Escherichia coli Hfr-DNA degradation in endonuclease I-deficient minicells.

[3H]Thymidine (dThd)-labelled Hfr DNA was transferred by conjugation into Escherichia coli F- minicells harvested from an endonuclease I-deficient (endI-) strain and its iosgenic wild type (endI+) parent. The susceptibility of this DNA to attack by DNAase was examined. The kinetics of in vivo conversion of [3H]dThd-labelled DNA into acid soluble radioactivity was examined. This activity, attributed to exonuclease action was the same for both strains. Contribution of endonuclease I was measured by an analysis of changes in weight-average (Mw) and number-average (Mn) molecular weight distribution of DNA molecules recovered from minicells. Reduction in Mw was greater in the endI-strain. The ratio Mn/Mw changed drastically during the incubation period of endI- minicells, but remained unchanged in the endI+ strain. These experiments suggest that the presence of the endI- mutation in minicell-producing strain chi1268 leads to a greater loss in M2 of Hfr DNA conjugally transferred into the minicells.

Interaction of T-2 toxin and murine lymphocytes and the demonstration of a threshold effect on macromolecular synthesis.

Although T-2 toxin intoxications have been described as radiomimetic, we find that T-2 toxin does not preferentially affect multiplying cells. Among the targets of T-2 toxin toxicity, DNA, RNA and protein synthesis inhibition are analysed. All three types of macromolecular syntheses are affected by a threshold dose of T-2 toxin which corresponds to the interaction of approx. 1 X 10(5) T-2 toxin molecules with the same number of T-2 toxin receptors (Gyongyossy-Issa, M.I.C. and Kachatourians, G.G. (1984) Biochim. Biophys. Acta 803, 197-202). Since toxic effects occur faster at higher toxin concentrations than at lower levels, the time-toxic effect relationship may be defined by a constant. Based on these observations, we hypothesize that complete receptor-occupation is the critical first step in the course of T-2 toxin toxicity events.

Interaction of T-2 toxin with murine lymphocytes.

T-2 toxin is taken up by lymphocytes in 10-15 min in a saturable manner. Uptake is dependent on temperature and partially on the availability of energy. Approx. 10(5) molecules of T-2 toxin are bound per cell, having a mean affinity constant, Ka = 1.6 X 10(7) M-1. The toxin is rapidly dissociated from the cell to leave approx. 10-15% of the original loading in 1 h. It is concluded that T-2 toxin uptake and release do not follow conventional mechanisms.

Characterisation of hemolysis induced by T-2 toxin.

The erythrocyte constitutes a good model system for the study of membrane-associated toxicity events caused by the trichothecene mycotoxin, T-2. This study confirms that T-2 has a direct lytic effect on erythrocytes. Lysis of guinea pig red cells requires approx. 10(10) molecules/cell and reaches plateau values after 4-6 h. An activation energy, Ea approximately equal to 4.5 kcal was derived from the Arrhenius equation. By use of osmotic blockers of differing Stokes' radii, the functional size of the membrane lesion caused by T-2 toxin was shown to be smaller than 5.5 A. It is concluded that T-2 toxin may exert its toxic effects via the cell membrane.

Cloning and analysis of five mitochondrial tRNA-encoding genes from the fungus Beauveria bassiana.

Five mitochondrial (mt) tRNA genes from the filamentous fungus, Beauveria bassiana, were cloned and sequenced. The genes encoding the Val-, Ile-, Ser-, Trp- and Pro-accepting tRNAs were found clustered in the region 5' to the lrRNA-encoding gene. The genes were 64-77% homologous with the equivalent genes from other filamentous fungi, 49-58% to yeasts with the exception of the Val-accepting tRNA-encoding gene which was 76%, and only slightly homologous with Escherichia coli. The B. bassiana mt genetic code was found to be similar to that of other fungal mitochondria in that the UGA codon is used as a signal for Trp rather than as a stop codon. Transcript analysis has revealed that the genes present in tRNA cluster are transcribed and processed into tRNA-size products. Secondary structure models proposed for the gene products show that conservation of tRNA secondary structure also exists. The presence of a GGC sequence rather than a GGU sequence in the D-loop of the tRNA(Trp)-encoding gene is a feature unique to the B. bassiana mt tRNA. An unconventional G-A base pair present in the D-stem of the tRNA(Ser)-encoding gene is a feature conserved in the mt tRNA of other filamentous fungi. Comparison of the B. bassiana tRNA-encoding genes with those of two other filamentous fungi and two yeasts revealed that the differences between closely related species favoured transition-type mutations.

The impact of biotechnology on hyphomycetous fungal insect biocontrol agents.

The potential for the control of insect pests by entomopathogenic fungi has been touted for decades, if not centuries. Only recently have advances in biotechnology provided the tools for indepth analysis of the mechanisms involved in pathogenesis and host death at the molecular level. This review outlines the current state of knowledge regarding the mode of infection and targets several key components that are amenable to improvement via biotechnology. Realization of the considerable economic potential of fungal bioinsecticides can occur only through a combined and coordinated effort involving fundamental science, formulation technology and field applications.

Expression of recombinant DNA functional products in Escherichia coli anucleate minicells.

This review covers the use of anucleate minicells of Escherichia coli for expressing the recombinant DNA encoded proteins. We briefly discuss the methods being used for preparation of anucleate minicells, incorporation of cloned DNA and assessment of gene expression. While the largest use has been that of microbially derived cloned functional DNA, examples of eukaryotic gene product synthesis have also been reviewed. This technology may represent some interesting commercial opportunities.

Constitutive and heat-inducible heat shock element binding activities of heat shock factor in a group of filamentous fungi.

This study represents the initial characterization of the heat shock factor (HSF) in filamentous fungi. We demonstrate that HSFs from Beauveria bassiana, Metarhizium anisopliae, Tolypocladium nivea, Paecilomyces farinosus, and Verticillium lecanii bind to the heat shock element (HSE) constitutively (non-shocked), and that heat shock resulted in increased quantities and decreased mobility of HSF-HSE complexes. The monomeric molecular mass of both heat-induced and constitutive HSFs was determined to be 85.8 kDa by UV-crosslinking and the apparent molecular masses of the native HSF-HSE complexes as determined by pore exclusion gradient gel electrophoresis was 260 and 300 kDa, respectively. Proteolytic band clipping assays using trypsin and chymotrypsin revealed an identical partial cleavage profile for constitutive and heat-induced HSF-HSE complexes. Thus, it appears that both constitutive and heat-inducible complexes are formed by trimers composed of the same HSF molecule which undergoes conformational changes during heat shock. The mobility difference between the complexes was not abolished by enzymatic dephosphorylation and deglycosylation, indicating that the reduced mobility of the heat-induced HSF is probably due to a post-translational modification other than phosphorylation or glycosylation.

Analysis of the chiB gene of Serratia liquefaciens.

A 4.6-kb EcoRI/BglII fragment of Serratia liquefaciens genomic DNA has been sequenced and within this fragment the chiB gene has been identified and characterized. The chiB ORF encodes a polypeptide with a deduced molecular mass of 52-kDa and the translational product in vitro has chitinase, but not chitobiase activity. Alignment of the predicted Chib 499 amino acid sequence indicated a chitin-binding and a catalytic domain that shares homology to the chitinase family 18 domain, to the Chib polypeptide of Serratia marcescens QMB1466 (93.6%), a human chitinase and several bacterial chitinases. This chiB gene sequence transcription/translation in Escherichia coli may be blocked by a RNA folding mechanism thus controlling the chitin utilization regulon in S. liquefaciens.

Toxic properties of Beauveria pigments on erythrocyte membranes.

The Beauveria pigments, tenellin, bassianin and oosporein, all inhibited total erythrocyte membrane ATPase activity in a dose-dependent manner by as much as 50% at 200 micrograms/ml. These pigments inhibited Ca(2+)-ATPases to a greater extent than Na+/K(+)-ATPase activity. The ATPase inhibitory activity for these pigments was not specific but was probably a consequence of membrane disruption, since pigments all caused alterations in erythrocyte morphology and promoted varying degrees of cell lysis.

Production of listeriolysin O by Listeria monocytogenes (Scott A) under heat-shock conditions.

Early stationary phase cells of Listeria monocytogenes (Scott A) were examined to determine the effect of heat-shock on the production of listeriolysin O (LLO) during and after resuscitation at 37 degrees C. Cells were subjected to a heat-shock at 48 degrees C for 1 h. Intracellular and extracellular proteins of the heat-shocked cells were assayed for LLO using a microtiter plate hemolysis assay and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Our results showed that significant amounts of LLO are synthesized under heat-shock conditions that are not detected in the extracellular medium by a functional assay. This situation is evident by the absence of hemolytic activity immediately after heat-shock, and may be due to either a lack of excretion or inactivation of the LLO at 48 degrees C once outside the cell. By studying the intracellular and extracellular proteins using SDS-PAGE and immunoblots of the heat-shocked cells, we substantiated an absence of excretion as an operating mechanism. Heat-shocked cells resumed LLO production within 2-4 h of resuscitation at 37 degrees C, achieving an activity level 2-fold higher compared to the controls and 4-fold higher compared to cells immediately after heat-shock. Most likely, the LLO excreted must have been from LLO accumulated in the cells during heat-shock.

Some effects of 1-(2,4-Dichlorophenyl)-4-dimethylamino-methyl-1-nonen-3-one hydrochloride on Escherichia coli GK-19.

1-(2,4-Dichlorophenyl)-4-dimethylaminomethyl-1-nonen-3-one hydrochloride (Id) was shown to inhibit the growth of Escherichia coli GK-19 at a concentration of 50 micrograms/mL in a medium of pH 6.5. Maximal antibacterial activity was found during the logarithmic growth phases rather than at the early stationary phase. Electron microscopy revealed that Id caused lysis, and inhibition of respiration and retardation of RNA and protein syntheses occurred in the bacteria with this compound at 50 micrograms/mL.

Changes induced by T-2 toxin in the erythrocyte membrane.

The interaction of the trichothecene mycotoxin T-2 with guinea-pig erythrocytes was studied. In a time- and dose-dependent manner, T-2 toxin showed a protective antihaemolytic effect in hypotonic solutions. In isotonic environments, T-2 toxin caused membrane changes resulting in an increase in cell volume and a dramatic alteration in red-cell morphology from the biconcave disc to an echinocyte. These results demonstrate that T-2 toxin, in line with other amphipaths, distributes into the outer half of the cell membrane's phospholipid bilayer. This constitutes the first direct demonstration of an initially amphipathic mechanism of action for this toxin. Therefore, it is suggested that the degree of the final toxic effect of T-2 may be influenced by the targeted cell's membrane.

Tumour minicells: single, large vesicles released from cultured mastocytoma cells.

We describe a new type of cell fragmentation in P815Y mastocytoma cells yielding one, large, enucleated 'minicell' at a time per parent tumour cell. These tumour minicells (TMC) arise spontaneously in semi-synthetic medium during early stationary phase of the growth curve. Their diameter comprises 21-30% of that of the parent cell line. Separated from parent tumour cells, they are non-tumorigenic. TMC can induce cytotoxic T cell activity against the parent tumour cell line greater cytotoxicity being observed against the P815Y line than an H-2-identical line, L1210. TMC may provide a new tool adaptable to the study of host-tumour relationships, cell size regulation or the mechanistic aspects of cell membrane function.

Larvicidal activity of blastospores and conidiospores of Beauveria bassiana (strain GK 2016) against age groups of Aedes aegypti.

Laboratory bioassays of two development stages, blastospores (BS) and conidiospores (CS) of Beauveria bassiana (strain GK 2016) against Aedes aegypti larvae were conducted at 27 degrees C. In Study 1, against 24 h post-hatched larvae, both BS and CS stages showed significant difference in their respective larvicidal efficacy over the control (P less than 0.0001). Larval mortality between 24 and 96 h post-exposure was significantly higher than any other time period investigated. Significantly higher larval mortality was observed with BS or CS at 10(8) ml-1 over lower concentrations (P less than 0.05). In Study 2, against different age groups, 12-24 h post-hatched larvae showed significantly higher mortality when treated with BS or CS than older age groups (P less than 0.05). A significant difference was found in the larvicidal potency of B. bassiana at different fungal stages.

Nucleotide excision repair and photoreactivation in the entomopathogenic fungi Beauveria bassiana, Beauveria brongniartii, Beauveria nivea, Metarhizium anisopliae, Paecilomyces farinosus and Verticillium lecanii.

AIMS: To compare the DNA repair capabilities of the entomopathogenic fungus (EPF) bassiana to the EPF Beauveria brongniartii, Beauveria nivea, Metarhizium anisopliae, Paecilomyces farinosus, Verticillium lecanii, and the fungi Aspergillus niger and Neurospora crassa. METHODS AND RESULTS: Germination of B. bassiana conidiospores following ultraviolet (UV) irradiation was used to show that nucleotide excision repair and photoreactivation decrease the post-UV germination delay. These two modes of repair were characterized and compared between the aforementioned EPF, A. niger and N. crassa using a physiological assay where per cent survival post-UV irradiation was scored as colony forming units. CONCLUSIONS: The results showed B. bassiana and M. anisopliae are the most UV-tolerant EPF. The DNA repair capabilities indicated that EPF do not have all DNA repair options available to fungi, such as A. niger and N. crassa. SIGNIFICANCE AND IMPACT OF THE STUDY: A key factor detrimental to the survival of EPF in agro-ecosystems is UV light from solar radiation. The EPF literature pertaining to UV irradiation is varied with respect to methodology, UV source, and dose, which prevented comparisons. Here we have characterized the fungi by a standard method and established the repair capabilities of EPF under optimal conditions.

Agricultural use of antibiotics and the evolution and transfer of antibiotic-resistant bacteria.

Microbial Resistance to antibiotics is on the rise, in part because of inappropriate use of antibiotics in human medicine but also because of practices in the agricultural industry. Intensive animal production involves giving livestock animals large quantities of antibiotics to promote growth and prevent infection. These uses promote the selection of antibiotic resistance in bacterial populations. The resistant bacteria from agricultural environments may be transmitted to humans, in whom they cause disease that cannot be treated by conventional antibiotics. The author reviews trends in antibiotic use in animal husbandry and agriculture in general. The development of resistance is described, along with the genetic mechanisms that create resistance and facilitate its spread among bacterial species. Particular aspects of resistance in bacterial species common to both the human population and the agrifood industry are emphasized. Control measures that might reverse the current trends are highlighted.

Chromosome replication following a temporary inhibition of DNA-synthesis by nalidixic acid in a temperature-sensitive dnaA mutant of Escherichia coli.

Chromosome replication cycle in a DNA initiation mutant of Escherichia coli (CRT-83, dnaAts) was blocked by nalidixic acid, an inhibitor of the A subunit of DNA gyrase. Following a period of inhibition of DNA synthesis, the drug was removed and "run-out" DNA synthesis was examined. It was found that the "capacity" for DNA synthesis was not affected by such a treatment.

Metabolic effects of trichothecene T-2 toxin.

Cereals and other agricultural products contaminated with trichothecene mycotoxins are unfit for consumption. Until recently, the metabolic effects of T-2 toxin (T-2) were thought to reside in its ability to inhibit protein synthesis. It is now clear that trichothecenes have multiple effects, including inhibition of DNA, RNA, and protein synthesis in several cellular systems, inhibition of in vitro protein synthesis, inhibition of mitochondrial functions, effects on cell division, normal cell shape, and hemolysis of erythrocytes. It is argued that these effects are pleiotropic responses of the cell's biosynthetic network to protein synthesis inhibition. However, in studies with erythrocytes, which lack nuclei and protein synthesis, changes in cell shape and lytic response towards T-2 are observed. Susceptibility to lysis is species dependent and correlates with the presence of phosphatidylcholine. Owing to their amphipathic nature, T-2 and other trichothecenes could exert their cytotoxicity by acting on cell membranes. As for cell energetics, T-2 inhibits the mitochondrial electron transport system, with succinic dehydrogenase as one site of action. Although initial investigations of the metabolic effects of T-2 mediated cytotoxicity suggested the inhibition of protein synthesis as the principal site of action, current thought suggests that the effects of trichothecenes are much more diverse.