an active 66 kDa toxin is brought about by exogenous proteases or larval gut enzymes. Under denaturing/reducing conditions this conversion is also mediated by endogenous protease(s) of the producer organism. This endogenous protease is identified as a metalloprotease as the activation process is inhibited by ethylenediamine tetraacetic acid at 2 mM concentration.

Intracellular proteases in sporulated Bacillus thuringiensis subsp. tenebrionis: detection and analysis by gelatin zymography.

Three intracellular proteases were identified from sporulated cultures of Bacillus thuringiensis subsp tenebrionis by fractionation with ammonium sulfate. In this study, we detected protease activities at M(r) 92 kDa, 81 kDa and 69 kDa employing gelatin zymography. The major proteolytic activity was due to the 81 kDa protease, which was identified as a metalloprotease being inhibited by both 1,10-phenanthroline and ethylenediamine tetraacetic acid. The proteases showed maximal azocasein hydrolytic activity at 60 degrees C and were heat-activated from 40 degrees C to 60 degrees C. The 69 kDa and 81 kDa proteases were thermo-inactivated at 70 degrees C and 80 degrees C respectively, while the 92 kDa protease was still active at 80 degrees C.

Simple method for the isolation of the antilepidopteran toxin from Bacillus thuringiensis subsp. kurstaki.

A protein with a molecular mass of 66 kDa was isolated by a simple, rapid, and inexpensive method, using 3-N-morpholinopropanesulfonic acid, potassium thiocyanate, and dithiothreitol, from a mixture of spores, parasporal crystals, and cell debris of Bacillus thuringiensis subsp. kurstaki. The protein was active against the third instar larvae of Trichoplusia ni, was soluble in 19 mM Na2CO3, and was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and confirmed as the insecticidal component of the 132-kDa protoxin of B. thuringiensis subsp. kurstaki by an enzyme-linked immunosorbent assay using antibodies prepared against the protoxin.

Copper and cobalt alter the cell wall composition of Cunninghamella blakesleeana.

Cunninghamella blakesleeana was highly sensitive to Cu and Co on a medium containing NaNO3 as the sole nitrogen source. The nitrate reductive pathway was altered by Cu and Co, and NO-2 accumulated in the medium. Under conditions of Cu toxicity, the mycelium and the cell walls acquired a blue color, and most of the Cu was located in the cell walls, which differed in several aspects from cell walls derived from Co-containing or control cultures. At half-maximal growth inhibition by Cu (2.5 micrograms/mL or 39.3 microM) or Co (3.5 micrograms/mL or 59.4 microM), the mycelia contained 1.5 micrograms Cu or 1.0 microgram Co/mg dry tissue, respectively, but the isolated cell walls contained 33.5 micrograms Cu or 1.8 micrograms Co/mg dry cell wall. The phosphorous content of mycelia from Co-containing cultures was the same as that from control cultures, whereas that of mycelia from Cu-containing cultures contained 36% less. However, the phosphorous content of the cell walls from mycelia cultured in the presence of Cu or Co was two- and three-fold higher, respectively, than that of cell walls from control cultures. The cell walls of Cu-containing cultures contained significantly less hexosamine than the control cell walls, and chitin and chitosan were present in equal quantities. The cell walls of Co-containing cultures had the same amount of hexosamine as the control cell walls, but 88% of the hexosamine was present as chitosan and bound very little Co. The control cell walls contained approximately 60% chitosan.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Wall Composition of Neurospora crassa Under Conditions of Copper Toxicity.

The mycelia of Neurospora crassa grown in the presence of high concentrations of copper were blue in color, but only on a medium containing inorganic nitrate and phosphate as the nitrogen and phosphate sources, respectively. The cell wall isolate of the blue mycelia contained large amounts (12%) of copper and higher amounts of chitosan, phosphate, and amino groups, with a 42% decrease in the chitin content. Although all the glucosamine of the cell wall of control cultures could be released within 6 h of hydrolysis with acid, that of the blue mycelium required prolonged hydrolysis for 24 h. On removal of copper, the cell wall of the blue mycelium could quantitatively bind again to copper as well as to zinc. Although zinc binding was fivefold greater, copper alone was preferentially bound from a mixture of the two metal ions. Supplementation of iron along with copper in the culture medium resulted in the disappearance of the blue color of the mycelium and restoration of normal growth and composition of the cell wall, probably by limiting the uptake of copper from the medium. The possibility of the cell wall being a specific site of lesion in copper toxicity in the mold is discussed.

Interrelationships in trace-element metabolism in metal toxicities in a cobalt-resistant strain of Neurospora crassa.

A strain of Neurospora crassa was isolated by training the mould to grow on media containing high concentrations of Co(2+). This strain, the Co(R) strain, exhibited approximately tenfold the resistance of the parent strain to Co(2+) and Ni(2+) but not to Zn(2+) or Cu(2+). Co(2+) toxicity in the Co(R) strain was reversed by Mg(2+) but not by Fe(3+). Also, Co(2+) did not affect iron metabolism in this strain. It is suggested that the mechanism of resistance in the Co(R) strain involves an alteration in the pattern of iron metabolism such that the latter is no longer adversely affected by toxic concentrations of Co(2+). The Co(R) strain is genetically stable and is most probably a result of a resistance mutation in N. crassa induced by Co(2+).

The mechanism of uptake of cobalt ions by Neurospora crassa.

Uptake of Co(2+) by 3-day-old mycelia of Neurospora crassa involves cell-surface binding as well as transport into the intracellular space. The surface binding is rapid and accounts for 30-40% of the total Co(2+) uptake. Transport of Co(2+) occurs at a rate of 40mug/h per 100mg dry wt. Surface binding and overall uptake show different temperature dependence. Metabolic inhibitors such as azide, dinitrophenol and fluoride depress transport of Co(2+). The overall uptake of Co(2+) exhibits a high K(m) value and hence the concentration mechanism is one of low ;affinity' for the metal. The uptake of Co(2+) varies linearly with pH in the range pH3 to pH6. Mg(2+) inhibits both surface binding and transport of Co(2+). It is suggested that the system that transports Mg(2+) is also involved in Co(2+) uptake by N. crassa.