Isolation,partial characterization and growth characteristics of a bacterium resistant to aflatoxin B1

A bacterium was isolated from the soil of dumping ground for cattle yard waste by enrichment culture containing aflatoxin B1. This bacterium was closely related to Bacillus firmus that was found to be a non-pathogenic bacterium. The minimum inhibitory concentration of aflatoxin B1 to the bacterium was found to be 80 microg ml(-1) as measured by total viable count and soluble protein content methods. The bacterium was sensitive to all the tested antibiotics. Plasmid curing by chemical agents did not show the resistance character residing in the plasmid. Protein profiles of cell extracts of aflatoxin B1 resistant bacterium grown in the presence and absence of the toxin showed 46 and 44 protein bands respectively in SDS-PAGE. It was observed that 39 bands were common in both the extracts and the remaining bands were showing differences near the high molecular weight range.     

Cry1A toxins of Bacillus thuringiensis bind specifically to a region adjacent to the membrane-proximal extracellular domain of BT-R(1) in Manduca sexta: involvement of a cadherin in the entomopathogenicity of Bacillus thuringiensis

Many subspecies of the soil bacterium Bacillus thuringiensis produce various parasporal crystal proteins, also known as Cry toxins, that exhibit insecticidal activity upon binding to specific receptors in the midgut of susceptible insects. One such receptor, BT-R(1) (210 kDa), is a cadherin located in the midgut epithelium of the tobacco hornworm, Manduca sexta. It has a high binding affinity (K(d) approximately 1nM) for the Cry1A toxins of B. thuringiensis. Truncation analysis of BT-R(1) revealed that the only fragment capable of binding the Cry1A toxins of B. thuringiensis was a contiguous 169-amino acid sequence adjacent to the membrane-proximal extracellular domain. The purified toxin-binding fragment acted as an antagonist to Cry1Ab toxin by blocking the binding of toxin to the tobacco hornworm midgut and inhibiting insecticidal action. Exogenous Cry1Ab toxin bound to intact COS-7 cells expressing BT-R(1) cDNA, subsequently killing the cells. Recruitment of BT-R(1) by B. thuringiensis indicates that the bacterium interacts with a specific cell adhesion molecule during its pathogenesis. Apparently, Cry toxins, like other bacterial toxins, attack epithelial barriers by targeting cell adhesion molecules within susceptible insect hosts.     

Cyt1A from Bacillus thuringiensis synergizes activity of Bacillus sphaericus against Aedes aegypti (Diptera: Culicidae)

Bacillus sphaericus is a mosquitocidal bacterium recently developed as a commercial larvicide that is used worldwide to control pestiferous and vector mosquitoes. Whereas B. sphaericus is highly active against larvae of Culex and Anopheles mosquitoes, it is virtually nontoxic to Aedes aegypti, an important vector species. In the present study, we evaluated the capacity of the cytolytic protein Cyt1A from Bacillus thuringiensis subsp. israelensis to enhance the toxicity of B. sphaericus toward A. aegypti. Various combinations of these two materials were evaluated, and all were highly toxic. A ratio of 10:1 of B. sphaericus to Cyt1A was 3, 600-fold more toxic to A. aegypti than B. sphaericus alone. Statistical analysis showed this high activity was due to synergism between the Cyt1A toxin and B. sphaericus. These results suggest that Cyt1A could be useful in expanding the host range of B. sphaericus.     

Aflatoxin B1 Uptake by Flavobacterium aurantiacum and Resulting Toxic Effects

Removal of aflatoxin B(1) from liquid cultures by resting and growing cells of Flavobacterium aurantiacum NRRL B-184 was studied. Spectrophotometic and thin-layer techniques served as aflatoxin assays. Cells grown in the presence of 5 ppm or higher levels of aflatoxin developed aberrant morphological forms. These toxin concentrations partially inhibited growth, and the nature of the inhibition suggested that aflatoxin interfered with cell wall synthesis. Incubation of 1.0 x 10(11) resting cells per milliliter with 7.0 mug/ml of aflatoxin B(1) during a 4-hr period facilitated complete toxin removal from a buffered aqueous medium. Autoclaved cells and cell wall preparations could remove a fraction of the aflatoxin of a test system. However, the toxin removed by autoclaved cells and cell walls could be extracted by washing with water but the aflatoxin B(1) removed by intact cells could not be extracted into the liquid phase. The uptake of aflatoxin B(1) by resting cells was sensitive to temperature and pH. Ruptured preparations of F. aurantiacum were not able to remove or modify the aflatoxin in an aqueous solution.     

Analysis of the physicochemical interactions between Clostridium difficile toxins and cholestyramine using liquid chromatography with post-column derivatization

A potential therapy for antibiotic-associated pseudomembranous colitis is to bind Clostridium difficile toxins A and B using cholestyramine, a hydrophobic anion exchange medium. Frontal analysis in isotonic phosphate buffer was studied using post-column derivatization with o-phthalaldehyde, which gave a highly sensitive (> or =30 ng) flow-through analysis. Following load (1.5-3.0 microg toxin/3.6 mg), toxin A was bound at a slightly higher capacity than B, due to slower kinetics. A salt gradient eluted roughly 20% of bound toxin A with 0.6 M NaCl and toxin B with 1.1 M NaCl, hence toxin A showed weaker electrostatic affinity. The remainder of toxin A (65%) and some of toxin B (10% out of 50%) were eluted using a subsequent gradient to 60% acetonitrile in normal saline, which measured predominantly hydrophobic binding. Low and high affinity populations of both toxins were observed. Glycocholic acid or amino acids were competitive binders, although these components had little effect on the toxin A population bound primarily through ionic interactions. Competitive protein constituents in hamster cecal contents were also profiled. These results help to explain the variable clinical response in using cholestyramine to treat colitis. Using quaternary amine-polyhydroxymethacrylate (PHM) ion exchange chromatography, a trend for increased binding at higher pH was observed, especially for toxin A. Binding to strong cation exchange resins (sulfonate-PHM) was not observed. A range of reversed phase media retained both toxins, although recovery was very poor relative to protein standards. Size exclusion chromatography with light scattering detection showed that toxin B exists in different aggregation states, while toxin A remains monomeric.     

Photorhabdus luminescens W-14 insecticidal activity consists of at least two similar but distinct proteins. Purification and characterization of toxin A and toxin B

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 (LD50) 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.     

Cloning and expression of the toxin B gene ofClostridium difficile

Results from our cloning studies on toxin A indicated that the gene for toxin B resided approximately 1 kb upstream of the toxin A gene. Clone pCD19, which contains the 5-end of the toxin A gene and a small open reading frame, was found to contain 1.2 kb of DNA which, when subcloned, expressed a nontoxic peptide that reacted with toxin B antibodies. The rest of the toxin B gene was located on the 6.8 kb cloned fragment of plasmid pCD19L. The two fragments overlapped 0.8 kb. Lysates containing protein expressed by the 6.8 fragment were cytotoxic and lethal, and were neutralized by toxin B antibody. The two fragments were ligated to give the complete toxin B gene. The protein expressed by the complete gene was cytotoxic and lethal, and showed complete immunological identity with toxin B. Further analysis of the expressed protein and the toxin B gene confirmed our earlier findings showing that toxin B has a molecular weight of 240,000 or greater.     

A novel approach for predicting the evolutionary time of acquisition of foreign genes by bacteria: Application of codon usage analyses

Lysogenic bacteriophages are considered as a major player for the introduction of foreign genes into bacterial strains. At the time of introduction foreign genes do not fit well into the translation system of the recipient host bacterium as they tend to retain the characteristics of the donor bacterium from which they have been transferred. Consequently foreign genes are poorly transcribed at the early phase of their evolution within the host bacterium. This is largely due to the difference in the codon usage pattern between the horizontally transferred genes and the host bacterium. In this study we present detailed analyses of various parameters of the codon usages such as codon adaptation index (CAI), mean difference (MD) of the relative adaptiveness, synonymous substitution rate (SSR) of six different phage encoded toxin genes (cholera toxin, shiga toxin, diphtheria toxin, neurotoxin C1, enterotoxin type A and cytotoxin), and proposed conceptual relationship between the evolutionary time of acquisition of the foreign genes and the selected set of parameters of the codon usage. On the basis of the observed data we hypothesize that CAI, MD and SSR of the phage encoded toxin genes are correlated with the evolutionary time of their acquisition, and have developed a novel approach based on the analyses of these parameters, which can be used to predict the evolutionary time of their acquisition by the corresponding host bacterium.     

The role of toxin A and toxin B in the virulence of Clostridium difficile

During the past decade, there has been a striking increase in Clostridium difficile nosocomial infections worldwide predominantly due to the emergence of epidemic or hypervirulent isolates, leading to an increased research focus on this bacterium. Particular interest has surrounded the two large clostridial toxins encoded by most virulent isolates, known as toxin A and toxin B. Toxin A was thought to be the major virulence factor for many years; however, it is becoming increasingly evident that toxin B plays a much more important role than anticipated. It is clear that further experiments are required to accurately determine the relative roles of each toxin in disease, especially in more clinically relevant current epidemic isolates.     

Two small c-type cytochromes affect virulence gene expression in Bacillus anthracis

Regulated expression of the genes for anthrax toxin proteins is essential for the virulence of the pathogenic bacterium Bacillus anthracis . Induction of toxin gene expression depends on several factors, including temperature, bicarbonate levels, and metabolic state of the cell. To identify factors that regulate toxin expression, transposon mutagenesis was performed under non-inducing conditions and mutants were isolated that untimely expressed high levels of toxin. A number of these mutations clustered in the haem biosynthetic and cytochrome c maturation pathways. Genetic analysis revealed that two haem-dependent, small c -type cytochromes, CccA and CccB, located on the extracellular surface of the cytoplasmic membrane, regulate toxin gene expression by affecting the expression of the master virulence regulator AtxA. Deregulated AtxA expression in early exponential phase resulted in increased expression of toxin genes in response to loss of the CccA-CccB signalling pathway. This is the first function identified for these two small c -type cytochromes of Bacillus species. Extension of the transposon screen identified a previously uncharacterized protein, BAS3568, highly conserved across many bacterial and archeal species, as involved in cytochrome c activity and virulence regulation. These findings are significant not only to virulence regulation in B. anthracis , but also to analysis of virulence regulation in many pathogenic bacteria and to the study of cytochrome c activity in Gram-positive bacteria.     

Determination of mycotoxins in bovine milk by liquid chromatography tandem mass spectrometry

Liquid chromatographic/tandem mass spectrometric methods using pneumatically assisted electrospray ionisation (LC-ESI-MS/MS) was developed for determination of 18 mycotoxins and metabolites-ochratoxin A, zearalenone, alpha-zearalenol, beta-zearalenol, alpha-zearalanol (zeranol), beta-zearalanol (taleranol), fumonisin B1, fumonisin B2, T-2 toxin, HT-2 toxin, T-2 triol, diacetoxyscirpenol (DAS), 15-monoacetoxyscirpenol (MAS), deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), 15-acetyldeoxynivalenol (15-AcDON), deepoxy-deoxynivalenol (DOM-1) and aflatoxin M1--in milk. The mycotoxins were extracted and cleaned up simultaneously. Extraction and removal of lipophilic compounds was performed at pH 2 using a two-phase mixture of acetonitrile and hexane. The acetonitrile concentration of the aqueous phase was reduced and the pH was adjusted to 8.5 before clean up by solid phase extraction (SPE) on Oasis HLB. The toxins DON, DOM-1, 3-AcDON, 15-AcDON, ochratoxin A, zearalenone, alpha-zearalenol, beta-zearalenol, alpha-zearalanol and beta-zearalanol were detected in negative ion mode after separation on a Hypersil ENV analytical column, while the toxins T-2 toxin, HT-2 toxin, T-2 triol, DAS, MAS, fumonisin B1, fumonisin B2 and aflatoxin M1 were detected in positive ion mode after separation on a Luna C18 column. Two transition products were monitored for each compound. The extraction and SPE conditions were optimised to obtain maximum recovery and minimum signal suppression/enhancement. The detection capabilities related to the transition products of lowest abundance were in the range 0.020-0.15 microg/l. The mean true recoveries were in the range 76-108% at levels of 0.2-10 microg/l.     

Production of monoclonal antibody to Clostridium difficile toxin A which neutralises enterotoxicity but not haemagglutination activity

Nine monoclonal antibodies (mAb) to Clostridium difficile toxin A were produced. The isotype of one mAb (37B5) was IgG2b, kappa, and that of the other eight mAbs was IgM, kappa. Immunoblot analysis after non-denatured PAGE showed that with the exception of one mAb (112G6) all mAbs gave a positive reaction with the 540 kDa band of toxin A. Immunoblot analysis showed that four mAbs (2E15, 3B4, 37B5 and 49C4) gave a positive reaction with the 240 kDa major band of toxin A. In neutralisation tests with these mAbs for enterotoxicity, mouse lethality, haemagglutination activity and cytotoxicity, 37B5 neutralised enterotoxicity in a rabbit ileal loop response test but did not neutralise any other biological activities. None of the other eight mAbs showed any neutralising activities at all.     

Chitinase system of Bacillus circulans WL-12 and importance of chitinase A1 in chitin degradation.

Bacillus circulans WL-12, isolated as a yeast cell wall-lytic bacterium, secretes a variety of polysaccharide-degrading enzymes into culture medium. When chitinases of the bacterium were induced with chitin, six distinct chitinase molecules were detected in the culture supernatant. These chitinases (A1, A2, B1, B2, C, and D) showed the following distinct sizes and isoelectric points: Mr 74,000, pI 4.7 (A1); Mr 69,000, pI 4.5 (A2); Mr 38,000, pI 6.6 (B1); Mr 38,000, pI 5.9 (B2); Mr 39,000, pI 8.5 (C); and Mr 52,000, pI 5.2 (D). Among these chitinases, A1 and A2 had the highest colloidal-chitin-hydrolyzing activities. Chitinase A1 showed a strong affinity to insoluble substrate chitin. Purified chitinase A1 released predominantly chitobiose [(GlcNAc)2] and a trace amount of N-acetylglucosamine (GlcNAc) from colloidal chitin. N-terminal amino acid sequence analysis of chitinases A1 and A2 indicated that chitinase A2 was generated from chitinase A1, presumably by proteolytic removal of a C-terminal portion of chitinase A1. Since chitinase A2 did not have the ability to bind to chitin, the importance of the C-terminal region of chitinase A1 to the strong affinity of chitinase A1 to substrate chitin was suggested. Strong affinity of the chitinase seemed to be required for complete degradation of insoluble substrate chitin. From these results, it was concluded that chitinase A1 is the key enzyme in the chitinase system of this bacterium.     

Identification, cloning and sequence analysis of the Bacillus sphaericus 1593 41.9 kD larvicidal toxin gene

A number of strains of the widespread aerobic soil bacterium, Bacillus sphaericus, possess crystalline inclusions of a toxin lethal to a variety of insect (larvae) which are vectors of major tropical diseases. Partial amino acid sequence data from one strain, B. sphaericus 2362 have permitted us to design oligonucleotide probes for identifying the toxin gene in the closely related B. sphaericus 1593. The gene was found to be contained within an EcoRI-HindIII fragment and was cloned in its entirety in the bacterial plasmid pUC12. The DNA sequence was determined together with the upstream and downstream controlling elements, and a sequence of 370 amino acids was deduced for the toxin protein. This is the first reported sequence of a B. sphaericus toxin gene and will facilitate further work in characterizing the genes from other strains of different virulence and host range. The data do not support the suggestion that the toxin is derived by proteolysis of a protoxin precursor.     

Enhanced toxicity of Bacillus thuringiensis japonensis strain Buibui toxin to oriental beetle and northern masked chafer (Coleoptera: Scarabaeidae) larvae with Bacillus sp. NFD2

Bacillus thuringiensisjaponensis strain Buibui (Btj) has the potential to be an important control agent for pest scarabs. Bioassays using autoclaved and nonautoclaved soil showed there were always lower LC, values associated with nonautoclaved soil. We identified five other bacteria found in the hemolymph of insects killed by Btj and used them in bioassays to see whether we could enhance the control achieved with Btj alone. One bacterium, designated NFD2 and later identified as a Bacillus sp., showed the greatest enhancement of Btj in preliminary experiments and was used in bioassays with Btj versus oriental beetle, Anomala orientalis (Waterhouse), and northern masked chafer, Cyclocephala borealis Arrow (Coleoptera: Scarabaeidae), larvae. This bacterium alone was nontoxic to grubs in bioassays. A combination of this bacterium with Btj in nonautoclaved soil resulted in a significantly lower LC50 value (0.23 microg toxin per g soil) from all other treatments for A. orientalis with one exception; the LC50 where NFD2 was added back into autoclaved soil (0.29 microg toxin per g soil). A combination of this bacterium with Btj in nonautoclaved soil resulted in a significantly lower LC50 value (48.29 microg toxin per g soil) from all other treatments for C. borealis with the exception of the treatment where Bacillus sp. NFD2 was added back to autoclaved soil (96.87 microg toxin per g soil) with Btj. This research shows that other soil bacteria can be used to enhance the toxicity of Btj and possibly other Bts.     

Use of a two-phase partitioning bioreactor for degrading polycyclic aromatic hydrocarbons by a Sphingomonas sp

A two-phase partitioning bioreactor (TPPB) utilizing the bacterium Sphingomonas aromaticivorans B0695 was used to degrade four low molecular weight (LMW) polycyclic aromatic hydrocarbons (PAHs). The TPPB concept is based on the use of a biocompatible, immiscible organic solvent in which high concentrations of recalcitrant substrates are dissolved. These substances partition into the cell-containing aqueous phase at rates determined by the metabolic activity of the cells. Experiments showed that the selected solvent, dodecane, could be successfully used in both solvent extraction experiments (to remove PAHs from soil) and in a TPPB application. Further testing demonstrated that solvent extraction from spiked soil was enhanced when a solvent combination (dodecane and ethanol) was used, and it was shown that the co-solvent did not significantly affect TPPB performance. The TPPB achieved complete biodegradation of naphthalene, phenanthrene, acenaphthene and anthracene at a volumetric consumption rate of 90 mg l(-1) h(-1) in approximately 30 h. Additionally, a total of 20.0 g of LMW PAHs (naphthalene and phenanthrene) were biodegraded at an overall volumetric rate of 98 mg l(-1) h(-1) in less than 75 h. Degradation rates achieved using the TPPB and S. aromaticivorans B0695 are much greater than any others previously reported for an ex situ PAH biodegradation system operating with a single species.     

Cyt1A from Bacillus thuringiensis Synergizes Activity of Bacillus sphaericus against Aedes aegypti (Diptera: Culicidae)

Bacillus sphaericus is a mosquitocidal bacterium recently developed as a commercial larvicide that is used worldwide to control pestiferous and vector mosquitoes. Whereas B. sphaericus is highly active against larvae of Culex and Anopheles mosquitoes, it is virtually nontoxic to Aedes aegypti, an important vector species. In the present study, we evaluated the capacity of the cytolytic protein Cyt1A from Bacillus thuringiensis subsp. israelensis to enhance the toxicity of B. sphaericus toward A. aegypti. Various combinations of these two materials were evaluated, and all were highly toxic. A ratio of 10:1 of B. sphaericus to Cyt1A was 3,600-fold more toxic to A. aegypti than B. sphaericus alone. Statistical analysis showed this high activity was due to synergism between the Cyt1A toxin and B. sphaericus. These results suggest that Cyt1A could be useful in expanding the host range of B. sphaericus.     

Activity of covalently cross-linked cholera toxin with the adenylate cyclase of intact and lysed pigeon erythrocytes.

Reaction of cholera toxin with NN'-bis(carboximidomethyl)tartaramide dimethyl ester produced several cross-linked species that had subunit B (which binds to the cell surface) and peptides A1 (which activates adenylate cyclase) and A2 all covalently joined together. This cross-linded material had activity with pigeon erythrocytes that was comparable in all respects with that of native toxin. It activated the adenylate cyclase of whole cells, showing a characteristic lag phase, and this activation was increased if the cells had been preincubated with ganglioside GM1, but abolished if the protein had been preincubated with the ganglioside. It activated the enzyme in lysed cells more strongly and without the lag phase. These results show that the toxin is active even when peptide A1 cannot be released from the rest of the molecule.