Tremorgenic mycotoxins produced by strains of Penicillium spp. isolated from toxic Poa huecu parodi

Seventeen strains of Penicillium spp. have been isolated from Poa huecu Parodi from the Zapala zone, exhibiting toxicity to sheet. The following strains have been identified: P. crustosum, cyclopium, notatum, palitans, puberulum, verrucosum, viridicatum and Penicillium spp. The toxigenic capacity of the strains was studied after growing them under suitable conditions. Toxins produced were analysed by thin layer chromatography (TLC). Penitrem A (PA) and Penitrem B (PB) neurotoxins were identified and quantitated in twelve strains; verruculogen (VERR) and fumitremorgen B (FTB) being present in one of them. The effect of these mycotoxins was studied in mice. Neurological symptoms characteristic of the intoxication by tremorgenic toxins and similar to those observed in sheep suffering from huecu's disease were observed. The possible role of these toxins as causative agents of huecu's disease is discussed.     

Increased yield of a lysozyme after self-cloning of the gene in Streptomyces coelicolor “Müller”

Summary Streptomyces coelicolor Müller DSM3030 excretes a lysozyme comprising both -1,4-N-acetyl-and -1,4-N,6-O-diacetyl muramidase activities. The lysozyme is named Cellosyl. Gene libraries have been established using genomic DNA from the wild-type strain, S. coelicolor DSM3030, and from an overproducing mutant, S. coelicolor HP1, which exhibits about a twofold increase in lysozome production. The lysozyme-encoding genes (cel) from both strains were detected by oligodeoxynucleotide hybridization. The nucleotide sequence of the cel genes isolated from both strains was shown to be identical. The different levels of lysozyme production could not be correlated with any mutations at the cel gene locus. The cel gene isolated from the wild-type strain could not be expressed in some other species of Streptomyces. However, self-cloning of the cel gene into S. coelicolor DSM3030 and HP1 resulted in a 2.5-fold increase in lysozyme production.     

Studies on Baker's yeasts of East Pakistan II

Summary 88 strains of yeast and 4 strains of fungi imperfecti have been isolated from dough from three more districts of East Pakistan. The yeasts comprise 53 strains ofSaccharomyces carlsbergensis, 15 strains ofCandida krusei, 8 strains ofCandida guilliermondii var.membranaefaciens, 6 strains ofTorulopsis colliculosa, 2 strains ofTorulopsis globosa, and 4 strains ofHansenula anomala. Of the four strains of fungi imperfecti, 3 belonged toCladosporium butyri and one toSporophora sp. TheSporophora sp. is considered to be a contaminant.The 92 isolates have been tested for their capacity to ferment -methyl glucoside. The possibility of the utilisation of the fermentation of -methyl glucoside as an additional character in yeast taxonomy has been discussed.Tests for the syntheses of various members of vitamin B-complex have shown that all the 92 isolates are more or less autotrophic.     

Screening for plasmids in the genus Clostridium

A plasmid screening was performed on 150 strains out of 75 clostridial species using a modification of the alkaline-lysis procedure. In 26 strains representing 21 species one or more plasmid bands were detected ranging in size from 3 to more than 100 kilobase pairs. Clostridium aceticum proved to contain a single small plasmid (pCA1) of 5.4 kbp as revealed by restriction analysis and electron microscopy. A physical map of pCA1 has been constructed. Spontaneous mutants of C. aceticum defective in autotrophic growth have been isolated. No direct correlation between plasmid content and autotrophy could be found.Abbreviations EDTA ethylenediaminetetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethane-sulfonic acid - TAE Trisacetate-EDTA - Tris tris-(hydroxymethyl)aminomethane     

Isolation and identification of the antialgal compound, harmane (1-methyl-β-carboline), produced by the algicidal bacterium, Pseudomonas sp. K44-1

Twelve strains of algicidal bacteria were isolated from the surfacewater of the pond Shinobazu and the moat Ote-bori (Tokyo, Japan). Nine of thesestrains were considered to be in the Pseudomonas group byanalyses of 16S rDNA sequences. The methanol extract ofPseudomonas sp. K44-1 showed marked antialgal activity bythe paper disk method. Harmane (1-methyl--carboline) was isolated fromtheethyl acetate extract of the whole culture broth ofPseudomonas sp. K44-1 by using silica gel columnchromatography and high performance liquid chromatography (HPLC). Harmaneshowedantagonistic activities against several cyanobacterial strains at aconcentration of 30 g disk^–1.     

Degradation of lignin and lignin model compounds by various mutants of the white-rot fungus Sporotrichum pulverulentum

In order to better understand which enzyme are of importance in lignin degradation, new cellulase deficient strains from Sporotrichum pulverulentum have been isolated by spontaneous and induced mutations from both wild type and from the earlier studied cellulase deficient strain 44. These new strains are xylanase positive (Xyl⁺), and produce considerably higher amounts of phenol oxidases (Pox) than either parent type. The new strains have been compared with the wild type and strain 44 with respect to their ability to release ¹⁴CO₂ from a) vanillic acid labelled in the carboxyl, methoxyl and ring carbons; b) the dimer (4-methoxy-¹⁴C)-veratryl-glycerol--guaiacyl ether; c) ¹⁴C-ring-labelled DHP and ¹⁴C[-carbon side chain] labelled DHP.The new strains, the wild type and strain 44 were compared with respect to their ability to cause weight losses in wood blocks and to delignify wood. One of the new strains, 63-2, caused a higher weight loss in wood than either the wild type or strain 44. Another strain, 44-2, produced a higher weight loss than strain 44. An increase in acid-soluble lignin was observed in wood blocks treated for two weeks with the two new mutant strains and wild type. After prolonged incubation for 6 and 8 weeks the amount of acid-soluble lignin decreased.Abbreviations DHP Dehydrogenation polymerizate - DMS 2,2-dimethylsuccinic acid     

Studies on dl-Alanine Formation by Thermophilic Bacteria

During the course of the screening of thermophilic microorganisms, several strains were found to accumulate amino-acids. Of those strains that were isolated from feces source with Bennett medium, one strain was found to produce a large amount of an amino acid. This amino acid was isolated in crystalline form and identified as dl-alanine by IR absorption spectrum, specific rotatory power and elementary analysis. The taxonomic studies were carried out and this strain was identified as Bacillus coagulans. The strain B. coagulans B₉-17 produced dl-alanine as much as 1000 mg/l after 24 hours at 50°C in shake culture.

The yield of dl-alanine was increased up to 16.5 g/l with some improvements.     

Isolation and Characterization of Bacteria Capable of Degrading Phenol and Reducing Nitrate Under Low-Oxygen Conditions

Nitrate-reducing bacteria capable of degrading phenol were isolated from natural and contaminated environments under low-oxygen conditions with nitrate-containing media, using phenol as a sole carbon and energy source. A total of 27 bacteria able to degrade phenol and reduce nitrate under low-oxygen conditions were isolated from all of the inoculum samples, regardless of previous phenol contamination. For all of these bacteria, oxygen was an essential requirement for phenol degradation. Nitrate reduction by 19 of the strains was insensitive to 10 mM sodium azide, and these strains were placed into the - and -subclasses of Proteobacteria and two were Gram-positive bacteria. To date, the order of Rhizobiales has hardly been reported to have an ability to degrade aromatic compounds. Interestingly, our study showed that all isolates that were placed into the -subclass of Proteobacteria are in the order of Rhizobiales. Furthermore, the genus Agrobacterium was isolated from most inoculum samples and one genus of Gram-positive bacteria, Staphylococcus, was also isolated. In the case of the remaining eight strains, nitrate reduction was inhibited by 10 mM sodium azide. Of these strains, seven were placed into the -subclass of Proteobacteria.     

Selection of a reserpine-producing cell strain using UV-light and optimization of reserpine production in the selected cell strain

Cultured Rauwolfia serpentina calluses consisted of cell colonies that had different fluorescences under 365 nm UV-light. Fluorescence was divisible into two main color categories, yellow-green and blue-white. Two cell strains of different fluorescence were selected from calluses cultured in the dark, by the fluorescence assay. Even after the selected strains had been transferred to liquid medium, they maintained similar fluorescence. HPLC analysis showed that the yellow-green fluorescent strains produced much reserpine, whereas the blue-white strains produced much 3,4,5-trimethoxy benzoic acid. A combination of 10M NAA and 10M BA enhanced production of reserpine in the yellow-green fluorescent cell strains.     

Temperature-sensitive mutations affecting the replication of F-prime factors in Escherichia coli K 12

Summary More temperature-sensitive mutants affecting the replication of the F-gal⁺ episome of Escherichia coli K12 have been isolated. Eight of the mutations were located on F itself and three were located on the chromosome.The temperature sensitive F-gal⁺'s have been integrated into the chromosome to produce Hfr strains. These Hfr strains have transfer origins similar to Hfr Cavalli, and all show aberrant excision and transfer of elongated segments of the chromosome including the integrated F-gal to generate long merodiploids.The chromosomal mutations that govern the replication of F have been termed seg (for segregation). Wild-type F-gal⁺ can be integrated into seg cells at 42° C to give Hfrs, in a process analogous to integrative suppression in the formation of Hfrs from cells carrying mutations that are temperature-sensitive for chromosomal DNA replication (dnaA). A curious feature of an Hfr derived from a seg strain is that it also shows F-genote enlargement as well as normal transfer of chromosomal genetic marker. Preliminary transductional mapping data show that the mutation seg-2 is linked to the threonine locus (minute 0).     

A translocation associated, loss-of-function mutation in the nitrogen metabolite repression regulatory gene of Aspergillus nidulans can revert intracistronically

Summary The areA ^r -18 mutation is a loss-of-function mutation in areA, the positive acting regulatory gene mediating nitrogen metabolite repression in Aspergillus nidulans. It results from a reciprocal translocation which splits the coding region into 5 and 3 moieties. Surprisingly, we have selected rare intracistronic revertants of areA ^r -18. From crosses heterozygous for areA ^r -18 revertant alleles, duplication-deficiency progeny containing two copies of a substantial portion of chromosome IV but lacking part of chromosome III, including the 5 moiety of areA, have been obtained. For all four revertants analysed genetically, growth properties of these duplication-deficiency strains indicate that the reversion events involve the 3 portion of areA and that the 5 portion of areA is unnecessary for the revertant phenotype. This conclusion was directly confirmed for one revertant using Southern blotting. As all four reversion events involve additional chromosomal rearrangements, they probably fuse functional promoters, ribosome binding sites and in frame initiation codons to the 3 portion of the gene. In the course of characterisation of these mutations, new mapping data for a large region of chromosome IV have been generated, and a new reciprocal translocation activating the cryptic regulatory gene areB, whose product can substitute for that of areA, has been identified.     

Genetic mapping by means of protoplast fusion in Bacillus subtilis

Summary A new mapping method involving protoplast fusion in Bacillus subtilis is described. Protoplasts from an isogenic standard marker strain containing purA and from a strain containing both purB and the marker, x, to be mapped were fused with polyethylene glycol, and purA ⁺ purB ⁺ fusants were selected. After isolation of single colonies and determination of unselected markers, marker x was mapped between two standard markers. This method was fully applicable to PBS1-resistant strains (e.g., lyt strains). The results obtained by protoplast fusion, conventional transformation and/or lysed protoplast transformation indicated that a lyt strain, Ni15, contained two new autolysin-minus mutations (lyt-151 and lyt-152). The properties of lyt-15 are also discussed.Abbreviations NTG N-methyl-N-nitro-N-nitrosoguanidine - SMM 0.5 M sucrose, 0.02 M MgCl₂, 0.02 M maleate buffer, pH 6.5     

The Genetic Basis of Malathion Resistance in Housefly (Musca domestica L.) Strains From Turkey

Organophosphate insecticide (parathion/diazinon) resistance in housefly (Musca domestica L.) is associated with the change in carboxylesterase activity. The product of MdE7 gene is probably playing a role in detoxification of xenobiotic esters. In our research, we have isolated, cloned and sequenced the MdE7 gene from five different Turkish housefly strains. High doses of malathion (600 g/fly) were applied in a laboratory environment for one year to Ceyhan1, Ceyhan2, Adana, and Ankara strains while no insecticide treatment was performed in the laboratory to Kirazli strain. Trp²⁵¹ Ser substitution was found in the product of MdE7 gene in all malathion-resistant and Kirazli stocks. In addition, we checked the malathion carboxylesterase (MCE), percent remaining activities in acetylcholinesterase (AChE), glutathion-S-transferase (GST), and general esterase activities in all five strains used in this study. In comparing with universal standard sensitive control WHO, a high level of MCE and GST activities were observed while lower level of general esterase activities was detected in the tested strains. In addition, a higher percent remaining activities in AChE than WHO susceptible strain were observed in all malathion-resistant strains.     

Isolation of auxotrophic and antimetabolite-resistant mutants of the hyperthermophilic bacterium Thermotoga neapolitana

Antimetabolite-resistant and auxotrophic mutants of the hyperthermophilic bacterium Thermotoga neapolitana were isolated to provide strains with genetic backgrounds amenable to genetic analyses. Norleucine, azaleucine, 4-nitropyridine-N-oxide, and 3-amino-1, 2, 4-triazole did not affect growth, while 5-fluorouracil (5 g/ml), 5-methyltryptophan (250g/ml), 6-azauracil (100 g/ml), and 4-fluorophenylalanine (30 g/ml) inhibited growth at the indicated minimum inhibitory concentrations. The effect of 5-fluorouracil was analyzed and found to be bacteriostatic. These inhibitors were used to select spontaneously arising resistant mutants. In addition, auxotrophic mutants requiring leucine, tryptophan, adenine, and histidine were isolated following mutagenesis with ethyl methanesulfonate. Six other auxotrophs with undefined growth requirements were also isolated. These strains will be useful for the development of genetic methods for T. neapolitana.     

Aflatoxin — induced alteration in the levels of membrane chemicals of subcellular organelles isolated from excised,incubated Glycine max,cv. ‘Essex’ roots

Isolates of aflatoxin-producing strains of Aspergillus grow on autoclaved and field-grown (lesser extent) Glycine max beans. Both mixed and aflatoxin B₁ inhibit G. max, cv. Essex bean germination and elongation of either attached or excised cultured roots. Because B₁ impairs the latter roots' ability to intracellularize [¹⁴C]-leucine, it may alter plasmalemma structure and/or function. To determine whether incubation of excised roots for 18 hours in toxin-containing medium could affect cellular membrane chemical content, organelles were isolated by differential centrifugation (1 000, 40 000, and 80 000 xg) of homogenates and characterized chemically. Statistically significant differences between treated and untreated roots in acid insoluble protein but not either sterol or lipid phosphorus levels were observed for both 40,000 and 80,000 xg pellets. Protein and sterol recoveries were 81 (treated) and 84 (untreated) % for the former and 77 (treated) and 79 (untreated) % for the latter. Lipid phosphorus recoveries were 87.3 (treated) and 136 (untreated) % with and 96 (treated) and 83 (untreated) without membrane stabilization. Protein:sterol:lipid phosphorus were 35.74.51 (1 000 xg), 18.93.61 (40000 xg), 26.34.61 (80 000 xg) and 1,010291 (80 000 xg supernatant) for untreated and 36.93.31 (1,000 xg), 23.13.81 (40 000 xg), 36.24.81 (80 000 xg) and 1,05321.71 (80 000 xg supernatant) for treated roots. Significant differences in RNA content between treated and untreated roots were found for both 1 000 and 40 000 xg pellets but not for the 80 000 xg pellet and its supernatant. Whereas a significant increase in the 1 000 xg pellet occurred upon treatment, a decrease was noted for the 40 000 xg pellet but not for the 80 000 xg pellet and its supernatant. Similar pH 6 (plasmalemma marker enzyme) and 9 (mitochondrial marker enzyme) K⁺-stimulated ATPase activities were demonstrated for 40 000 and 80 000 xg pellets. The 1 000 xg pellet contained greater than 50% of the NADH-cytochrome c-reductase activity (endoplasmic reticulum marker enzyme) recovered from fractions examined for this activity which was absent from the 40 000 xg pellet. Both the 80 000 xg pellet and its supernatant possessed equivalent reductase activities. Inosine diphosphatase activity (dictyosome marker enzyme) was not present in 1 000 xg pellets obtained from either treated or untreated roots but was in both 40 000 and 80 000 xg pellets. Based on these results, a tentative assignment of organelles to each fraction (xg force) is reported.Abbreviations used AFB₁ aflatoxin B₁ - AFB₂ aflatoxin, B₂ - AFG₁ aflatoxin G₁ - AFG₂ aflatoxin G₂ - ATPase adenosine triphosphatase - IDPase inosine diphosphatase - NADH reduced nicotinamide-adenine dinucleotide - PCA perchloric acid - TCA trichloroacetic acid - 2, 4-D 2,4-dichlorophenoxyacetic acid Aided by grant IN-127 from the American Cancer Society to WVD and funds from the Departments of Biology, West Virginia University and Virginia Commonwealth University as well as a Sigma Xi award to JMD.     

Agrobacterium-mediated transformation of Arabis gunnisoniana

An efficient method for adventitious shoot regeneration for Arabis drummondii and a transformation protocol for A. gunnisoniana from hypocotyl explants are described. Hypocotyl explants from 7-day-old aseptically grown seedlings were cultured on MS medium containing plant growth regulators (6-benzylaminopurine, 1-phelyl-3- (1,2,3-thiadiazol-5-yl) urea, -naphthaleneacetic acid and 2,4-dichlorophenoxy-acetic acid). After 4 weeks in culture, high frequency of adventitious shoot regeneration was observed. Regenerated shoots were rooted on half-strength MS basal medium supplemented 1% (w/v) sucrose, with or without NAA. This protocol was then used to produce transformed Arabis gunnisoniana plants. A. gunnisoniana hypocotyl explants were co-cultivated with Agrobacterium tumefaciens strain GV3101 harbouring pBJ40. Transgenic shoots were selected on MS 21 medium supplemented with 50 mg l kanamycin. PCR analysis verified the presence of the nptII gene in the plant DNA isolated from kanamycin resistant shoots.     

Development of gene transfer methods forRubrivivax gelatinosus S1: construction, characterization and complementation of apuf operon deletion strain

Gene transfer systems were developed inRubrivivax (Rx.) gelatinosus S1. First, a system for conjugative transfer of mobilizable plasmids fromEscherichia coli toRx. gelatinosus S1 was established. Secondly, optimal conditions for the transformation ofRx. gelatinosus S1 by electroporation were determined. A puf strain was constructed. Complementation with thepuf operon from a wild-type strain cloned in a replicative plasmid restored photosynthetic growth. Two insertion strains were also selected. All the strains constructed were green, due to a change in carotenoid content. Characterization of these strains provides genetic evidence for a superoperon organization in this bacterium.     

Fertility ofFusarium moniliforme from maize and sorghum related to fumonisin production in Italy

Forty-three strains ofFusarium moniliforme isolated from infected maize and sorghum plants in Italy were assayed for their ability to produce fertile crosses with A and F mating population tester strains, in relation to their ability to produce fumonisins on maize substrate. Most of the strains isolated from maize (ear and stalk rot and maize-based feed), producing fumonisin B₁ (FB₁) and B₂ (FB₂) (up to 4,100 and 855 mg/kg, respectively), belonged to the A mating population. All of the strains isolated from sorghum belonged to the F mating population and produced little or no FB₁ and FB₂. This is the first report of the occurrence of mating population F in Europe. Our data on strains from Italy are consistent with previous studies from the United States that found significant differences in sexual fertility and fumonisin production between strains from maize and sorghum.     

Agrobacterium tumefaciens-mediated transformation of Vigna mungo (L.) Hepper

Transformed Vigna mungo (blackgram) calli were obtained by cocultivating segments of primary leaves with Agrobacterium tumefaciens vir helper strains harbouring the binary vector pGA472 having kanamycin resistance gene as plant transformation marker. Transformed calli were selected on Murashige and Skoog medium supplemented with 50 mg/l kanamycin and 500 mg/l carbenicillin. Transformed calli were found to be resistant to kanamycin up to 900 mg/l concentration. Expression of kanamycin resistance gene in transformed calli was demonstrated by neomycin phosphotransferase assay. Stable integration of transferred DNA into V. mungo genome was confirmed by Southern blot analysis.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - 2iP 6-(,-dimethylallylamino)purine - Kn kinetin - nptII neomycin phosphotransferaseII - MS Murashige and Skoog (1962) medium     

Susceptibility of clinical isolates of yeasts to anti-fungal agents

The antimicrobial activity of amphotericin B, 5-fluorocytosine, nystatin, clotrimazole and miconazole were compared in vitro against 244 strains of yeasts that had been isolated from clinical specimens. The yeasts used in this study included 20 species of Candida, Cryptococcus, Saccharomyces Geotrichum, Rhodotorula, Torulopsis and Trichosporon. The majority of the strains (78%) had an MIC of 0.5 g/ml for amphotericin B, 81% an MIC of 1 g/ml for 5-fluorocytosine, 99% 8 g/ml for nystatin, 91%, 8.0 g/ml for clotrimazole and 98% had an MIC of 4.0 for miconazole. Of the anti-fungal agents tested, 5-fluorocytosine and nystatin were found to have the greatest antifungal activity.